JP2000060585A - Production of ethanol by culture conversion of carbon dioxide into biomass - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for producing ethanol by conversion of CO2 into a biomass, having high productivity, capable of carrying out the conversion into the biomass in an outdoor land. SOLUTION: In this method for producing ethanol through conversion of CO2 into a biomass, collection of a crude extracted solution of D-glucose from the biomass after the conversion and production of ethanol from the crude extracted solution of, D-glucose, the conversion of CO2 into the biomass is performed by culturing a blue-green algae in a minimum medium comprising three different kinds of inorganic salts or the salts as main components under aeration of CO2-containing gas.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for producing ethanol by converting CO ₂ culture biomass. More specifically, the invention of this application is to selectively fix CO ₂ from exhaust gas from a thermal power plant and to produce ethanol from this fixed CO ₂ more efficiently and as a highly practical system. The present invention relates to a new method for producing ethanol by converting CO ₂ culture biomass that can be used.

[0002]

2. Description of the Related Art As one of global environmental problems, coping with global warming by CO ₂ (carbon dioxide) has become a major issue worldwide, and at the Kyoto Conference held in 1996, Japan Since they also promise to cut CO ₂ emissions by 6%, measures to achieve this target have become an urgent technical issue.

Regarding CO ₂ reduction, various approaches have been proposed so far, and specific research and development have been underway, but thermal power plants have become a large emission source of CO ₂ emissions. It is not practically easy to collect CO ₂ emitted from various plants including the above-mentioned by the conventional technology. Under such circumstances, the biomass conversion technology proposed by the inventors of the present application has been attracting attention as a technology for recovering and recycling CO ₂ . In this technology, exhaust gas emitted from a thermal power plant or the like is ventilated to photosynthetically grow cyanobacteria (cyanobacteria) to fix CO ₂ and convert it into cultured biomass, and then the cultured biomass is converted into D-glucose. A crude extract is obtained, and further ethanol is produced by enzymatically fermenting the crude D-glucose extract, which is characterized by obtaining ethanol useful as a fuel or the like by collecting exhausted CO _2. There is. This makes it possible to convert CO _{2 into} a re-energy resource.

FIG. 1 shows an example of the configuration of this technique, and more specifically, it is characterized by producing ethanol through the following process (Energy Vol. 2).
2, No. 2/3, pp 353-356, 1997; En
Energy Convers. Magnt Vol.38, Suppl.pp. S523
-S528, 1997). <1> Biomass conversion that fixes CO ₂ by photosynthetic growth of Synechococcus leopoliensis <2> Acid solubilization of biomass Hydrolysis, neutralization, desalination to obtain crude D-glucose extract <3> As biomass extract Yeast of crude D-glucose extract of :; Production of ethanol by fermentation with Saccharomyces cerevisiae Proposed by the inventors, for example, fixation by conversion of culture biomass of CO ₂ as described above and conversion to ethanol re-energy resource The method is for photosynthetic growth of cyanobacteria by ventilating exhaust gas from a thermal power plant to an outdoor open pond, mild operation conditions such as reaction by yeast, and relatively small equipment burden, and It is expected to be developed in the future as having characteristics such as efficient ethanol production.

[0005] However, the technology for converting CO ₂ culture biomass and ethanol production had further problems to be investigated in order to make the method more practical. One of them is that the composition of the medium for the photosynthetic growth of cyanobacteria requires the preparation of many components, and for cultivation in an open outdoor pond, the medium composition should be as few components as possible to reduce the risk of contamination by bacteria. There is a need for a large-scale open pond that can be reduced and have a larger liquid depth, and thus a smaller light irradiation area, and next, the D-glucose production efficiency of biomass can be further increased. It was desired to further increase the ethanol production capacity.

[0006]

Therefore, the invention of this application was made in order to solve the problems as described above, and it is the conversion of CO ₂ into biomass and the subsequent D-from biomass. Acquisition of crude glucose extract and D
In a method for producing ethanol via production of ethanol from a crude glucose extract, CO ₂ is produced by using three different inorganic salts or a minimal medium containing the inorganic salts as a main component.
A method of ethanol production by the culture biomass conversion of CO _2, characterized in that by culturing the cyanobacteria open to air containing gas performs biomass conversion CO _2.

Further, the invention of this application is the above method, wherein CO ₂ is converted into biomass of cyanobacteria by using an outdoor open pond, and the biomass is acid-solubilized and hydrolyzed to perform crude D-glucose extraction. There is also provided a method characterized in that a liquid is obtained, ethanol is produced from a crude D-glucose extract by fermentation with yeast, and the CO ₂ -containing gas is exhaust gas.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION The invention of this application has the characteristics as described above, and the embodiments of the invention will be described in more detail below. First, in the method for producing ethanol by CO ₂ biomass conversion of the invention of this application, in the same manner as the process along the above-mentioned FIG. 1, <1> CO ₂ is immobilized and converted to biomass <2> Biomass is converted from biomass. Acquisition of D-glucose crude extract <3> Configured as a process of ethanol production from D-glucose crude extract. In the <1> CO ₂ biomass conversion of the invention of this application, as described above, three different kinds of inorganic salts or a minimal medium containing the same as the main component is used to indigo a cyanobacteria under aeration of a CO ₂ -containing gas. The major feature is that CO ₂ is converted into biomass by culturing.

The present invention is also characterized in that CO ₂ can be converted into cyanobacterial biomass using an outdoor open pond. The present invention provides guidelines for designing and operating conditions such as location conditions, aeration conditions, and deepening of the liquid that enable cultivation in the outdoor open pond. Synechococcus leopoliens, which the inventors have already proposed, is the cyanobacteria in this case.
is (Anacystis nidulans IAMM6, Synechococcus
PCC6301) is a preferred representative.
Of course, other things will naturally be considered.

Biomass conversion by fixing CO ₂ is C
O ₂ -containing gas, for example, exhaust gas from a thermal power plant, an incinerator, a chemical plant, or the like is aerated to perform photosynthetic growth. More preferably, it is a bubble-disturbed flow high-depth culture by blowing a mixed gas of exhaust gas and compressed air. It is considered that the culturing is carried out at room temperature to about 50 ° C., and the culturing in an outdoor open pond is carried out at room temperature accompanying seasonal changes or under slightly heated conditions.
The concentration of CO _{2 in} the blown gas and the gas flow rate are not particularly limited, but it is considered to be about 1 to 15 vol% of the total amount including air and other components, for example. Considering that the gas flow rate is preferably 12 to 15 m / h, the gas line concentration based on the superficial column is preferably 12 to 15 m in order to carry out bubble submerged flow type deep liquid culture.

The light for photosynthesis is provided by light irradiation from a bioreactor sunlight or a lamp, but in an open outdoor pond, efficient use of sunlight is considered.
There are no particular restrictions on the location of outdoor open ponds using sunlight, and any location that can be attached to a CO ₂ emission source such as a thermal power plant, an incinerator, or a chemical plant can be used. Utilization of land and marshes is possible. The activity of CO ₂ fixation reaction in an open outdoor pond is highly dependent on the duration of sunshine. For this reason, the temperate zone or the subtropical zone where the sunshine duration is about 9 hours is considered as a preferable location condition for the outdoor open pond. In order to attach an outdoor open pond to a CO ₂ emission source, it is important to secure a light irradiation area that is as small as possible for planned implementation. If this is possible, it is desirable to set the liquid depth as large as possible. In the outdoor culture technique of the invention, a deep liquid depth is achieved by performing bubble disturbed flow culture with compressed air accompanied by exhaust gas.

In the present invention, the medium for culturing is
Is composed of 3 different inorganic salts or the main component
Use the minimum medium. As the inorganic salt in this case,
For example, metal cations such as K, Na, Mg, Ca, Fe and Mn.
Salt of anion and the same metal cation
Used as a kind of different inorganic salt if different
To be done. The same applies to the same anion. Anion
For example, NO₃ ^-, HPO_Four ^2-, SO_Four ^2-etc
Is.

The three different salts that make up the medium include
For example, KNO ₃ / K ₂ HPO ₄ / MgSO ₄ · 7H ₂ O KNO ₃ / Na ₂ HPO ₄ · 12H ₂ O / MgSO ₄ ·
7H ₂ O K ₂ HPO ₄ / MgSO ₄ .7H ₂ O / NaNO _{3 and the} like are exemplified. More preferably, the metal is K or M
g is an essential component and is an inorganic salt with different anions (acid components).

The concentration of the three kinds of inorganic salts constituting the medium is 1.46 to 1.50 g / dm ³ as a whole, which constitutes the minimum medium of the present invention. The fact that biomass conversion is possible by fixing CO _{2 in} a minimal medium consisting of only three different inorganic salts is first presented in this invention, but in practice, these three inorganic salts are the main components. In addition, a trace amount of another inorganic salt may be further added. In each of these cases, 3 as the minimum medium
By clarifying the composition of inorganic salts of different species, it becomes possible to reduce the amount of inorganic salts and reduce the risk of contamination by various bacteria. For example, as a trace component, CaCl ₂ ·
_{2H 2 O, FeSO 4 · 7H} 2 O, MnSO 4 · 7H 2
O etc. are mentioned.

More specifically, in the present invention, <A> three different inorganic salts, for example, KNO ₃ / K ₂ HPO ₄ / MgSO ₄ · 7H ₂ O <B> three different inorganic salts. , For example, KNO ₃ / Na ₂ HPO ₄ · 12H ₂ O / MgSO ₄ ·
7H ₂ O and the smallest trace components, eg CaCl ₂ .2H ₂ O / FeSO ₄ .7H ₂ O / MnS
A combination of O ₄ .7H ₂ O, etc. is shown. Trace components, as a whole,
0.92 g / dm ³ or less is sufficient, and the amount can be made extremely smaller than 1/100 or less than that of MDM which is a medium which has been often used conventionally, and the total inorganic salt is 1.46 to A preferable amount is 1.50 g / dm ³ and 90% or less of MDM. These culture media of the present invention do not require Zn and Cu cation salts which have been often used conventionally.

The minimum medium, which is a feature of the present invention, will be further described. For example, M3 is used in Examples described later.
And CS medium have been used, which are essentially different from conventional MDM (Modified Detmer Medium). Known technology MDM (Modified Detmer Medium)
Is intended for use as a medium for a wide range of cyanobacterial photosynthetic growth. It is perfect as a medium candidate for the storage and growth of newly isolated cyanobacteria, but it has sugars (for MDM Although it does not contain sugar, it is judged as unsuitable as a medium for outdoor open pond culture, because sugar is produced from cell lysates when cyanobacteria are killed) and bacterial contamination easily occurs. In this medium, the rate at which CO ₂ is taken into the dry solid weight (CUR) is extremely high, and 0.00619 to 0.0706 per culture unit volume in photosynthesis with a liquid depth of 0.02 to 0.0742 m.
Reach g / (dm ³ · h).

On the other hand, the M3 medium of the present invention (Minimal 3 c
omponents Medium) is used for: cyanobacteria 240 hours photosynthetic reaction (activity: MD
It is a minimum medium for about 1/3 of the activity in M) and has a very small number of components that are not known in the art. Since this solution does not contain trace components at all, it is suitable for outdoor cultivation because it is unlikely to contaminate bacteria. Further, the medium exchange interval for outdoor culture is about 24 to 60 hours, and the above-mentioned time limit of 240 hours is a sufficient time interval and no problem occurs in the culture operation. Also, 24
If a trace component (described in CS medium) is administered to the open pond every 0 hours, the cyanobacteria 240 hours photosynthesis can be continued semipermanently.

Further, the CS medium of the present invention (Cyanobacteria
l Synthetic Minimal Medium) is the minimum medium for the purpose of use: cyanobacterial photosynthesis reaction (similar to the activity in active MDM), and can maintain the activity similar to that in MDM for a long time, and The medium has a very small number of components and a very small amount of trace components, which are not known in the art.

[0019] MDM is a nutrient solution listed for all kinds of cyanobacteria and for multipurpose use without failing to contain necessary trace components, but CS medium is a cyanobacteria Synechococcus
For photosynthesis for leopoliensis and outdoor open ponds, unnecessary trace components are excluded as much as possible, and it is composed of the minimum necessary trace component number and content. This difference may be emphasized.

In the present invention, the CO ₂ -immobilized culture biomass is then acid-solubilized and hydrolyzed to produce D-glucose. Regarding the hydrolysis at this time, the concentration is 1
It is considered to be carried out at a temperature of 40 to 90 ° C. for 10 to 60 g / dm ³ biomass (hydrochloric acid aqueous solution) using ˜6 mol / dm ³ mineral acid, more preferably HCl (hydrochloric acid). This allows the maximum amount of D-glucose production.

Next, after the production of D-glucose by hydrolysis, alkali neutralization and desalting are carried out. Then, in the method of the present invention, D as a biomass extract was used.
-Fermentation of glucose with yeast produces ethanol. As yeast, Saccharomyces cerevisiae IFO
2347 (Institute for Fermentation (Osaka, Japa
n)) is shown as a preferred representative. Aerobic culture
It is considered to be performed at a temperature of room temperature to 40 ° C.

In the ethanol fermentation method using the yeast of the present invention, the production efficiency of ethanol becomes higher. That is, since the D-glucose sugar extract derived from cyanobacterial biomass prepared by the present invention contains a fermentation raw material that can be converted into ethanol in addition to D-glucose, the yield of ethanol is commercially available. It is higher than when D-glucose is used as the fermentation raw material. Further, since the crude D-glucose extract also has an effect of increasing the ethanol tolerance of yeast, it is considered to use the D-glucose sugar extract as a raw material for high-concentration ethanol fermentation.

Therefore, the method of the present invention will be described in more detail with reference to the following examples.

[0024]

[Example] Biomass conversion was carried out by fixing CO ₂ using each of the media shown in Table 1. Synechococcus leopoliens
is (Anacystis nidulans IAMM-6) (Microbialand
Microalgal Research Center University of Tokyo)
Photosynthetic culture with hotobioreactor was performed while aeration of a mixed gas of CO ₂ 6% -air. Gas flow rate is 1
It was 2.0 m / h. Light was irradiated using a 10 kl _x fluorescent lamp.

[0025]

[Table 1]

The medium MDM is known (A. Watanabe, J. Gen. A.
ppl.Microbiol.6,283 (1960)), and the M3 medium is the minimum medium of the present invention (three inorganic salts).
Then, the CS medium is the minimum medium to which the trace components of the present invention are added. The total amount MDM is 1.62g / dm ^3, M3 medium 1.50g / dm ^3, CS medium 1.46g / dm ³
And M3 medium and CS medium were 92.6% of MDM and 9%.
The total amount is about 0.1% lower.

The number of inorganic salt components was 11 for MDM, while it was extremely reduced to 3 for M3 and 8 for CS. The number of trace components other than nitrates, phosphates, and magnesium salts and The total amount of trace components is 8 kinds of MDM 125 mg / d
m ³ medium, while M3 medium is 0 species 0 mg / dm ³ ,
The CS medium is overwhelmingly reduced to 5 kinds of 0.918 mg / dm ^3, and the total amount of the minor components of the CS medium obtained by supplementing the M3 medium with the minor components is about 0.63% of the total amount of the CS medium. It is a "minor component", which is less than 0.1% of the total amount of minor components of MDM, and the number of metal cations is 8 in MSM, while 2 in M3 medium.
The number of seeds and CS medium has reached a breakthrough of 6 types.

Thus, M3 medium and CS medium are
It is a nutrient solution that is composed of only essential components (but not CO ₂ ) for the photosynthetic growth of cyanobacteria and contains the required amount of each component. FIG. 2 shows the above S. leop in the case of the medium M3.
It shows the proliferation of oliensis, and Passage in the figure shows the number of subcultures using only M3 medium, that is, the number of passages. It can be seen that even with the minimal medium of the present invention, CO ₂ -fixed photosynthetic growth can be performed without lowering the activity for 240 hours or more, which is the second passage number.

Table 2 shows MDM medium, M3 and CS medium.
CO in each case of the ground₂It shows the immobilization ability,
From Table 2, it can be confirmed by the M3 and CS medium of the present invention.
Indeed CO₂It can be seen that immobilization is being performed. MDM
Compared to the medium, CO in the M3 medium depends on the cells₂Intake rate
Is CO₂ uptake rate (CUR) and biomass growth
Quantity X_fAlthough it is low, in the CS medium of this invention, MDM
Comparable CO ₂Do you know that fixation can be achieved?
It

[0030]

[Table 2]

In Table 2, μ indicates the specific growth rate of biomass, and Y _{noa / x} indicates the yield of nitrate ion by the biomass. It can be seen that the nitrate ion yield of M3 and CS medium of the present invention is higher than that of MDM. Figure 3
Shows the evaluation results of contamination of various bacteria in the media M3 and CS of the present invention. From this FIG. 3, E. coli (carbon source, 0.311 g / dm ³ D-gl
It can be seen that there is almost no growth (X) of ucose) and that it has an excellent effect of reducing the risk of contamination. On the other hand, it has been confirmed that in the case of the known medium MDM, Escherichia coli remarkably grows over time, and after 10 hours, for example, grows up to 3 times as much as in the case of M3 and CS medium.

From this, it can be seen that the culture medium of the present invention has an extremely low risk of biomass growth by CO ₂ fixation due to contamination of various bacteria and can be effectively used even in an open pond outdoors. The effectiveness of biomass growth in outdoor open ponds is also supported, for example, by the results shown in Figure 4 with CS medium without pH control and under 9-hour and 24-hour illumination, respectively.
This is because, in any case, almost the same biomass growth (X _f ) is observed at 120 hours after the growth has stopped.

Further, FIG. 5 shows the results when the pH was controlled in the CS medium so that the pH was 7.8 and the lighting time per day was changed. Even with just the lighting, a considerable proliferation effect is recognized. FIG. 6 shows the CUR in relation to the lighting time per day and the number of operating days. CO ₂ consumption rate CU
The maximum value of R is obtained when the sunshine duration is 9 hours, and it is understood that the location conditions of the outdoor open pond of the present invention are preferably temperate and subtropical.

From the above results, it can be seen that the medium of the present invention can effectively grow biomass in an open outdoor pond. Furthermore, FIG. 7 shows the relationship between the cell concentration (X _f ) and the culture solution depth ( _HL ) for biomass growth in the medium of the present invention, which is 0.4 m.
It has a concentration of approximately 0.4 g / dm ³ even at a liquid depth of more than two times, and the liquid depth is more than double that of the open-air pond for chlorella that has been reported in many cases by the introduction of the bubble-disturbed flow high-depth culture method. It can be seen that the light irradiation area can be made large and therefore the light irradiation area can be made small.

Next, 3 mol / dm ³ HCl (50 g / dm ³ biomass-HCl aqueous solution) was added to the biomass obtained by the culture, and the mixture was treated at a temperature of 80 ° C. for 20 minutes to solubilize and hydrolyze the biomass. Was saccharified by. It was neutralized with ³ mol / dm ³ of NaOH and desalted with a cation conversion resin and an anion conversion resin.

By this operation, 0.501 g of D-glucose could be produced per 1 g of dry weight of biomass. D-glucose was obtained in a very high yield. Then Saccharomyces cerevisiae
Using IFO2347, aerobic culture was performed at a temperature of 30 ° C. to produce ethanol.

FIG. 8 is a diagram showing the relationship between the yeast cell concentration (X) and the produced ethanol concentration (C _p ). FIG. 9 shows the results of an experiment in which ethanol having a relatively high concentration for yeast was added to yeast in the logarithmic growth phase, and the effect of ethanol on the specific growth rate of yeast was examined. The horizontal axis shows the ethanol concentration after addition, the vertical axis shows the specific growth rate (μ) after addition, and the intersection of the straight line and the horizontal axis in the figure is the ethanol concentration at the time when the yeast growth is stopped. Indicates the value used to judge the size. In all the figures, control indicates the conversion of D-glucose to ethanol when using commercially available purified D-glucose as a carbon source for yeast culture. Also, biomass
Extract indicates the conversion of D-glucose in the crude extract into ethanol when the crude D-glucose extract derived from cyanobacteria was used as the carbon source. From FIGS. 8 and 9,
It can be seen that according to the example of the present invention, ethanol production becomes more efficient and ethanol tolerance can be improved (FIG. 9).

According to the above example, the same amount of fuel ethanol can be produced by converting the biomass in the open open pond area of about 1/25 of the cultivated area of sugar cane as biomass.

[0039]

INDUSTRIAL APPLICABILITY As described in detail above, the invention of the present application enables efficient and highly productive production of ethanol by CO ₂ biomass conversion, and provides a highly practical method. More specifically, the biomass conversion using the culture medium of the present invention enables efficient biomass conversion in an open outdoor pond and suppression of contamination of various bacteria, and further, the productivity of D-glucose production and ethanol production is also high. Will be improved. By recovering CO ₂ emitted from a thermal power plant or the like, the fuel ethanol can be recycled as an energy resource.

[Brief description of drawings]

FIG. 1 is a block diagram showing a process of CO ₂ biomass conversion and ethanol production.

FIG. 2 is a diagram showing an example of biomass growth in the M3 medium of the present invention over time.

FIG. 3 is a diagram showing the growth of Escherichia coli over time in the case of the medium M3 and CS of the present invention.

FIG. 4 is a view showing biomass conversion in the CS medium of the present invention with time as a case of changing the illumination time per day.

FIG. 5 is a diagram showing the dependency on illumination time, as in FIG.

FIG. 6 is a diagram showing a relationship between CUR and illumination time and operation time.

FIG. 7 is a diagram showing the relationship between cell concentration and liquid depth.

FIG. 8 is a diagram showing the relationship between the cell concentration (X) and the produced ethanol concentration (C _p ) during ethanol production.

FIG. 9 is a diagram showing the relationship between the ethanol concentration and the growth rate (μ) during ethanol production.

Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) (C12P 19/00 C12R 1:01)

Claims (5)

[Claims] 1. A method for producing ethanol by converting CO ₂ into biomass and then obtaining a crude D-glucose extract from the biomass and producing ethanol from the crude D-glucose extract is different in 3 Inorganic salts of the species or CO
_A method for producing ethanol by converting CO ₂ culture biomass, comprising culturing cyanobacteria under aeration of a gas containing ₂ to convert CO ₂ into biomass. 2. The method for producing ethanol according to claim 1, wherein CO ₂ is converted into a cyanobacterial biomass using an outdoor open pond. 3. D- by acid-solubilizing and hydrolyzing biomass
The method for producing ethanol according to claim 1 or 2, wherein a crude glucose extract is obtained. 4. The method for producing ethanol according to claim 1, wherein ethanol is produced from the crude D-glucose extract by fermentation with yeast. 5. The method for producing ethanol according to claim 1, wherein the CO ₂ -containing gas is exhaust gas.