DESCRIPTION
Title of Invention
METHOD FOR PRODUCING SUGAR, METHOD FOR PRODUCING ETHANOL,
METHOD FOR PRODUCING LACTIC ACID, AND METHOD FOR PRODUCING
STARTING MATERIAL FOR ENZYMATIC SACCHARIFICATION USED
THEREIN
Technical Field
The present invention relates to a method for producing a sugar, a method
for producing ethanol, and a method for producing lactic acid, all of which utilizes a
biomass material, and also relates to a method for producing a material for
enzymatic sacchrification that is used in the method for producing a sugar, the
method for producing ethanol, and the method for producing lactic acid.
Background Art
Recently, as one of the countermeasures for global warming, it has been
widely attempted to use, as various fuels or chemical feedstocks, ethanol produced
from a material containing cellulose such as woody biomass, herbaceous biomass,
and the like. The production of ethanol from a biomass material can be done, for
example, by decomposing the collected biomass material into a sugar in a
saccharification step, followed by turning the sugar into ethanol using
micro-organisms such as yeast, and the like in a fermentation step.
Meanwhile, use of biodegradable polymers has been increased for reducing
environmental loads, and lactic acid has been used as one of the materials therefor.
This lactic acid can be also produced by saccharification of a biomass material,
followed by fermentation thereof.
The saccharification has conventionally been carried out using concentrated
sulfuric acid, but the reduction in the amount of sulfuric acid for use has been
desired for reducing environmental loads. To this end, saccharification of a biomass
material using an enzyme has been widely studied as a means for replacing
saccharification by concentrated sulfuric acid. The saccharification by an enzyme is
a desirable method in view of an influence to the environment, but a pretreatment
needs to be performed on a biomass material in advance for enzymatic
saccharification so that the enzyme efficiently acts on the biomass material.
Various methods have been known as a pretreatment method of the biomass
material, but among these methods, steaming using diluted sulfuric acid,
compressed hot water, or the like is common (for example, see PTL 1 to PTL 4).
This method has, however, problems that it is not desirable to use sulfuric acid as
mentioned earlier, and in the case where the biomass material is subjected to the
pretreatment disclosed in any of the patent literatures and the obtained processed
product is provided to enzymatic saccharification, the pretreatment needs to be
performed in several steps, or at the high temperature of 200°C or higher, for
securing the desirable degree of the enzymatic saccharification efficiency.
Although it has been known that chemical and biochemical reactivity of a
biomass material is improved by finely grinding the biomass material by physical
means, if it is attempted to achieve sufficient enzymatic saccharification efficiency
only by the grinding, a large scale of energy is required for the grinding process,
which may impair the economic rationality.
Moreover, it has been known that chemical and biochemical reactivity of a
biomass material is improved by pretreating the biomass material with ammonia or
an organic amine (see, for example, PLT 5). However, the pretreated biomass does
not yet have sufficient enzymatic saccharification efficiency. Accordingly, it is the
current situation that the development of an enzymatic saccharification technique
capable of enhancing enzymatic saccharification efficiency, and the development of a
pretreatment technique of a biomass material suitable for the enzymatic
saccharification have been still desired.
Citation List
Patent Literature
PTL 1 Japanese Patent Application Laid-Open (JP-A) No. 2006-075007
PTL 2 Japanese Patent Application Laid-Open (JP-A) No. 2004-121055
PTL 3 Japanese Patent Application Laid-Open (JP-A) No. 2002-541355
PTL 4 Japanese Patent Application Laid-Open (JP-A) No. 2002-159954
PTL 5 European Patent Publication No. 77287
Disclosure of Invention
Technical Problem
The present invention aims to solve the various problems in the art, and
achieve the following object. An object of the present invention is to provide a
method for producing a sugar, a method for producing ethanol, and a method for
producing lactic acid, in all of which enzymatic saccharification can be efficiently
performed to thereby respectively improve the production efficiency of sugar, the
production efficiency of ethanol, and the production efficiency of lactic acid, as well
as providing a method for producing a material for enzymatic saccharification that is
effectively used in the method for producing a sugar, the method for producing
ethanol, and the method for producing lactic acid.
Technical Solution
As a result of the diligent studies conducted by the present inventors to solve
the problem mentioned above, the present inventors have reached the following
findings. Specifically, it is the findings that enzymatic saccharificationcan be
efficiently performed by processing a biomass material containing cellulose I, which
is natural cellulose, with a processing agent containing ammonia and/or an organic
amine to obtain a modified biomass material, grinding the modified biomass
material, and providing the ground modified biomass material to enzymatic
saccharification, and as a result of this, production efficiency of a sugar, production
efficiency of ethanol, and production efficiency of lactic acid can be significantly
improved.
Note that, the present inventors previously applied a patent describing that
enzymatic saccharification can be efficiently performed by using, as a target for
enzymatic saccharification, cellulose (e.g. cellulose IIIi) having the lower crystalline
density than that of cellulose I, which is natural cellulose, and cellulose for
enzymatic saccharification containing cellulose IIIi can be efficiently obtained by
processing a biomass material containing cellulose I with ammonia, especially
supercritical ammonia (see JP-ANo. 2008-161125).
Conventionally, it has not been known that significant improvement of
enzymatic saccharification of a biomass material is achieved by processing a biomass
material with a processing agent containing ammonia and/or an organic amine,
grinding the processed product, and providing the ground product to enzymatic
saccharification, which is novel insights of the present inventors.
The present invention is based upon the insights of the present inventors,
and means for solving the problem mentioned above are as follows:
<1> A method for producing a sugar, containing:
(a) processing a biomass material containing cellulose I with a processing
agent containing ammonia and/or an organic amine to obtain a modified biomass
material,
(b) grinding the modified biomass material to obtain a material for enzymatic
saccharification, and
(c) enzymatically saccharifying the material for enzymatic saccharification,
to thereby obtain a sugar.
<2> The method for producing a sugar according to <1>, wherein the processing
agent used in (a) is ammonia.
<3> The method for producing a sugar according to any of <1> or <2>, wherein
the biomass material containing cellulose I is woody biomass.
<4> The method for producing a sugar according to any one of <1> to <3>,
wherein the material for enzymatic saccharification obtained in (b) has average
particle diameter of 5 µm to 80 µm in the median size.
<5> A method for producing ethanol, containing:
fermenting the sugar obtained in the method for producing a sugar as
defined in any one of claims <1> to <4>, to thereby obtain ethanol.
<6> A method for producing lactic acid, containing-'
fermenting the sugar obtained in the method for producing a sugar as
defined in any one of <1> to <4>, to thereby obtain lactic acid.
<7> A method for producing a material for enzymatic saccharification,
containing-'
(a) processing a biomass material containing cellulose I with a processing
agent containing ammonia and/or an organic amine to obtain a modified biomass
material, and
(b) grinding the modified biomass material to obtain a material for enzymatic
saccharification.
Advantageous Effects
According to the present invention, the object mentioned above can be
achieved, the various problems in the conventional art can be solved, and a method
for producing a sugar, a method for producing ethanol, and a method for producing
lactic acid, in all of which enzymatic saccharification can be efficiently performed to
thereby improve production efficiency of the sugar, production efficiency of the
ethanol, and production efficiency of the lactic acid, and a method for producing a
material for enzymatic saccharification that is effectively used in the method for
producing a sugar, the method for producing ethanol, and the method for producing
lactic acid, can be provided.
Brief Description of Drawings
FIG. 1 is an X-ray diffraction diagram of the coarsely ground eucalyptus.
FIG. 2 is an X-ray diffraction diagram of a sample obtained by grinding the
coarsely ground eucalyptus.
FIG. 3 is an X-ray diffraction diagram of a sample obtained by processing the
coarsely ground eucalyptus with ammonia.
FIG. 4 is an X-ray diffraction diagram of a sample obtained by grinding the
coarsely ground eucalyptus, and further treated with ammonia.
FIG. 5 is an X-ray diffraction diagram of a sample obtained by processing the
coarsely ground eucalyptus with ammonia, and further grinding the treated coarsely
ground eucalyptus.
FIG. 6 is an X-ray diffraction diagram of coarsely ground Salix schwerinii.
FIG. 7 is an X-ray diffraction diagram of a sample obtained by grinding the
coarsely ground Salix schwerinii.
FIG. 8 is an X-ray diffraction diagram of a sample obtained by processing the
coarsely ground Salix schwerinii with ammonia.
FIG. 9 is an X-ray diffraction diagram of a sample obtained by grinding the
coarsely ground Salix schwerinii, and further processing with ammonia.
FIG. 10 is an X-ray diffraction diagram of a sample obtained by processing
the coarsely ground Salix schwerinii with ammonia, and further grinding the
treated coarsely ground Salix schwerinii.
FIG. 11 is an X-ray diffraction diagram of coarsely ground Japanese cedar.
FIG. 12 is an X-ray diffraction diagram of a sample obtained by grinding the
coarsely ground Japanese cedar.
FIG. 13 is an X-ray diffraction diagram of a sample obtained by processing
the coarsely ground Japanese cedar with ammonia.
FIG. 14 is an X-ray diffraction diagram of a sample obtained by grinding the
coarsely ground Japanese cedar, and further processing with ammonia.
FIG. 15 is an X-ray diffraction diagram of a sample obtained by processing
the coarsely ground Japanese cedar with ammonia, and further grinding the treated
coarsely ground Japanese cedar.
Best Mode for Carrying out the Invention
(Method for Producing Sugar)
The method for producing a sugar of the present invention contains: (a)
processing a biomass material containing cellulose I with a processing agent
containing ammonia and/or an organic amine to obtain a modified biomass material."
(b) grinding the modified biomass material to obtain a material for enzymatic
saccharification; and (c) enzymatically saccharifying the material for enzymatic
saccharification, to thereby obtain a sugar, and may further contain other steps, if
necessary.

In the (a), a modified biomass material is obtained by processing a biomass
material containing cellulose I with a processing agent containing ammonia and/or
an organic amine.
-Biomass Material Containing Cellulose I-
The biomass material containing cellulose I is appropriately selected
depending on the intended purpose without any restriction. For example,
"waste-origin biomass" obtained as a residue from the production activities such as
agriculture, and forestry, and "resources crop-origin biomass" obtained by
intentionally growing for the purpose of attaining energy, or the like, can be used.
Examples of the "waste-origin biomass" include building material waste, waste from
tree thinning, rice straw, wheat straw, chaff, and bagasse, and examples of the
"resources crop-origin biomass" include: glucide or starch crops, which are also
grown as food, such as sugar cane, and corn; and eucalyptus, poplar, acacia, willow,
Japanese cedar, switchgrass, napier grass, Erianthus, Miscanthus, and Japanese
pampas grass grown for the purpose of utilizing cellulose. Moreover, the biomass
material containing cellulose I is also classified into "woody biomass" that uses wood
as a material, and "herbaceous biomass" that uses grass. In the present invention,
both the woody biomass and herbaceous biomass can be used, but use of the woody
biomass is preferable as the effect of the present invention is obtained remarkably.
Furthermore, the biomass material containing cellulose I may cellulose I itself,
which is obtained by purification or the like of various biomass mentioned above.
The biomass material containing cellulose I may be used independently, or two or
more thereof may be used in combination. Note that, although cellulose I, which is
natural cellulose, is classified into cellulose Ia and cellulose Ib, the cellulose I
contained in the biomass material may be either of them, or both of them.
In the processing using the processing agent containing ammonia and/or an
organic amine, the biomass material containing cellulose I may be used in the state
as collected, but it is preferably used after sizing the biomass material containing
cellulose I into a certain size or smaller by cutting, grinding, or the like. The size of
the biomass material is appropriately adjusted depending on the intended purpose
without any restriction, and for example, the opening size of the mesh through which
the biomass material go through is preferably 5 mm or smaller, more preferably 3
mm or smaller, and even more preferably 2 mm or smaller. When the opening size
of the mesh is larger than 5 mm, the processing using the processing agent may be
insufficient. When the size thereof is within the even more preferable range, by
contrast, the processing duration can be shortened, and an amount of the processing
agent used is kept small, and thus it is advantageous. Note that, the step of cutting
or grinding the collected biomass material may be merely referred to as "coarse
grinding," hereinafter.
By performing the coarse grinding in advance, the processing with the
processing agent containing ammonia and/or an organic amine is efficiently
progressed, and the material for enzymatic saccharification that is in the form of the
finer particles, and has the excellent enzymatic saccharification efficiency can be
obtained when the modified biomass material is ground. The grinder used for the
coarse grinding is appropriately selected depending on the intended purpose without
any restriction, and examples thereof include a Wiley mill, a cutter mill, a hammer
mill, and a pin mill.
-Processing with Ammonia and/or Organic Amine-
In the case where the biomass material containing cellulose I is processed
using ammonia as the processing agent, the method of the processing is
appropriately selected depending on the intended purpose without any restriction.
For example, it can be performed by introducing the biomass material containing
cellulose I and the ammonia into a pressure vessel, setting the inner pressure and
temperature of the pressure vessel to desirable pressure and temperature, and
processing for a desirable period. The ammonia may be in the liquid phase, in the
gas phase, or in the supercritical state. As a result of the processing with ammonia,
at least part of the cellulose I contained in the biomass material is transformed into
cellulose III1 that has the high enzymatic saccharification efficiency, and the
ammonia for use is suitably liquid ammonia or supercritical ammonia in view of the
improvement in the transformation efficiency. Nevertheless, a processing at the
conditions suitable in each situation can be selected under the consideration of the
intended saccharification rate, energy consumption, and the like. The conditions
for the processing with ammonia are not restricted, but the generally preferable
conditions are the temperature of-35°C to 140°C, and the pressure of 0 MPa to 12.5
MPa.
In the case where the biomass material containing cellulose I is processed
using an organic amine as the processing agent, the organic amine for use is
appropriately selected depending on the intended purpose without any restriction.
For example, ethylene diamine, monomethyl amine, monoethyl amine are preferably
used as the organic amine, and ethylene diamine is preferable as the organic amine.
In the case where the biomass containing the cellulose I is processed with the
organic amine listed above, the conditions thereof are not particularly restricted, but
the processing temperature and pressure thereof can be set the same as in the
processing with the ammonia.
In the present invention, ammonia is preferably used as the processing agent
in view of the transformation efficiency from the cellulose I into cellulose III1,
easiness of removal of the processing agent after the processing, and the like.
The duration of the processing with the processing agent containing the
ammonia and/or an organic amine is appropriately selected within the range where
the transformation from cellulose I into cellulose III1 is progressed to a desirable
extend depending on the amount of the biomass material containing cellulose I for
use, the processing pressure mentioned above, the processing temperature
mentioned above, and the like without any restriction, but it is preferably 10
minutes to 10 hours, more preferably 30 minutes to 8 hours, and particularly
preferably 30 minutes to 5 hours. When the duration of the processing is shorter
than 10 minutes, the transformation from cellulose I into cellulose IIII may not be
progressed to the desirable extend, and when the duration thereof is longer than 10
hours, the transformation from cellulose I into cellulose IIII is not progressed any
further, and therefore the processing is inefficient as a whole. When the duration of
the processing is within the aforementioned more preferable range, by contrast, it is
advantageous, as the transformation from cellulose I into cellulose IIII is efficiently
progressed.
An amount of the ammonia and/or an organic amine used for the processing
with the processing agent containing the ammonia and/or an organic amine is
appropriately selected depending on the intended purpose without any restriction,
and for example, it is preferably 10 mg to 300 g, more preferably 100 mg to 150 g,
and particularly preferably 1 g to 50 g, relative to 1 g of the biomass material
containing cellulose I. When the amount of the ammonia and/or an organic amine
used is less than 10 mg relative to 1 g of the biomass material containing cellulose I,
there may be a case where the processing is insufficiently performed. When the
amount thereof is more than 300 g, the efficiency of the processing may be poor.
When the amount thereof is within the particularly preferable range, by contrast,
the processing duration can be shorten, and the amount of the processing agent used
can be kept small, and thus it is preferable.
Note that, at the time of the processing using the processing agent
containing ammonia and/or an organic amine, other compounds are further used in
combination, provided that the processing agent contains ammonia and/or an
organic amine, examples of other compounds mentioned above include carbon
dioxide, nitrogen, ethylene, methane, ethane, propane, butane, pentane, hexane,
toluene, benzene, phenol, dioxane, xylene, acetone, chloroform, carbon tetrachloride,
ethanol, methanol, propanol, and butanol. Note that, it is preferred that water be
not used as other compounds mentioned above. If water is used, there are cases
where the obtained IIII may be returned into cellulose I.
-Modified Biomass Material-
As a result of the processing with the processing agent containing the
ammonia and/or an organic amine, a modified biomass material is obtained. The
processing can make at least part of cellulose I contained in the biomass material
transformed into cellulose IIII that has the lower crystalline density. The cellulose
IIII has an advantage that an enzyme easily acts thereon because of its low
crystalline density. Furthermore, large part of hemicellulose contained in the
biomass material is decomposed into around the size of oligosaccharides, and
therefore it becomes soluble to water. Therefore, by processing the biomass
material in the manner mentioned above, cellulose I or hemicellulose contained in
the biomass material can be changed into the state that can be easily affected by an
enzyme, i.e. cellulose IIII or oligosaccharide derived from hemicellulose, respectively,
so that the enzymatic saccharification efficiency can be improved. Note that, the
fact that at least part of cellulose I is transformed into cellulose IIII by the processing
can be confirmed, for example by X-ray diffraction, FT-IR, solid-state NMR, and the
like. In the present specification, the term "modified biomass material" means the
biomass material containing cellulose I which has been processed with the
processing agent containing ammonia and/or an organic amine, and it is preferably
the biomass material containing cellulose I in which at least part of the cellulose I
contained in the biomass material is transformed into cellulose IIII.
In the modified biomass material, cellulose may contain another compound
between the molecular structures thereof. For example, the modified biomass
material can be obtained by processing the biomass material containing cellulose I
(which is natural cellulose) with the processing agent containing ammonia and/or an
organic amine as mentioned earlier, but the modified biomass material may be in the
state of the complex composed of the cellulose and the ammonia and/or an organic
amine (may also be referred to as "a cellulose, ammonia, etc. complex" hereinafter).
However, using the cellulose, ammonia, etc. complex makes it difficult to control pH
at the time of enzymatic saccharification, and moreover the complex has a
characteristic that it is returned into cellulose I by the influence of water.
Therefore, it is preferred that the modified biomass material in the state where the
ammonia and/or an organic amine is removed from the cellulose, ammonia, etc.
complex be used at the time of enzymatic saccharification.
Accordingly, it is preferred that a removal step of removing the ammonia
and/or an organic amine from the cellulose, ammonia, etc. complex be provided after
the processing of the biomass material with the ammonia and/or an organic amine.
The removal method of the ammonia and/or an organic amine is appropriately
selected depending on the intended purpose without any restriction, and examples
thereof include: a method in which the obtained modified biomass material
containing the cellulose, ammonia, etc. complex is washed with methanol, ethanol,
acetone, or the like, after the processing with the ammonia and/or an organic amine;
a method in which drying of the obtained modified biomass material containing the
cellulose, ammonia, etc. complex is performed under reduced pressure; and a method
in which the modified biomass material containing the cellulose, ammonia, etc.
complex is dried at the temperature equal to or higher the boiling point of the
processing agent. In the case where ammonia is used as the processing agent, the
removal method thereof is preferably a method in which drying is performed at the
temperature equal to or higher than the boiling point of ammonia (e.g. normal
temperature to 50°C) under normal pressure or reduced pressure, because an
organic solvent is not used in this method and thus this method is excellent in safety.
The modified biomass material preferably contains cellulose IIII, and the
proportion of cellulose IIII in the modified biomass material is not restricted, but it is
preferably as large as possible because better enzymatic saccharification efficiency
can be achieved, as larger the proportion of the cellulose IIII is. Moreover, the
modified biomass material may contain, other than the cellulose IIII mentioned
above, for example, cellulose I (cellulose Ia, cellulose Iß), or other substances, such as
hemicellulose, lignin, and the like. However, in view of the improvement of the
enzymatic saccharification efficiency, it is preferred that lignin be not contained, or
an amount of lignin contained be small.

In the step (b), a material for enzymatic saccharification is obtained by
grinding the modified biomass material obtained in the step (a).
-Grinding-
In the step (b), the modified biomass material contained in the step (a) is
ground.
The method of grinding the modified biomass material is appropriately
selected depending on the intended purpose without any restriction, and for example,
the grinding can be performed by means of a grinder such as a horizontal stone mill,
a planetary ball mill, a vibration ball mill, a bead mill, a jet mill, and the like.
Among them, the horizontal stone mill is preferable as the grinder, as the fine
powdery material for enzymatic saccharification having the excellent enzymatic
saccharification efficiency can be obtained with relatively low energy. By the
grinding, the modified biomass material can be turned into the material for
enzymatic saccharification of the present invention. By grinding the modified
biomass material, it is possible to further improve the enzymatic saccharification
efficiency.
The conditions for the grinding are appropriately selected depending on the
grinder for use, the biomass material for use, the intended average particle diameter
of the ground product, and the like, without any restriction. In the case where the
horizontal stone mill is used as the grinder, the ground biomass material is
discharged from the mill, and therefore for further grinding the ground biomass
material, the discharged ground product is collected, and supplied to the mill again,
and this may be repeated a few times. In the case where the grinding is repeated,
the number of the grinding performed is appropriately selected depending on the
grinder for use, the duration for a single grinding operation, loaded energy, and the
like, without any restriction, but the grinding performed repeatedly twice or more
times is advantageous because it can give the finer powdery material for enzymatic
saccharification having the excellent enzymatic saccharification efficiency. The
more the grinding is performed is more advantageous because the finer powdery
material for enzymatic saccharification having the excellent enzymatic
saccharification efficiency can be obtained, but the number of the grinding
performed is more than necessary, the finer powder and further improvement of the
enzymatic saccharification efficiency cannot be expected, which may cause
inefficiency as a whole, and therefore the number of the grinding performed is
preferably 4 times or less.
-Material for Enzymatic Saccharification-
The term "material for enzymatic saccharification" for use in the method of
the present invention means the modified material which has been ground. By
grinding the modified biomass material, further improvement of the enzymatic
saccharification efficiency is possible. The size of the particles of the material for
enzymatic saccharification obtained by the grinding is not particularly restricted,
and cannot be unconditionally defined as it varies depending on the biomass
material for use, but as the size, the average particle diameter thereof is preferably 5
µm to 80 µm, more preferably 5 to 50 µm, and even more preferably 5 µm to 30
µm. In the case where it is attempt to make the average particle diameter of the
material for enzymatic saccharification smaller than 5 urn, large energy and long
time are required for the grinding, and thus the economic rationality thereof is lost.
In the case where the average particle diameter thereof is larger than 80µm, the
enzymatic saccharification efficiency may not be sufficiently improved. When the
average particle diameter of the material for enzymatic saccharification is within the
further more preferable range mentioned above, by contrast, it is advantageous
because of the desirable balance between the energy and time required for the
grinding and the obtainable enzymatic saccharification efficiency. Note that, in the
present specification, the average particle diameter of the material for enzymatic
saccharification is based upon a median size measured by a laser
diffraction-scattering method. In the present specification, the median size means
a certain particle diameter at which the total volume of the particles having the
particle diameter equal to or larger than the certain particle diameter and the total
volume of the particles having the particle diameter equal to or smaller than the
certain particle diameter become equal.
The material for enzymatic saccharification obtained by grinding the
modified biomass material can be, for example, provided to the enzymatic
saccharidication of the step (c) mentioned later, as it is, or after appropriately going
through other steps. The aforementioned other steps may be appropriately selected
depending on the intended purpose without any restriction, and examples thereof
include: a pH adjustment step for adjusting pH of the powdery material for
enzymatic saccharification obtained by the grinding to the pH thereof suitable for
the enzymatic saccharification described later.

In the step (c), the material for enzymatic saccharification obtained in the
step (b) is subjected to enzymatic saccharification, to thereby produce a sugar.
-Enzymatic Saccharification-
The method for enzymatic saccharification is not particularly restricted, and
for example, it can be carried out at the conditions mentioned as follows.
The enzyme used for the enzymatic saccharification is appropriately selected
depending on the intended purpose without any restriction, and examples thereof
include cellulase, and cellobiase (6-glucosidase).
An amount of the enzyme used for the enzymatic saccharification is
appropriately selected depending on the intended purpose without any restriction,
and for example, it is preferably 0.001 mg to 100 mg, more preferably 0.01 mg to 10
mg, and even more preferably 0.1 mg to 1 mg, relative to 1 g of the material of the
enzymatic saccharification. When the amount of the enzyme for use is less than
0.001 mg relative to 1 g of the material for enzymatic saccharification, the enzymatic
saccharification may be progressed insufficiently. When the amount thereof is
more than 100 mg, inhibition of saccharification may be caused. When the amount
of the enzyme for use is within the further more preferable range, by contrast, it is
advantageous because an amount of the sugar obtained increases relative to an
amount of the enzyme added.
The temperature for the enzymatic saccharification is appropriately selected
depending on the intended purpose without any restriction, and for example, it is
preferably 10°C to 70°C, more preferably 20°C to 60°C, and even more preferably
30°C to 50°C. When the temperature is lower than 10°C, the enzymatic
saccharification may not be sufficiently progressed. When the temperature is
higher than 70°C, the enzyme may be deactivated. When the temperature is within
the further more preferable range, by contrast, it is advantageous because an
amount of the sugar obtained increases relative to an amount of the enzyme added.
The pH at the time of the enzymatic saccharification is appropriately
selected depending on the intended purpose, and for example, it is preferably 3.0 to
8.0, more preferably 3.5 to 7.0, and even more preferably 4.0 to 6.0. When the pH is
lower than 3.0, or higher than 8.0, the enzyme may be deactivated. When the pH
thereof is within the more preferable range mentioned above, by contrast, it is
advantageous because a large amount of a sugar is obtained relative to the amount
of the enzyme added.
-Sugar-
As a result of the enzymatic saccharification, for example, a carbohydrate
solution containing glucose, which is a sugar derived from cellulose IIII contained in
the material for enzymatic saccharification obtained in the step (b), can be obtained.
Moreover, the carbohydrate solution obtained by the enzymatic saccharification may
contain, other than the one mentioned above, for example, glucose derived from the
cellulose I, and/or a sugar derived from the hemicellulose. The sugar derived from
the hemicellulose includes at least one of pentose such as xylose, and arabinose; and
hexose such as glucose, galactose, and mannose.
The carbohydrate solution may be subjected to the method for producing
ethanol or method for producing lactic acid of the present invention as it is, or after
going through other steps mentioned below.
The aforementioned other steps are appropriately selected depending on the
intended purpose without any restriction, and examples thereof include a pH
adjustment step for adjusting the pH of the carbohydrate solution to the pH thereof
suitable for each of fermentation steps described later.
(Method for Producing Ethanol)
The method for producing ethanol of the present invention contains:
fermenting the sugar obtained in the method for producing a sugar of the present
invention, to thereby obtain ethanol (fermentation step), and may further contain
other steps, if necessary.

In the method for producing ethanol, the method for fermenting the sugar is
appropriately selected depending on the intended purpose without any restriction,
and for example, a method in which an alcohol fermentation micro-organism, such
as yeast and the like is added to the solution containing the sugar to thereby subject
the sugar to alcohol fermentation is particularly preferable. The yeast is
appropriately selected depending on the intended purpose without any restriction,
and examples thereof include yeast of Saccharomyces group. Note that, the yeast
may be natural yeast, or genetically engineered yeast. Specific examples of the
ethanol fermentation micro-organism include: yeast, such as Saccharomyces
cerevisiae, Kluyveromyces fragilis, K. lactis, K. marxianus, Pichia stipitis, P.
pastoris, Pachysolen tannophilus, and Candida Glabrata, and genetically modified
organisms thereof; bacteria such as Zymomonas mobilis, Zymobacter palmae,
Clostridium thermocellum, and C. ljungdahlii, and genetically modified organisms
thereof.
At the time of the fermentation, the amount of the yeast for use, the
fermentation temperature, pH, the fermentation duration, and the like are
appropriately selected depending on the amount of the sugar subjected to the alcohol
fermentation, the yeast for use, and the like, without any restriction.

The aforementioned other steps are appropriately selected depending on the
intended purpose without any restriction, and examples thereof include a step of
separating and purifying the ethanol obtained in the fermentation step. The step of
separating and purifying is appropriately selected depending on the intended
purpose without any restriction, and examples thereof include distillation.
The ethanol obtained by the method for producing ethanol can be suitably
used, for example, as ethanol fuel, industrial ethanol, and the like. Since the
ethanol can be obtained from the biomass material, it can be reproduced as long as
plants that will be used as the biomass material can be grown. Moreover, as the
plants absorb carbon dioxide in the atmosphere during the cultivation of the plants,
and therefore the concentration of the carbon dioxide in the atmosphere does not
increase even though carbon dioxide is generated by combustion of the ethanol.
Accordingly, the ethanol is a desirable energy source for preventing the global
warming. Moreover, such ethanol has been currently especially expected to be used
as environmentally friendly vehicle fuels by mixing with gasoline.
Alcohols other than ethanol can be produced by fermenting the sugar
obtained from the method for producing a sugar of the present invention using
microorganism which produces the intended alcohol, instead of the yeast or the like
that produces the ethanol. For example, buthanol can be produced by fermentation
using acetone-butanol bacteria.
(Method for Producing Lactic Acid)
The method for producing lactic acid of the present invention contains:
fermenting the sugar obtained by the method for producing a sugar of the present
invention, to thereby obtain lactic acid (fermentation step); and may further contain
other steps, if necessary.

The method of fermenting the sugar in the method for producing lactic acid is
appropriately selected depending on the intended purpose without any restriction,
and for example, a method in which lactic acid fermentation micro-organism, such as
lactic acid bacteria, is added to a solution containing the sugar to carry out lactic
acid fermentation is particularly preferable. The lactic acid bacteria is
appropriately selected depending on the intended purpose without any restriction,
and examples thereof include Lactobacillus manihotivorans, Lactobacillus
plantarum, Streptococcus thermophilus, and Lactobacillus bulgaricus. Note that,
the lactic acid bacteria may be natural lactic acid bacteria, or genetically engineered
lactic acid bacteria.
An amount of the lactic acid bacteria for use, fermentation temperature, pH,
fermentation duration, and the like for the fermentation are not particularly
restricted, and appropriately selected, for example, depending on the sugar provided
for lactic acid fermentation, the lactic acid bacteria for use, and the like.

The aforementioned other steps are appropriately selected depending on the
intended purpose without any restriction, and examples thereof include a step of
separating and purifying the lactic acid obtained in the fermentation step. The
method of separating and purifying is appropriately selected depending on the
intended purpose without any restriction.
The lactic acid obtained by the method for producing lactic acid can be
suitably used, for example, for the production of polylactic acid by chemically
polymerizing the lactic acid. It is desired that lactic acid, which is currently often
produced from starch of corn or the like, can be produced from a biomass material
containing cellulose that cannot be supplied as food, and the method for producing
lactic acid can efficiently produce polylactic acid from such biomass material
containing cellulose.
Organic acids other than lactic acid, such as citric acid, succinic acid, malic
acid, oxalic acid, and the like, can be produced by fermenting the sugar obtained by
the method for producing a sugar of the present invention using micro-organism that
produces the indented organic acid, instead of the lactic acid bacteria.
(Method for Producing Material for Enzymatic Saccharification)
The method for producing a material for enzymatic saccharification of the
present invention contains: (a) processing a biomass material containing cellulose I
with a processing agent containing ammonia and/or an organic amine to obtain a
modified biomass material; and (b) grinding the modified biomass material to obtain
a material for enzymatic saccharification; and may further contain other steps, if
necessary.
The step (a) and the step (b) of the method for producing a material for
enzymatic saccharification are each as described in the respective description in the
method for producing a sugar of the present invention.
It has been commonly known that the chemical and biological reactivity of
the biomass material containing cellulose are improved by grinding the biomass
material containing cellulose. Moreover, the present inventors have already
disclosed that the enzymatic saccharification efficiency is improved by processing
the biomass material containing cellulose I with ammonia or the like to transform
cellulose I into cellulose IIII.
The method for producing a sugar of the present invention can perform
enzymatic saccharification with the significantly excellent efficiency, compared with
the case where enzymatic saccharification is performed after only processing of the
biomass material with the processing agent containing ammonia and/or an organic
amine, the case where enzymatic saccharification is performed after only grinding of
the biomass material is performed, and the case where enzymatic saccharification is
performed after grinding of the biomass material is performed, followed by
processing with the processing agent containing ammonia and/or an organic amine.
The functional mechanism thereof has not been clearly known, but the present
inventors infer that the mechanism thereof is as follows.
In the method for producing a sugar of the present invention, it has been
found out that an X-ray diffraction pattern of the material for enzymatic
saccharification obtained in the step (b) is significantly changed from that of the
modified biomass material obtained in the step (a). Specifically, there is a
possibility that a structure of the cellulose is changed in some way as a result of
grinding of the modified biomass material. Meanwhile, in the case where grinding
of the biomass material is performed in the same manner as in the step (b) of the
method of the present invention, followed by the processing with the processing
agent containing ammonia and/or an organic amine, an X-ray diffraction pattern of
the resultant does not have a significant change from that of a product which has
been processed with the processing agent containing ammonia and/or an organic
amine without performing grinding. Based on these facts, there is a possibility that
a structural change reflected to a change in the X-ray diffraction pattern caused by
the grinding of the modified biomass material relates to high enzymatic
saccharification efficiency of the material for enzymatic saccharification for use in
the present invention.
Examples
Examples of the present invention will be explained hereinafter, but these
examples shall not be construed as to limit the scope of the present invention in any
way.
(Example 1)
-Biomass Material-
As the biomass material containing cellulose I, eucalyptus was used.
-Coarse Grinding-
The provided eucalyptus was coarsely ground by means of a Wiley mill into
the intended average particle diameter of 200 µm.
-Ammonia Processing-
The coarsely ground eucalyptus was provided for the processing with the
supercritical ammonia in the following manner.
A sample of the coarsely ground eucalyptus (4 g) which had been dried in an
oven of 60°C for 24 hours was placed in a TVS-N2 type portable reactor (a product of
TAIATSU TECHNO CORPORATION, may also referred to as "a vessel" hereinafter)
having the inner volume of 120 mL and the vessel was sealed, and ammonia was
allowed to flow into the vessel at the pressure of 0.5 MPa for 30 minutes while
cooling the vessel to -13°C using a cooling device. Thereafter, a PC-V type heater
(manufactured by TAIATSU TECHNO CORPORATION) was attached to the vessel,
and a heat-pressure treatment was performed at 140°C for 1 hour. During the
heat-pressure treatment, it was confirmed that the pressure inside the vessel was 11
MPa or higher, at which ammonia was in supercritical state. After the
heat-pressure treatment, ammonia was removed by returning the pressure inside
the vessel to atmospheric pressure, and the temperature was decreased till room
temperature to thereby collect a solid sample yielded in the vessel. The sample was
left to stand over night without sealing to thereby sufficiently volatillize ammonia.
-Grinding-
By using a horizontal ceramic mill, a home-use mortar type tea leaf mill
"Marugoto (Entirely) Green Tea EU6820"(product name), manufactured by
Panasonic Corporation, 2 g of the ammonia processed sample was ground three
times at the setting of "fine", and in each time grinding was performed for 10
minutes.
-X-ray diffraction analysis-
The ground sample (100 mg) was compressed and molded at the pressure of
200 kg/cm2, and the molded sample was provided for X-ray diffraction analysis. For
the X-ray diffraction analysis, a bulb-type X-ray generator, RINT2200 (product
name), manufactured by Rigaku Corporation was used, and the X-ray diffraction
analysis was carried out in accordance with X-ray diffractometry. For the
measurement, CuKa rays (wavelength: 0.15418 nm) monochromatized by a
monochrometer was used at the voltage of 38 kV, and current of 50 mA, and the
measurement was performed in accordance with a step-scan method at the
conditions where the operation angle range 29 of 5° to 30°, the step width of 0.1°, and
the time of 20 seconds in total. Moreover, an X-ray diffraction analysis was
performed on both of a sample after the coarse grinding, and a sample after the
ammonia processing in the same manner as mentioned above. The diffraction
pattern of the sample after the coarse grinding is presented in FIG. 1, the diffraction
pattern of the sample after the ammonia processing is presented in FIG. 3, and the
diffraction pattern of the sample after the ammonia processing and the grinding is
presented in FIG. 5.
-Enzymatic Saccharification Reaction-
The sample on which the ammonia processing and grinding had been
performed as mentioned above was subjected to an enzymatic saccharification
reaction in the following manner.
A sample (10 mg) was weight and placed in a microtube having an inner
volume of 1.5 mL, and an enzymatic saccharification reaction liquid was prepared so
that the reaction liquid had a sample concentration of 1%(wt/vol), each enzyme
concentration of 0.01%(wt/vol), 0.02%(wt/vol) in total, and pH of 4.5 (with acetic acid
buffer solution), where as the enzyme, Celluclast® 1.5L and Novozyme® 188 (both
product names, and both manufactured by Novozymes). The prepared reaction
liquid was shaken by an end-over-end mixer (15 rpm) for 24 hours in a constant
temperature room at 37°C to be thereby subjected to an enzymatic saccharification
reaction. After the reaction, a concentration of glucose contained in a supernatant
liquid obtained through centrifugal separation of the reaction liquid was measured
by Glucose CII-Test Wako (product name, manufactured by Wako Pure Chemical
Industries, Ltd.), and the glucose yield was calculated from the glucose
concentration. The result is presented in Table 1.
Note that, the glucose yield is defined by the following formula.
Glucose yield (%)=[an amount of glucose in an enzymatic saccharification reaction
solution /(an amount of a material for enzymatic saccharificationxthe total
transformation rate to glucose/100)]x100
The total transformation rate to glucose (%):(an amount of glucose obtained
by completely chemically hydrolyzing the biomass material independently /an
amount of the biomass material)x100 (equivalent to theoretical yield of the biomass
material standard)
Note that, the total transformation rate to glucose of the used eucalyptus
was 43.3%.
(Comparative Example 1-1- Enzymatic Saccharification of Non-Processed Sample)
The coarsely ground eucalyptus used in Example 1 was directly subjected to
an enzymatic saccharification reaction in the same manner as in Example 1 without
performing ammonia processing and grinding. The result is shown in Table 1.
(Comparative Example 1-2: No Ammonia Processing, Enzymatic Saccharification of
Ground Sample)
-Grinding-
The coarsely ground eucalyptus used in Example 1 was ground in the same
manner as the grinding of the sample after the ammonia processing in Example 1,
without performing the ammonia processing.
-X-ray diffraction analysis-
An X-ray diffraction analysis was performed on the ground sample in the
same manner as in Example 1. The diffraction pattern obtained is presented in
FIG. 2.
-Enzymatic Saccharification Reaction-
The ground sample was subjected to an enzymatic saccharification reaction
in the same manner as the operations of the enzymatic saccharification reaction
conducted in Example 1. The glucose yield was calculated in the same manner as
in Example 1. The result is shown in Table 1.
(Comparative Example 1-3: Enzymatic Saccharification of Ammonia Processed
Sample)
A sample prepared by processing the coarsely ground eucalyptus obtained in
Example 1 with ammonia was subjected to an enzymatic saccharification reaction in
the same manner as the enzymatic saccharification reaction conducted in Example 1,
without grinding the sample after the ammonia processing. The glucose yield
thereof was calculated in the same manner as in Example 1. The result is shown in
Table 1.
(Comparative Example 1-4: Enzymatic Saccharification of Sample That Is Processed
With Ammonic After Grinding)
-Grinding-
The coarsely ground eucalyptus in Example 1 was ground in the same
manner as the grinding operation of the sample performed after the ammonia
processing in Example 1.
The ground sample was processed with ammonia in the same manner as in
Example 1.
-X-Ray Diffraction Analysis-
An X-ray diffraction analysis was performed on the sample prepared by the
grinding followed by the ammonia processing, in the same manner as in Example 1.
The diffraction pattern thereof is presented in FIG. 4.
-Enzymatic Saccharification Reaction-
The sample prepared by the grinding followed by the ammonia processing
was subjected to an enzymatic saccharification reaction by the same operations of
the enzymatic saccharification reaction conducted in Example 1. The glucose yield
was calculated in the same manner as in Example 1. The result is shown in Table
1.
(Example 2)
Coarse grinding, an ammonia processing, grinding, and an enzymatic
saccharification reaction were performed in this order in the same manner as in
Example 1, provided that eucalyptus used in Example 1 was replaced with Salix
schwerinii. The result is shown in Table 1. Moreover, an X-ray diffraction
analysis was performed on the sample of each stage in the same manner as in
Example 1. X-ray diffraction patterns of the sample were corresponded to
respective eucalyptus samples in Example 1. The diffraction pattern of the sample
after the coarse grinding is presented in FIG. 6, the diffraction pattern of the sample
after the ammonia processing is presented in FIG. 8, and the diffraction pattern of
the sample after the ammonia processing and grinding is presented in FIG. 10.
Note that, the total transformation rate to glucose of the Salix schwerinii
used was 45.1%, and the coarsely ground Salix schwerinii had the average particle
diameter of 252 µm.
(Comparative Examples 2-1 to 2-4)
A pretreatment was performed in the same manner as in Comparative
Examples 1-1 to 1-4, respectively, provided that the coarsely ground eucalyptus used
as a material in each of Comparative Examples 1-1 to 1-4 was changed to the
coarsely ground Salix schwerinii used in Example 2, and the prepared samples were
each subjected to an enzymatic saccharification reaction in the same manner as in
Example 2. In each Comparative Example, the glucose yield was calculated. The
results are shown in Table 1. Moreover, the sample after the pretreatment in each
of Comparative Examples 2-2 to 2-4 was subjected to an X-ray diffraction analysis.
X-ray diffraction patterns of the samples were similar to the respective eucalyptus
samples of Comparative Examples 1-2 to 1-4. The diffraction pattern of the sample
after the grinding is presented in FIG. 7, the diffraction pattern after the grinding
followed by the ammonic processing was presented in FIG. 9.
(Example 3)
Coarse grinding, an ammonia processing, grinding, and an enzymatic
saccharification reaction were performed in this order in the same manner as in
Example 1, provided that eucalyptus used in Example 1 was replaced with Japanese
cedar. The result is shown in Table 1. Moreover, an X-ray diffraction analysis was
performed on the sample of each stage in the same manner as in Example 1. X-ray
diffraction patterns of the sample were similar to the respective eucalyptus samples
in Example 1. The diffraction pattern of the sample after the coarse grinding is
presented in FIG. 11, the diffraction pattern of the sample after the ammonia
processing is presented in FIG. 13, and the diffraction pattern of the sample after
the ammonia processing and grinding is presented in FIG. 15.
Note that, the total transformation rate to glucose of the Japanese cedar
used was 42.7%, and the coarsely ground Japanese cedar had the average particle
diameter of 207 µm.
(Comparative Examples 3-1 to 3-4)
A pretreatment was performed in the same manner as in Comparative
Examples 1-1 to 1-4, respectively, provided that the coarsely ground eucalyptus used
as a material in each of Comparative Examples 1-1 to 1-4 was changed to the
coarsely ground Japanese cedar used in Example 3, and the prepared samples were
each subjected to an enzymatic saccharification reaction in the same manner as in
Example 3. In each Comparative Example, the glucose yield was calculated. The
results are shown in Table 1. Moreover, the sample after the pretreatment in each
of Comparative Examples 3-2 to 3-4 was subjected to an X-ray diffraction analysis.
X-ray diffraction patterns of the samples were similar to the respective eucalyptus
samples of Comparative Examples 1-2 to 1-4. The diffraction pattern of the sample
after the grinding is presented in FIG. 12, the diffraction pattern after the grinding
followed by the ammonic processing was presented in FIG. 14.
From the results presented in Table 1, it was found that by subjected a
biomass material to enzymatic saccharification after processing the biomass
material with ammonia, followed by grinding, the enzymatic saccharification
efficiency was improved compared to the non-processed biomass material, or the
biomass material which had been processed with ammonia or ground, and moreover
compared to the case where the biomass material had been ground and then
processed with ammonia. From the results presented in FIGs. 1 to 15, it is clearly
shown that by processing the biomass material with ammonia, the X-ray diffraction
peaks attributed to cellulose IIII appear at 26 of 12°, 17°, and 21°; by grinding this
biomass material, the diffraction pattern is significantly changed; and the diffraction
pattern of the case where the grinding was performed after the ammonia processing
was different from the diffraction pattern of the sample where the biomass material
was ground and then processed with ammonia. Based upon these results, it is
suggested that by processing the biomass material with ammonia, and then grinding
the same, the resulting biomass material has an unique structure.
Industrial Applicability
The method for producing a sugar, method for producing ethanol, and
method for producing lactic acid of the present invention can significantly improve
production efficiency of a sugar, ethanol, and lactic acid, respectively. Moreover, the
method for producing a material for enzymatic saccharification of the present
invention can efficiently produce a material for enzymatic saccharification, which is
suitable for the method for producing a sugar, method for producing ethanol, and
method for producing lactic acid of the present invention. Accordingly, the method
for producing a sugar, method for producing ethanol, and method for producing lactic
acid, and moreover the method for producing a material for enzymatic
saccharification can be suitably used in productions of ethanol from biomass
materials for the purpose of providing environmentally friendly fuels, which have
recently attracted attentions, and productions of environmentally friendly
biodegradable plastics, and the like.
CLAIMS
1. A method for producing a sugar, comprising:
(a) processing a biomass material containing cellulose I with a processing
agent containing ammonia and/or an organic amine to obtain a modified biomass
material,
(b) grinding the modified biomass material to obtain a material for enzymatic
saccharification, and
(c) enzymatically saccharifying the material for enzymatic saccharification,
to thereby obtain a sugar.
2. The method for producing a sugar according to claim 1, wherein the
processing agent used in (a) is ammonia.
3. The method for producing a sugar according to any of claim 1 or 2, wherein
the biomass material containing cellulose I is woody biomass.
4. The method for producing a sugar according to any one of claims 1 to 3,
wherein the material for enzymatic saccharification obtained in (b) has an average
particle diameter of 5 µm to 80 µm in the median size.
5. A method for producing ethanol, comprising:
fermenting the sugar obtained in the method for producing a sugar as
defined in any one of claims 1 to 4, to thereby obtain ethanol.
6. A method for producing lactic acid, comprising:
fermenting the sugar obtained in the method for producing a sugar as
defined in any one of claims 1 to 4, to thereby obtain lactic acid.
7. A method for producing a material for enzymatic saccharification,
comprising:
(a) processing a biomass material containing cellulose I with a processing
agent containing ammonia and/or an organic amine to obtain a modified biomass
material, and
(b) grinding the modified biomass material to obtain a material for enzymatic
saccharification.

To provide a method for producing a sugar, a method for producing ethanol,
and a method for producing lactic acid, in all of which enzymatic saccharification can
be efficiently performed to thereby respectively improve the production efficiency of
sugar, the production efficiency of ethanol, and the production efficiency of lactic acid,
as well as providing a method for producing an effective material for enzymatic
saccharification used in the methods as mentioned.
Provided is a method for producing a sugar, which contains: (a) processing a
biomass material containing cellulose I with a processing agent containing ammonia
and/or an organic amine to obtain a modified biomass material, (b) grinding the
modified biomass material to obtain a material for enzymatic saccharification, and
(c) enzymatically saccharifying the material for enzymatic saccharification, to
thereby obtain a sugar.