JPS6258361B2 - - Google Patents

Description

[Detailed description of the invention]

In biologically decomposing cellulosic substances, the present invention specifically decomposes cellulosic substances such as rice straw, wheat straw, bagasse, waste paper, and wood chips using cellulose-degrading bacteria or cellulase (cellulose hydrolase). The present invention relates to a pretreatment method capable of improving the biological degradability of cellulosic materials. Cellulose is reduced in molecular weight to cellulose, glucose, and the like by cellulose hydrolase (cellulase) produced by cellulolytic bacteria.
This cellulase is a complex enzyme, and its mechanism of action can generally be shown by the model below.

[Table] However, most natural cellulosic substances, with the exception of cotton, contain lignin. Since the cellulose in this material is covered with persistent lignin, cellulose-degrading bacteria or cellulases cannot directly act on the cellulose. Furthermore, since cellulose has a structure in which cotton-like homopolymers of glucose are coordinated in parallel to each other, that is, a crystalline structure, even if lignin is removed, the action of cellulose-degrading bacteria or cellulases is weak, i.e.
_C1 enzyme activity is weak. Therefore, in order to efficiently decompose cellulosic materials such as rice straw, wheat straw, bagasse, waste paper, and wood chips using cellulolytic bacteria or cellulases, it is necessary to decompose lignin and destroy the crystal structure of cellulose. For this purpose, pretreatment such as chemical or physical treatment is performed. Chemical treatments include alkaline treatment with sodium hydroxide or the like and acid treatment with phosphoric acid or the like, but a common method is to add a cellulosic substance to an aqueous sodium hydroxide solution and heat it. This is due to the action of extracting and removing lignin and swelling the crystal structure of cellulose. However, this method has problems such as consuming a large amount of water, loss of active ingredients such as cellulose, hemicellulose, and protein, and the need to treat wastewater containing persistent lignin and the like. On the other hand, as a physical treatment, pulverization using a ball mill, a vibration mill, etc. is common. This method involves crushing cellulosic materials into approximately 400 mesh pieces to directly destroy the crystalline structure of cellulose. However, this method has drawbacks such as the need to thoroughly dry the cellulosic material and the significant power costs. An object of the present invention is to overcome the drawbacks of the prior art described above and to provide an efficient method for pre-treating cellulosic materials. To summarize the present invention, the pretreatment method of the present invention is characterized by bringing a water-containing cellulosic material into contact with ozone in biologically decomposing the cellulosic material. Lignin is decomposed by ozone treatment of lignin waste liquid discharged from the wood pulping process in the paper industry (Specific research by the Ministry of Education in 1975,
Takamasa Higuchi, “Decomposition of waste liquid lignin by ozone and microbial treatment”: Research report on environmental purification by microorganisms) and the fact that ultraviolet decomposition of cellulose is better in an air atmosphere than in a nitrogen atmosphere (published by Asakura Shoten, Dai Organic Chemistry) , Natural Polymer Compounds,
(See page 42) is known. As a result of various studies regarding the ozone treatment of cellulosic materials, the present inventors have discovered that by pre-treating the water-containing cellulosic materials by contacting them with a specific amount of ozone or more, the biological degradability of the cellulosic materials, i.e. The present invention was achieved by discovering that decomposition by cellulose-degrading bacteria or cellulase can be improved. The present inventors investigated various conditions for ozone treatment of cellulosic substances from various angles using the following experimental apparatus. Next, the present invention will be specifically explained based on an overview of the apparatus and operation and the experimental results obtained. Figure 1 is a schematic cross-sectional view of the equipment used in the experiment, where 1 is an ozone reaction tank, 2 is a gas injection port, and 3 is a cross-sectional diagram of the equipment used in the experiment.
4 is a moving bed, 4 is a raw material storage tank, 5 is a raw material, 6 is a discharge feeder, 7 is an ozone generator, and 8 is air. As shown in the figure, this apparatus is comprised of an ozone reaction tank 1 that brings a cellulosic material raw material 5 into contact with ozone, and an ozone generator 7 that produces ozone. In the ozone reaction tank 1, the cellulosic material raw material 5 supplied from the raw material storage tank 4 and the ozone supplied from the gas blowing port 2 come into countercurrent contact and react. Here, the ozone supplied from the gas blowing port 2 may be a mixed gas with other gases. The raw material 5 for cellulosic material in the ozone reaction tank 1 is
It is extracted by the discharge feeder 6 and descends as a moving layer 3, coming into contact with the rising ozone. On the other hand, ozone produced by the ozone generator 7 is consumed within the ozone reaction tank 1 and discharged to the outside of the system. Although this ozone reaction tank 1 is of a countercurrent contact type using a moving bed 3, it may also be of a type in which mechanical stirring is performed to bring the cellulosic material into contact with ozone. EXAMPLES The present invention will be explained below with reference to Examples, but the present invention is not limited to these in any way. Example 1 (Effect of moisture content of raw material on ozone reaction amount and influence of ozone reaction amount on enzymatic decomposition) Rice straw cut into lengths of 5 to 10 mm was used as a cellulosic material, and ozone concentration was determined depending on the initial moisture content. The fluctuation of the reaction amount was investigated. Using the above-mentioned experimental apparatus, the treatment was carried out with the ozone concentration blown into the ozone reaction tank 1 constant at 12 g/m ³ . The reaction temperature was set at room temperature, but it is not particularly limited as long as it is in the range of 0 to 70°C. The amount of ozone reaction was calculated from the difference in ozone concentration between the inlet and the outlet, the ozone blowing rate, and the contact time. The unit of the amount of ozone reaction was the weight (g) of ozone reacted per 1 g of dry rice straw. The results obtained are shown in FIG. That is, FIG. 2 is a graph in which the amount of ozone reaction and water content are plotted against contact time. A ₀ , A ₅ , A ₁₀ , A ₁₅ , A ₂₀ , A ₃₈ and A ₅₅
have an initial moisture content of 0% by weight, 5% by weight, respectively.
10wt%, 15wt%, 20wt%, 38wt% and 55
The change in water content in weight % is shown. _B0 , _B5 ,
B ₁₀ , B ₁₅ , B ₂₀ , B ₃₈ and B ₅₅ have initial moisture contents of 0% by weight, 5% by weight, 10% by weight, 15% by weight, 20% by weight, 38% by weight and 55% by weight, respectively. This shows the change in the amount of ozone reaction. As is clear from FIG. 2, the amount of ozone reaction increases with contact time, but forms a saturation curve, and the saturation value is proportional to the initial moisture content. However, when the initial water content is 0% by weight, ozone does not substantially react. Note that the moisture content of rice straw decreases as the contact time increases because ozone gas is dry. Comparing this moisture content decreasing curve with the saturation curve of ozone reaction amount, it is found that at initial moisture content of 15, 20, 38, and 55% by weight, when the moisture content of rice straw becomes about 10% by weight or less, the ozone reaction amount decreases. It is saturated and the reaction rate is significantly reduced. Therefore, it can be seen that in order to carry out the reaction efficiently, the moisture content of rice straw should be about 10% by weight or more, particularly 15% by weight or more. Furthermore, as a result of conducting the same experiment as above with wood chips and newspaper as cellulosic substances, the second
We obtained results similar to those shown in the figure. From the above, it has been found that in order to cause the cellulosic material to react with ozone, it is necessary to impregnate the cellulosic material with water. Furthermore, in order to carry out the reaction efficiently, it is desirable that the moisture content of the rice straw be approximately 10% by weight or more. Next, the results of evaluating the biological degradability of rice straw treated with ozone using the above method using cellulase are shown. When cellulase is applied to cellulosic substances, the amount of glucose produced by one unit (U) of enzyme in one hour is defined as enzymatic degradability, and this is used as an indicator of the difficulty of decomposing cellulosic substances by cellulase. . That is, the larger the enzymatic decomposition value, the easier the decomposition of the cellulosic substance by cellulase. As a result, the relationship between enzymatic decomposition and the amount of ozone reaction was investigated through experiments. As cellulase, commercially available Trichoderma
The one originating from viride (manufactured by Amano Pharmaceutical Co., Ltd.) was used. The enzymatic decomposition properties of rice straw treated by the above method were determined. The measurement conditions for enzymatic decomposition are PH4.5 and temperature 50.
℃. In addition, the enzyme unit of the enzyme solution was measured using carboxymethylcellulose (CMC),
One unit (U) corresponds to 1 micromol (μmol) of glucose produced per minute with 1ml of enzyme solution. The results obtained are shown in FIG. That is, the third
The figure is a graph showing the relationship between the amount of ozone reaction and enzymatic decomposition. As is clear from FIG. 3, the enzymatic decomposition property is improved when the ozone reaction amount is 0.005 (g-ozone/g-dried rice straw) or more. Therefore, when treating cellulosic materials by contacting them with ozone, the amount of ozone reaction is reduced to 0.005 (g-ozone/
It has been found that the effect can be exerted by using a dry rice straw (g-dry rice straw) or more. Example 2 (Effects of ozone treatment on various cellulosic substances) In addition to rice straw, wheat straw, bagasse, wood chips, and newspaper were used as cellulosic substances to determine their enzymatic decomposition properties (cellulase originating from Trichoclerma viride) after ozone treatment. . Regarding ozone treatment,
In both cases, the ozone reaction amount was 0.02 (g-ozone/g-
(dried cellulosic material), and the measurement conditions were the same as in Example 1 above. Rice straw and wheat straw are cut into 5-10 mm pieces, bagasse and wood chips are crushed into 2-5 mm pieces, and newspaper is 2 mm pieces.
A size of 5 mm was used. The results obtained are summarized in the table below. For comparison, a case without ozone treatment is also shown.

【table】

[Table] As is clear from the table, enzymatic decomposition of wheat straw, bagasse, wood chips, and newspaper can be improved by ozone treatment in the same manner as rice straw. Based on the above examples, the pretreatment method of the present invention can be applied to a hydrous cellulosic material, preferably with a water content of about
By contacting 10% by weight or more of a cellulosic material with ozone at an ozone reaction amount of 0.005 (g-ozone/g-dry cellulosic material) or more, the cellulosic material can be efficiently biologically decomposed. In this case, it is appropriate that the upper limit of the water content is below the saturated water content (approximately 80 to 85% by weight), and when the cellulosic material is immersed in water, lignin and organic substances elute. This is undesirable because the efficiency decreases as a result of reaction with ozone. Here, the ozonated cellulosic material is
Since it can be efficiently decomposed by cellulase (cellulose hydrolase), an ozonated cellulosic material is mixed with a general medium from which carbon sources have been removed by a known method, and then cellulose-degrading bacteria are inoculated and the mixture is heated at an appropriate temperature and pH. It has been experimentally confirmed that cellulosic substances can be efficiently decomposed by cellulose-degrading bacteria if the control is controlled as follows. This will be shown below as an application example. Application example (degradation of ozonated cellulosic materials by cellulose-degrading bacteria) As a cellulose-degrading bacterium, Trichoderma
viride [American Type Culture Collection (ATCC) No. 13631], Cellulomonas
flavigena (ATCC No. 484) and a strain isolated from compost (unidentified, tentative name CNY-266 strain) were pretreated with ozone using the same raw materials and conditions as in Example 1, and then treated with cellulose-degrading bacteria. The relationship between assimilation and ozone reaction amount was determined. The composition of the medium is sodium chloride 6.0g/, ammonium sulfate 1.0g/, phosphate-potassium 0.5
g/, dipotassium phosphate 0.5 g/, magnesium sulfate 0.2 g/, calcium chloride 0.3 g/, and yeast extract 1.0 g/. In addition, the concentration of cellulosic material (rice straw) was 10 g/. In addition, each culture temperature is Trichoderma
viride and Cellulomonas flavigena at 30℃;
For strain CNY266, the temperature was 50°C. In addition, the culture time was 10 days. As an index of the assimilation ability of cellulose-degrading bacteria, the amount of protein produced by each cellulose-degrading bacteria was determined.
The amount of protein produced was determined by subtracting the amount of protein contained in the rice straw from the amount of protein contained in the total solid content in the culture solution. The culture results obtained are shown in FIG. In other words, Figure 4 is a graph showing the relationship between the amount of ozone reaction and the amount of protein produced, and C is
Trichoderma viride, D is Cellulomonas
flavigena, E indicates the case of CNY-266 strain. Fourth
As is clear from the figure, the amount of each protein produced is approximately 0.005 (g-ozone/g-
Dry rice straw) increases rapidly from about 0.02 to
It became saturated from around 0.03 (g-ozone/g-dry rice straw). Moreover, according to the observation of the culture results, when the ozone-treated rice straw was inoculated with cellulose-degrading bacteria, the rice straw collapsed, and the cellulose-degrading bacteria were found to be growing well. From the above results, when a hydrous cellulosic material (rice straw) is brought into contact with ozone and cellulose-degrading bacteria are cultured using this material as a carbon source, the amount of ozone reaction of the cellulosic material is reduced to approximately 0.005 (g- ozone/g of dry cellulosic material) or more, it is possible to improve the protein produced by cellulose-degrading bacteria, that is, the amount of growth. That is,
It can be seen that the cellulose material was efficiently decomposed by cellulose-degrading bacteria. As explained above, when biologically decomposing a cellulosic material, a cellulosic material containing water, preferably one with a water content of about 10% by weight or more, is brought into contact with ozone, and the amount of ozone reaction at that time is reduced to about 10% by weight.
By setting the concentration to 0.005 (g-ozone/g-dry cellulosic material) or more, the cellulase enzyme 1
The amount of glucose produced per unit or the amount of growth of cellulose-degrading bacteria can be improved. Therefore, according to the present invention, the biological degradability of cellulosic substances can be improved, and it is extremely effective in biologically degrading cellulosic substances to obtain useful substances. For example, as shown in the examples and application examples above, in addition to producing glucose and microbial proteins from cellulosic materials, ozone-pretreated cellulosic materials may be used directly as roughage, or they may be applied to methane fermentation to produce methane. The present invention is extremely effective when converting cellulosic substances into biologically useful substances, such as when recovering cellulosic substances or producing organic acids by cellulose-degrading bacteria that produce organic acids such as acetic acid.

[Brief explanation of the drawing]

Figure 1 is a cross-sectional schematic diagram of the experimental apparatus of the present invention, Figure 2 is a graph showing changes in ozone reaction amount and water content with respect to contact time, and Figure 3 is a graph showing the relationship between ozone reaction amount and enzymatic decomposition. Graph, 4th
The figure is a graph showing the relationship between the amount of ozone reaction and the amount of protein produced. 1... Ozone reaction tank, 2... Gas blowing port,
3...Moving bed, 4...Raw material storage tank, 5...Raw material,
6...Discharge feeder, 7...Ozone generator,
8...Air.

Claims (1)

[Scope of Claims] 1. A method for pretreatment of a cellulosic material, which comprises bringing a hydrous cellulosic material into contact with ozone in biologically decomposing the cellulosic material. 2 The water content of the hydrated cellulosic material is approximately 10% by weight
The method for pre-treating cellulosic substances according to claim 1, which is the above. 3 Cellulosic material and ozone are mixed in an ozone reaction amount of about 1 g of dry weight of the cellulosic material.
3. The method for pre-treating a cellulosic material according to claim 1 or 2, wherein the cellulosic material is brought into contact at a rate of 0.005 g or more.