JPS6319150B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a method for culturing cellulose-degrading bacteria using cellulosic materials such as rice straw, wheat straw, bagasse, waste paper, and wood chips as a carbon source. This invention relates to a method for culturing cellulose-degrading bacteria to enable cellulose assimilation.

When cellulose-degrading bacteria are allowed to act on cellulosic substances and the cellulase (cellulose hydrolase) and organic acids secreted by the cellulose-degrading bacteria are grown, or the cellulose-degrading bacteria themselves are grown to produce microbial proteins, the culture of cellulose-degrading bacteria is used. In addition, it must be possible for cellulose-degrading bacteria to efficiently utilize cellulose in cellulosic substances. However, cellulose in cellulosic materials such as rice straw, wheat straw, bagasse, waste paper, and wood chips has a chemical structure that makes it difficult for cellulose-degrading bacteria to act on it. Cellulose is
It is a homopolymer in which glucose is linearly arranged with β-1,-4 bonds, but the cellulose in the above substance has a structure in which the linear homopolymers are coordinated in parallel to each other, that is, a crystal structure, and furthermore, It is covered with persistent lignin. By the way, the ability of cellulose-degrading bacteria to use cellulose as a nutrient source is due to the action of cellulase (cellulose hydrolase) secreted by the same bacteria. Cellulase is
_C1 enzyme that amorphizes crystalline cellulose, Cx enzyme that decomposes amorphous cellulose into cellobiose, and β- enzyme that decomposes cellobiose into glucose.
It is a complex enzyme composed of glucosidase.
Cellulose-degrading bacteria convert cellulose into cellobiose and glucose using the cellulase. However, Cerase is C ₁
It has weak activity and no lignin decomposition ability. Therefore, cellulose-degrading bacteria cannot directly assimilate cellulose contained in cellulosic substances having the above-mentioned structure. Therefore, in order to cultivate cellulose-degrading bacteria using cellulosic materials as a substrate,
When preparing the substrate, it is necessary to decompose lignin and destroy the crystal structure of cellulose. Chemical or physical treatment is used as a method for this purpose. Chemical methods include alkaline treatment with sodium hydroxide or the like and acid treatment with phosphoric acid, etc., but a common method is to add a cellulosic substance to an aqueous sodium hydroxide solution and heat it.
This has the effect of extracting and removing lignin and swelling the crystal structure of cellulose. However, this method consumes large amounts of water and there is a loss of active ingredients such as cellulose, hemicellulose and protein. Additionally, it is necessary to treat wastewater containing persistent lignin. On the other hand, as a physical treatment, pulverization using a ball mill, vibration mill, etc. is common. This method crushes cellulosic materials into approximately 400 meshes and directly destroys the crystalline structure of cellulose. However, this method has disadvantages, such as the need to thoroughly dry the cellulosic material and the significant power costs.

An object of the present invention is to provide a method for culturing cellulose-degrading bacteria that eliminates the drawbacks of the conventional methods described above and allows cellulose-degrading bacteria to efficiently utilize cellulose in cellulose materials. It is something to do.

To summarize the present invention, the method for culturing cellulose-degrading bacteria of the present invention involves mixing a cellulosic material with an aqueous solution containing inorganic nitrogen, inorganic salts, etc. so that the water content becomes 90% or less, and then adding ozone and gas-solidified material. By contacting it, the ability of cellulose-degrading bacteria to assimilate cellulose in cellulose substances is improved.

It has been reported that lignin is decomposed by ozone treatment of the lignin waste liquid discharged from the wood pulping process in the paper industry (1975 Ministry of Education specific research, Takamasa Higuchi, “Reducing waste liquid lignin by ozone and microbial treatment”). "Decomposition": see research report on environmental purification using microorganisms). Therefore, the inventors thought that this method could be applied to a substrate preparation method for culturing cellulose-degrading bacteria, and carried out ozone treatment by suspending a cellulosic substance in water and blowing ozone into it. However, cellulose-degrading bacteria hardly utilized cellulose in cellulosic substances. This is because the lignin in cellulosic substances has a three-dimensional tertiary structure, unlike soluble lignin such as waste liquid lignin, so it is assumed that the lignin in cellulosic substances is difficult to be decomposed by ozone using the method reported above. Ta. Therefore, as a result of further studies on ozone treatment of cellulosic materials, the inventors discovered that cellulose decomposition can be prevented by immersing cellulosic materials in water, bringing them into gas-solid contact with ozone, and reacting with more than a specific amount of ozone. The present invention was achieved by discovering that cellulose in cellulosic substances can be effectively decomposed.

The present invention will be described below with reference to Examples.

The present inventors conducted a multifaceted study on ozone treatment of cellulosic materials and assimilation of the ozonated cellulosic materials by cellulose-degrading bacteria using the following apparatus.

FIG. 1 is a schematic diagram of the culture apparatus used in the present invention, in which 1 is a cellulosic substance, that is, a raw material, 2 is a water-soluble medium containing other than 1, 3 is a mixer, 4 is an ozone treatment tank, 5 1 is a moving bed, 6 is an ozone inlet, 7 is an ozone generator, 8 is air, 9 is a discharge feeder, 10 is a sterilization tube, 11 is steam, 12 is a culture tank, and 13 is a discharge port.

In this apparatus, first, as shown in the figure, a raw material 1 which is a cellulosic substance and a medium 2 containing no carbon source are mixed in a mixer 3, and then reacted with ozone in an ozone treatment tank 4. Here, the medium 2 is an aqueous solution containing inorganic nitrogen such as ammonium sulfate and ammonium chloride, and inorganic salts such as phosphate, magnesium salt, and potassium salt. Further, the medium 2 may be simply water, but in this case, the above-mentioned medium composition may be added again at a later stage. Note that if cellulose-degrading bacteria require organic nitrogen or vitamins, they may be added after the ozone reaction. In the mixer 3, the moisture content of the cellulosic substance raw material 1 is
Prepared to 90% or less. This raw material 1 comes into contact with ozone supplied from an ozone blowing port 6 at the bottom of the tank in the ozone treatment tank 4 . Note that ozone is prepared by an ozone generator 7 using air 8 as a raw material. The inlet ozone concentration at this time is 0.1 to 20g/
m ³ is sufficient, but there is no particular limitation.
The raw material 5 in the ozone reaction tank 4 is extracted by a discharge feeder 9 provided at the bottom of the tank, and thus becomes a moving tank 5 and descends. At this time, it comes into countercurrent contact with the rising ozone. Note that the ozone treatment tank 4 may be a device that mechanically stirs the raw material 5 and brings it into contact with ozone.

The raw material 5 that has been ozonated in the ozone treatment tank 4 is
It is extracted by the discharge feeder 9 and enters the sterilization tube 10. The sterilization tube 10 is heated to about 100° C. by the steam 11. Then, the raw material 5 passed through the sterilization pipe 10
is sterilized and at the same time unreacted ozone is decomposed. Note that the sterilization method is not particularly limited.

The sterilized raw material 5 is supplied to the culture tank 12. In the culture tank 12, the raw material 5 and cellulose-degrading bacteria are mixed. The cellulose in the raw material 5 is reduced in molecular weight by cellulase (cellulose hydrolase) secreted by cellulose-degrading bacteria.
The culture method may be either a solid culture method or a submerged culture method. At this time, conditions in the culture tank 12 that are suitable for the growth of cellulose-degrading bacteria, such as temperature and pH, must be controlled to optimal values. In addition, cellulose-degrading bacteria are Cellulomans
Genus, Cellribrio, Pseudomonas, Clostrdium
Genus, Actinomyces, Aspergillus,
Bacteria capable of decomposing cellulose, such as those of the genus Penicillium, cannot be used, and there are no particular limitations.

A mixture of decomposition products, decomposition residues, and cellulose-degrading bacteria is taken out from the outlet 13.

Next, the present invention will be explained with reference to Examples, but the present invention is not limited thereto.

Example 1 Sun-dried rice straw was cut into 5-10 mm pieces and mixed with a culture medium to prepare rice straw with a moisture content of 5-85% by weight. The medium contains 6.0g of sodium chloride/
, ammonium sulfate 1.0g/, monopotassium phosphate 0.5g/, dipotassium phosphate 0.5g/, magnesium sulfate 0.2g/, calcium chloride 0.3
g/aqueous solution. This rice straw was placed in the ozone treatment tank and subjected to gas-solid contact reaction with ozone. Note that the ozone concentration blown was 12 g/m ³ . Moreover, the reaction temperature was room temperature. 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 ozone reaction amount is the weight of ozone reacted per 1 g of dry rice straw.

Figure 2 shows the measurement results. Plot A is the result of this example. Even when a water-soluble medium containing inorganic nitrogen and inorganic salts was added to rice straw, the rice straw reacted with ozone, and the amount of ozone reaction increased as the water content increased. As a comparative example, rice straw soaked with water was subjected to the same treatment. The results are also shown in Figure 2. Plot B is the result of a comparative example. From the results of the comparative example and the above example,
It was found that almost the same results as with water can be obtained using a water-soluble medium. The reason why the amount of ozone reaction decreased when the water content was 70% by weight or more was because the rice straws adhered to each other, reducing the efficiency of contact with ozone.

Example 2 Cellulose-degrading bacteria were cultured using the ozonated rice straw obtained in Example 1 as a substrate.

Trichoderma is a cellulose-degrading bacterium.
viride (American Type Culture Collection: ATCC No. 13631) and a bacterial strain isolated from compost (final identification, tentative name CNY-
226 strains) were used. Cultivation is done using a solid culture method.
That is, ozonated rice straw and cellulose-degrading bacteria were mixed aerobically in a moist state. In addition, both strains are
Since the yeast requires vitamins, a previously sterilized yeast extract solution was added to the ozone-treated rice straw for cultivation. In addition, the culture temperature is
30℃ for Trichoderma viride, 50℃ for CNY-266 strain
℃.

FIG. 3 shows the moisture content and the amount of bacterial cells produced. The culture time was 8 days. C is for Trichoderma
viride, D shows the case of CNY-226 strain. In addition,
The plot with a moisture content of 0% by weight is the case without ozone treatment. From the figure, as the water content increases,
The amount of bacterial cells produced increased, and the same results as in Example 1 were obtained. From this point of view, the amount of ozone reaction is considered to be a factor that influences the amount of bacterial cell production. In other words, it is thought that as the amount of ozone reaction increases, the cellulose in rice straw becomes more easily assimilated by cellulose-degrading bacteria, and when the water content is 70% by weight or more, the amount of ozone reaction is greater than the water content of 10% by weight. Regardless, the amount of bacteria produced is small. In particular, when the water content was around 90% by weight, the amount of bacterial cells produced was almost the same as that of the untreated sample. The reason for this is thought to be as follows. When rice straw with a moisture content of 70% by weight or more was prepared, there was a large amount of free water, and the free water contained soluble substances and had a color. However, when treated with ozone, most of the color was removed. From this, the above cause is that ozone does not react directly with rice straw,
It is assumed that this is because the ozonation reacts with the eluate of the rice straw, and as a result, the effect of ozonation treatment is virtually ineffective.

Judging from the above results, in order to substantially obtain the effect of ozone treatment, the water content should be 90% by weight or less.

Example 3 Rice straw with an ozone reaction amount of 0 to 0.04-ozone/g-dried rice straw was prepared, and Trichoderma viride,
CNY-226 strain and Cellulomonas flavigena
(ATCC No. 484) was cultured. In addition,
Tichoderma viride and CNY-226 strains were cultured in solid state, and Cellulomonas flavigena was cultured in liquid culture.

In addition, in the culture of Cellulomonas flavigena,
The temperature was 30°C and the pH was controlled at 6.8.

When the results on the 10th day of culture were organized by the amount of ozone reaction, the results were as shown in Figure 4. E is for Trichoderma
viride, F is CNY-226 stock, and G is
The case of Cellulomonas flavigena is shown.

From Figure 4, as the amount of ozone reaction increases,
It can be seen that the amount of bacterial cells produced is increasing. In other words, it was found that as the amount of ozone reaction increased, cellulose in rice straw became more easily decomposed by cellulose-degrading bacteria. In particular, the amount of ozone reaction
The amount of bacteria produced increased significantly with rice straw containing 0.005g-ozone/g-dry rice straw or higher.

From the above examples, in order to substantially obtain the effect of ozone treatment, the water content of the cellulosic material should be 90% or less, and a gas-solid contact reaction with ozone should be carried out.
It was also concluded that the amount of ozone reaction should be set to 0.005 g/g of dry rice straw or more.

If the present invention is applied, for example, when cellulose substances are used as a carbon source and cellulose-degrading bacteria are cultured to produce microbial proteins from the bacterial cells, or when cellulase or organic acids secreted by cellulose-degrading bacteria are produced, etc. The cellulose in the cellulosic material is efficiently decomposed by cellulose-degrading bacteria, improving the production rate. Furthermore, since the present invention involves a gas-solid contact reaction between a cellulosic substance and ozone, there is no problem in waste liquid treatment such as in caustic soda treatment. Furthermore, since the treatment effect is obtained with a small amount of consumed ozone reaction, it is economical.

[Brief explanation of the drawing]

Figure 1 is a schematic diagram of the culture apparatus used in the present invention, Figure 2 is a graph showing the relationship between ozone reaction amount and water content, Figure 3 is a graph showing the relationship between water content and bacterial cell production, and Figure 4 is a graph showing the relationship between water content and bacterial cell production. The figure is a graph showing the relationship between the amount of ozone reaction and the amount of bacterial cell production. 1... Raw material, 2... Culture medium, 3... Mixer, 4...
... Ozone treatment tank, 5 ... Mobile tank, 6 ... Ozone blowing port, 7 ... Ozone generator, 8 ... Air,
9... Discharge feeder, 10... Sterilization tube, 11...
Steam, 12...Culture tank, 13...Outlet.

Claims (1)

[Claims] 1. When culturing cellulose-degrading bacteria using a cellulosic substance as a carbon source, the cellulosic substance is mixed with an aqueous solution containing inorganic nitrogen and inorganic salts so that the water content is 90% or less, and then A method for culturing cellulose-degrading bacteria, which is characterized by bringing the cellulose-degrading bacteria into gas-solid contact with ozone. 2 Cellulosic material and ozone are mixed in an amount of ozone reaction per 1 g of dry weight of the cellulosic material.
The method for culturing cellulose-degrading bacteria according to claim 1, wherein the cellulose-degrading bacteria are brought into contact at a rate of 0.005 g or more.