JPH0365155B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a pretreatment method for cellulosic materials, and in particular, after pretreatment in which cellulosic materials such as straw, bakasu, and wood chips are impregnated with water or an alkaline aqueous solution and subjected to a gas-solid contact reaction with ozone, then biological This invention relates to a pretreatment method for cellulosic materials that eliminates the inhibition of subsequent biological reactions caused by ozone reaction products during pretreatment when the ozonated cellulosic materials are chemically converted into useful substances such as microbial proteins and ethanol. It is. As a method of decomposing cellulosic substances to obtain various useful substances, cellulosic substances are
Cellulomonas flavigena, Trichoderma
reesei, Ruminococus albs, and Clostridium
A method of directly producing microbial cells, cellulase, ethanol, organic acids, etc. by using cellulose-degrading bacteria such as thermocellulum, or a method of decomposing the contained cellulose into glucose by cellulase, and then decomposing the microbial cells by the action of yeast or bacteria. There are methods for producing ethanol and ethanol, but in these cases, cellulosic substances usually have a structure that makes it difficult for cellulose-degrading bacteria or cellulases to act on them, which causes a reduction in the rate of decomposition. This is because cellulose has a strong crystalline structure and is covered with persistent lignin, which makes it possible to biologically produce useful substances such as microbial proteins and ethanol from cellulosic materials. In order to do this, it is necessary to destroy the above-mentioned structure in advance, that is, so-called pretreatment. As the above-mentioned pretreatment method, there is a method using ozone. Arthur Binder et al. found that when crushed wheat straw was suspended in water (about 5%) and ozone was aerated for 2 to 8 hours, approximately 50% of the lignin was decomposed and cellulose-degrading bacteria (Trichoderma reesei) and Chaetomium
reported that the degradability by cellulase and cellulase was improved. In addition, ozone reaction products dissolved in the solution from which treated wheat straw is removed are yeasts (Cardida utilis, Hansenula).
polymorpha) and activated sludge. [European J. Appl.Microbiol.
Biotechnol. Vol 11 , 1-5 (1980)] However, the disadvantage of this method is that the processing time is long. This is due to the low solubility of ozone in the liquid to be treated, and the reaction of dissolved ozone with lignin or lignin decomposition products dissolved in the liquid, resulting in less ozone reacting with cellulosic substances. -ing On the other hand, a cellulosic material is impregnated with water or an alkaline aqueous solution, and ozone is added to the impregnated cellulose material.
According to the pretreatment method (Japanese Unexamined Patent Publication No. 57-29293) that involves solid contact, the treatment time is significantly shortened to 20 to 60 minutes. On the other hand, Takamasa Higuchi et al. aerated ozone into a 5% ligninsulfonic acid aqueous solution (ozone concentration 7.2 g/m ³ ,
When the ozone supply rate was 1 g/h for 5 hours), the liquid
It has been reported that the pH decreases from 5.2 to 2.8, and organic acids such as oxalic acid and maleic acid are produced (1975 Ministry of Education, Culture, Sports, Science and Technology specific research "Report on research on environmental purification using microorganisms", pages 308-315). Masaaki Kuwahara, Arthur Binder, and others have reported that ozone reaction products containing these lignin decomposition products can be used as carbon sources for yeast and bacteria (Masaaki Kuwabara, "Growth of yeast in ozonolysis lignin sulfonic acid waste fluid") : 1975 Ministry of Education specific research “Report on environmental purification research using microorganisms”). However, due to the studies conducted by the present inventors,
Cellulosic substances treated with ozone in a solid contact reaction were saccharified with cellulase and concentrated, and then yeast (Saccharomyces cerevisiae) was cultured and alcohol fermented, and growth and fermentation inhibition by ozone reaction products was observed. Ta. Similarly, when cellulose-degrading bacteria were cultured using this ozone-treated cellulosic material, growth inhibition by the ozone reaction product was observed. In ozone treatment in a gas-liquid-solid system, ozone reaction products mainly elute to the liquid side, so the amount of ozone reaction products remaining in the raw material is small.
In solid catalytic reactions, all ozone reaction products remain in the raw material. Furthermore, since the cellulose content of the cellulosic material is about 40%, it is necessary to feed raw materials at a high concentration in order to obtain the desired product at a high concentration. Therefore, when a cellulosic material pretreated by a gas-solid contact reaction with ozone is used as a raw material, the concentration of ozone reaction products that become reaction inhibiting factors in the subsequent biological reaction utilization process becomes high. For this reason, when an ozone pretreatment method using a gas-solid contact reaction is adopted, it is necessary to eliminate the inhibitory effect on biological reactions caused by ozone reaction products. Based on this recognition, the present inventors have attempted various studies as described below. First, when a cellulosic material impregnated with water or an aqueous alkaline solution undergoes a gas-solid contact reaction with ozone, and then is suspended in an aqueous solution, the pH value of the liquid is
The value was extremely low at 1.2-1.4. If cellulase or cellulose-degrading bacteria are allowed to act directly on the cellulosic material in this state, the activity of each will decrease, and in the worst case, it will be deactivated. Therefore, after neutralizing with alkali, we tried using cellulase or cellulose-degrading bacteria. first,
The ozone-treated cellulosic material was neutralized and saccharified using a commercially available cellulase, and the saccharified liquid was concentrated and then subjected to alcoholic fermentation. As a result, the ethanol production rate decreased significantly and required a long fermentation time. When the cause of this was analyzed, it was thought that it was due to an increase in the concentration of salt produced by neutralization. Therefore, when alcohol fermentation was carried out in a synthetic medium containing the same salt concentration (NaCl) as the concentrate, although a decrease in the rate of ethanol production was observed to some extent, the production rate was lower than in the case of the concentrate. It was a large value. This suggests that the ozone reaction product itself inhibits alcohol fermentation. Therefore, after removing the ozone reaction product from the azan-treated cellulosic material, the same operation as above was performed. As a result, inhibition of alcoholic fermentation was eliminated, and fermentation time was shortened to about 1/4. Next, cellulose-degrading bacteria isolated from the compost were cultured in a synthetic medium containing the ozone-treated cellulosic material. As a result, as the amount of cellulosic material added increased, the growth rate of the cellulose-degrading bacteria decreased. This is also,
It turns out that this is because ozone reaction products inhibit the growth of cellulose-degrading bacteria. From the above, it has become clear that the subsequent microbial reaction is inhibited depending on the concentration of the ozone reaction product in the ozone-treated cellulosic material. Therefore, in order to find a way to eliminate the inhibitory effect caused by such ozone reaction products, we first analyzed the ozone reaction products. The ozone reaction product was extracted from the ozone-treated cellulosic material, and the composition was then examined using liquid chromatography. As a result, a variety of organic acids such as oxalic acid, glyoxylic acid, formic acid, and acetic acid, as well as acidic substances that absorb ultraviolet light, were detected. Therefore, it is thought that these substances, singly or in combination, inhibit microbial reactions. Furthermore, the inhibitory substance is also considered to vary depending on the type of microorganism used. Therefore, in order to establish a method for eliminating inhibition, it is necessary to identify the inhibitory substance for each microorganism used and to elucidate its mechanism. However, this requires effort and effort, and even if individual inhibitors are identified,
In some cases, it may be difficult to selectively remove the substance. Rather, even without such an analysis, the above study results show that even if the inhibitor and its inhibition mechanism are unknown, as long as the ozone reaction product is removed from the ozonated cellulosic material, the ozone reaction product can be removed. It is very clear that inhibition of microbial reactions can be overcome. The present invention was achieved based on such studies and the knowledge that removing ozone reaction products during pretreatment of cellulosic materials with ozone improves the reactivity of the subsequent microbial reaction. It is something. The purpose of the present invention is to eliminate the drawbacks of the conventional method and to
A pretreatment of cellulosic substances that can eliminate reaction inhibition and obtain industrially useful substances when subjected to subsequent biological reactions by pretreating cellulosic substances with ozone in a solid contact reaction system. The purpose is to provide a method. The present invention provides a method for pretreatment of cellulosic materials in which a cellulosic material impregnated with water or an alkaline aqueous solution undergoes a gas-solid contact reaction with ozone when the cellulosic material is decomposed by a biological reaction. A method for pretreatment of a cellulosic material, characterized by removing an ozone reaction product from an ozonated cellulosic material obtained by a contact reaction, and recovering the ozone reaction product removed from the ozonated cellulosic material. The gist of the present invention is a method for pretreating a cellulosic substance, which is characterized by: The present invention will be explained in detail below with reference to the drawings. FIG. 1 shows a system diagram of an apparatus suitable for carrying out the method of the present invention, including a humidity control mixing step in which a cellulosic material 1 is impregnated with water or an alkaline aqueous solution, and a gas-solid mixture of the cellulosic material and ozone. It consists of an ozone treatment step for causing a contact reaction, and a step for removing ozone reaction products. Cellulosic substances 1 include straw, bagasse, corn stover (corn cob),
Plant fibers containing lignin such as hay and wood chips are used. The crushed raw materials obtained by crushing these materials are mixed with water or alkaline aqueous solution sent from a storage tank 3 in a humidity control mixing tank 2. The mixing time at this time is preferably set to a time until the water content of the cellulosic material 1 becomes uniform. Then, the valve 4 at the bottom of the mixing tank 2 is opened, and the cellulosic material 1 is sent to the ozone reaction tank 6 through the feeder 5 . The ozone reaction tank 6 is not particularly limited as long as it has a tank structure that allows a gas-solid contact reaction between the cellulosic substance 1 and ozone. In this example, a packed tank type reaction tank was used as the ozone reaction tank 6. The cellulosic material forms a packed bed within the ozone reaction tank 6.
At this time, ozone prepared by an ozone generator 7 was vented from the bottom of the reaction tank 6. Ozone rises in the packed bed of cellulosic material and undergoes a gas-solid contact reaction with the cellulosic material, and unreacted ozone is discharged from the top of the tank to the outside of the tank and treated with activated carbon or the like. Note that a distribution in the height direction occurs within the filled tank. Therefore, in this example, the packed bed is divided in the height direction, and the ozone reaction amount of the cellulosic material in the section at the bottom of the tank is measured from the change in ozone concentration.When the value reaches the set value, the ozone ventilation was stopped, and the cellulosic material in that section was extracted by an extraction conveyor 8. Then, the cellulosic material was additionally supplied through the supply feeder 5, ozone was again blown into the container, and the ozone treatment was repeated. Through such an operation, the cellulosic substance descends in the ozone reaction tank 6 and undergoes a gas-solid contact reaction with ozone. In addition, the ozone inlet is
It is installed so that it does not interfere with the descent of cellulosic material. The ozonated cellulosic material extracted by the extraction conveyor 8 is mixed with water sent from the settling tank 11 within the same conveyor 8. For mixing here, a dedicated mixing tank may be provided, and the mixing method is not particularly limited. Further, the amount of water to be mixed is preferably 2 to 15 in terms of the ratio of water to the dry weight of the ozonated cellulosic material, that is, mixing is preferably carried out under kneading conditions. The thus mixed cellulosic material was sent to the solid-liquid separator 10 by opening the discharge damper 9. This solid-liquid separator 10 separated the cellulosic substance and the liquid. In this example, a centrifugal dehydrator was used as the solid-liquid separator, but a filter such as an olive filter or a filter press, or a compressor such as a roll mill may also be used. In particular, in the present invention, it is effective to perform compression dehydration using a roll mill or the like. This water addition-mixing-dehydration operation is performed in order to increase the removal rate of ozone reaction products.
May be repeated. Next, the separated liquid obtained by dehydration was sent to a precipitation tank 11 and neutralized with an alkali agent sent from an alkali agent aqueous solution storage tank 12. The alkaline agent may be an aqueous solution such as caustic soda or slaked lime, and is not particularly limited. The precipitate 13 here is collected from the bottom of the tank, and the supernatant liquid is extracted and recycled to the conveyor 8. The resulting precipitate 13 is an organic acid salt such as oxalic acid, which is advantageous as an organic substance. In the present invention, the ozone reaction product is recovered by a method in which the separated liquid obtained by dehydration in the solid-liquid separator 10 is sequentially added to an anaerobic fermentation tank such as a methane fermentation tank and the resulting product is recovered. You can also do it. For example, if separated water is added to a methane fermenter, methane useful for organic synthesis will be recovered. By such a method, in the present invention, the pretreated cellulosic material 14 from which the ozone reaction product has been recovered from the ozonated cellulosic material
is taken out from the solid-liquid separator 10 and sent to the subsequent biological reaction process. EXAMPLES The present invention will be explained in more detail by Examples below, but the present invention is not limited to the following Examples unless the gist thereof is exceeded. Example 1 Using bagasse as the test cellulosic substance,
It was saccharified with cellulase and subjected to alcoholic fermentation. The sample dried bagasse was mixed with a 0.1% NaOH aqueous solution at a weight ratio of 1:1, and then brought into gas-solid contact with ozone in an ozone reaction tank. The ozone concentration at the inlet was reduced to 11g/
m ³ and the ventilation amount was 2.4 m ³ /h. After a contact time of 40 minutes, the ozonated bagasse was extracted. The amount of ozone reaction at this time was 0.06Kg-O ₃ /Kg-bagasse. This was mixed with twice the amount of water and then dehydrated to separate a separated liquid. The bagasse subjected to the above pretreatment was saccharified by acting on commercially available cellulase. The saccharification conditions were: bagasse concentration 20%, pH 4.8, temperature 50°C, and saccharification time 38 hours. As a result, glucose concentration
A 4.5% sugar solution was obtained. At the same time, as a comparative example, the same saccharification was carried out on bagasse which was not subjected to the water addition/dehydration operation. Note that NaOH was used as a neutralizing agent at this time. The glucose concentration of the obtained sugar solution was 4.3%, which was almost the same as that of the bagasse treated to remove ozone reaction products. Both sugar solutions were concentrated three times and then subjected to alcoholic fermentation using yeast. In addition, 2.5 g, 1.0 g, and 3.0 g of asparagine, potassium phosphate, and magnesium sulfate were added per fermentation, respectively. Furthermore, the pH was 5.0 and the temperature was 30°C. As a control, fermentation was also carried out using a synthetic medium in which the above-mentioned additives and 130 g of glucose were dissolved in 1 part of water and the glucose concentration was the same as that of the above-mentioned concentrate. Figure 2 shows the fermentation results. From Figure 2, fermentation A when ozone reaction products are removed by the method of the present invention is almost the same as fermentation C using a synthetic medium, whereas fermentation when ozone reaction products are not removed. It is clear that fermentation of B is significantly inhibited. Comparing the concentration of ozone reaction products in the liquid at this time as soda salt, the concentration of ozone reaction products removed by the method of the present invention was about 25 g/, whereas the concentration of ozone reaction products removed by the method of the present invention was about 25 g/. The weight of those without was approximately 50g/. Note that the concentration of each ozone reaction product before saccharification is approximately
8.5g/and about 40g/. Therefore, by concentrating three times, the concentration of the ozone reaction product is approximately 25 g/(approximately 8.5 g/×3) when the ozone reaction product is removed by the method of the present invention, but the concentration of the ozone reaction product is Approximately if the object is not removed
It is 120g/ which does not match with about 50g/. This seems to be because ozone reaction products precipitate as soda salts due to pH adjustment during saccharification and fermentation. Reference Example 1 Most of the ozone reaction product is dissolved as soda salt. Therefore, we investigated the effect of salt concentration on alcohol fermentation. Using NaCl as a salt, synthetic media with NaCl concentrations of 25 g/ and 50 g/ were prepared, and alcoholic fermentation was carried out in the same manner as in Example 1. The fermentation results are shown in Table 1. Table 1 also shows the results of Example 1 when the ozone reaction product was removed, when it was not removed, and the control results.

[Table] As can be seen from Table 1, the influence of salt concentration on alcohol fermentation was observed, but when the ozone reaction product concentration is large and the ozone reaction product is not removed, /3 is small. On the other hand, when comparing the case where the ozone reaction product is removed and the NaCl concentration of 25 g/, the former is larger. These results show that alcohol fermentation is inhibited not only by the salt concentration but also by the concentration of the ozone reaction product. Example 2 Yeast was cultured using the sugar solutions prepared in Example 1 (one from which ozone reaction products were removed and one from which ozone reaction products were not removed).
As a control, culture was performed in a synthetic medium containing glucose as the carbon source. For further reference
Culture was carried out in a synthetic glucose medium with a NaCl concentration of 50 g/g. The culture conditions were pH 5.0, temperature 30°C, and shaking culture. Table 2 shows the bacterial concentration after the completion of culture. However, the initial bacterial concentration was 2 g/.

[Table] From Table 2 above, it can be seen that the bacterial concentration is highest when ozone reaction products are removed, and lowest when ozone reaction products are not removed. From this, it is clear that the ozone reaction product inhibits the growth of yeast. Example 3 A fermentation test similar to Example 1 was conducted using rice straw, wheat straw, and corn cob as cellulosic materials. The fermentation results are shown in Table 3.

[Table] The same results as in Example 1 were obtained for all three types of cellulosic substances. Example 4 Bagasse from which ozone reaction products were removed after ozone treatment in Example 1 and bagasse from which ozone reaction products were not removed from ozone treatment were used as carbon sources, respectively.
Cellulose-degrading bacteria isolated from compost were cultured and their growth rates were compared. The composition of the medium other than the above bagasse is: sodium chloride 6.0g/, ammonium sulfate 1.0g/, monopotassium phosphate 0.5g/, dipotassium phosphate 0.5
g/, magnesium sulfate 0.5 g/, calcium chloride 0.3 g/, and yeast extract 1.0 g/. In addition, the medium pH was set to 6.0. The bagasse concentration is
Initially, 20 g/bagasse was added, and the total amount of bagasse added was 100 g/medium. Note that the culture temperature was 50°C. Figure 3 shows the culture results. As can be seen from Figure 3, when the ozone reaction product is not removed, E
In this case, as the added bagasse concentration increases, the amount of bacteria produced decreases, and the growth is poorer than in case D when the ozone reaction product is removed. From the above, it is clear that according to the present invention, it is possible to eliminate the inhibition of ozone reaction products on microbial reactions after ozone treatment. Example 5 An organic acid recovery test from the separated liquid obtained in Example 1 was conducted. In Example 1, when slaked lime was added to the separated liquid obtained by the water addition mixing-dehydration treatment, a white precipitate was obtained. When this was analyzed, it was found to be calcium oxalate. Therefore, it can be seen that oxalic acid can be recovered by removing the soluble ozone-reactive substance from the ozone-treated cellulosic material. Example 6 When the separated liquid obtained in Example 1 was added to a methane fermentation tank, methane was recovered. According to Examples 5 and 6, it is clear that useful substances such as oxalic acid and methane can be obtained from the ozone reaction product. Example 7 The degradability of cellulosic materials by cellulase was compared when a roll mill and a centrifugal dehydrator were used to remove ozone reaction products. In Example 1, when ozonated bagasse was mixed with water and then dehydrated, bagasse dehydrated by a roll mill and bagasse dehydrated by a centrifugal dehydrator were used, and the saccharification rate with commercially available cellulase was investigated. The results are shown in Table 4.

[Table] As can be seen from Table 4, the method of removing ozone reaction products affects the biodegradability of cellulosic materials. This is thought to be because in roll meal, destruction of the fiber structure of the cellulosic substance progresses simultaneously with the removal of ozone reaction products. Therefore, the pressing dehydration method using a roll mill as in this example is extremely advantageous because it provides a pressing effect and a fiber structure destruction effect, and improves the saccharification rate. According to the present invention, the biodegradability of cellulosic substances is improved because the inhibition of biological reactions caused by ozone reaction products can be eliminated. In the process of producing ethanol, microbial proteins, etc. from cellulosic substances, it becomes possible to feed ozone-treated cellulosic substances at a high concentration, thereby improving the efficiency of the process in which a high-concentration target product is obtained. In addition, when removing ozone reaction products, simply leaving the ozonated cellulosic material in water does not provide a sufficient elution rate, and a large amount of water is required (this is because the ozone reaction products are impregnated with the cellulosic material). (This is thought to be due to the substance remaining in the fibers of the substance.) On the other hand, the ozonated cellulosic substance and water are mixed, and preferably, water is added so that the mixture is in a kneaded state. Ozone reaction products can be removed even better by applying physical force to dehydrate, such as by post-squeezing. Moreover, this ozone reaction product can be effectively recovered as a useful substance according to the present invention.

[Brief explanation of drawings]

FIG. 1 is a system diagram showing an apparatus suitable for carrying out the method of the present invention. FIG. 2 is a graph showing the fermentation results according to Example 1. Figure 3 shows Example 4
It is a graph showing the culture results. 1... Cellulose material, 2... Humidity control mixing tank,
3... Storage tank, 4... Valve, 5... Supply feeder, 6... Ozone reaction tank, 7... Ozone generator,
8...Extraction conveyor, 9...Discharge damper,
10... Solid-liquid separator, 11... Sedimentation tank, 12...
Alkaline agent aqueous solution storage tank, 13...Sediment, 14...
...pretreated cellulosic material.

Claims (1)

[Scope of Claims] 1. A pretreatment method for a cellulosic material in which a cellulosic material impregnated with water or an alkaline aqueous solution is subjected to a gas-solid contact reaction with ozone when the cellulosic material is decomposed by a biological reaction, which comprises the following steps. A method for pretreating a cellulosic material, comprising the steps of: (i) A step of adding and mixing water to the cellulosic material subjected to ozone treatment, and then dehydrating it by a squeezing operation to elute and remove the organic acid of the lignin-degradable component, and then subjecting it to the subsequent biological reaction step; (b) A step of adding an alkali to the eluate separated liquid to precipitate and recover the organic acid, or a step of adding the separated liquid to a methane fermentation tank to convert it into methane. 2. The method for pre-treating a cellulosic material according to claim 1, wherein the amount of water added is in the range of 2 to 15 in terms of weight ratio to the dry weight of the ozonated cellulosic material. . 3. Pretreatment of cellulosic material according to claims 1 and 2, characterized in that the aqueous solution obtained by precipitating the organic acid is recycled as water to be added to the ozonated cellulosic material. Method.