TWI405852B - Method of cultivating fermentative bacterium used in non-detoxified xylossolution - Google Patents

Abstract

A new fermentative bacterium, Pichia stipitis INER 1128, is cultivated according to the present invention. The fermentative bacterium can effectively convert xylose in a solution, which is not even detoxified. The conversion rate is high while xylose is not wasted and cost is reduced.

Description

Xylose fermentation method for removing toxic fiber raw material hydrolyzate

The invention relates to a method for fermenting xylose of a hydrolyzate of a non-toxic fiber raw material, in particular to a domesticated culture of xylose fermentation strain of a fiber raw material hydrolyzate prepared by a high temperature and high pressure reaction process of an extruded mixed acid and a hot water washing process, and According to the method, the xylose fermentation method for the undecomposed fiber raw material hydrolyzate can be applied to the xylose fermentation of the fiber raw material hydrolyzate without detoxification, and the yield of xylose converted to alcohol can reach 90. %the above.

Biomass alcohol has been regarded as a substitute fuel for replacing the potential of gasoline. At this stage, the raw materials for commercial mass production are mainly cereals and sugar cane. The main components that can be converted into alcohol are starch and sucrose, respectively. After simple pretreatment, the fermentation technology can be used to produce alcohol. Since the fermentation process of starch alcohol and sucrose alcohol mostly uses Saccharomyces cerevisiae and other Saccharomyces cerevisiae, it usually obtains a very good alcohol yield, so the fermentation technology has been developed to a fairly mature stage.

>> However, due to the use of cereal crops and sugar cane as raw materials for the production of alcohol, there has always been a dispute over the struggle for food, which may affect the supply and demand of food. Therefore, fiber-based raw materials based on wood, bagasse, Rice Straw, Corn Stover, Wheat Straw, Silvergrass, and paper waste. High global storage, diversified sources and no food The advantages of production conflicts, the conversion of alcohol into fiber raw materials has been regarded as the most promising bio-alcohol production technology in the future. Generally speaking, general fiber raw materials mainly contain 60-80% cellulose, hemicellulose and 15~25% lignin, wherein cellulose and hemicellulose are converted into six carbon by Saccharification Process respectively. After sugar (mainly glucose) and five-carbon sugar (mainly xylose), these monosaccharides can be further converted into alcohol by bio-fermentation technology.

>> On the other hand, in the process of fiber-converting alcohol, high temperature and high pressure thermochemical pretreatment technology such as Dilute-acid Hydrolysis or Acid-Catalyzed Steam Explosion is used at this stage. Cellulose is decomposed into xylose. Due to the reaction process of such thermal chemical pretreatments, a certain proportion of raw materials and aqueous solution are usually loaded into the reactor, and then under high temperature and high pressure reaction conditions. 1~2% (w/w) of dilute sulfuric acid will be added. Therefore, the liquid obtained after the reaction, that is, the hydrolyzate, will contain a higher concentration of sulfate ion due to the addition of dilute sulfuric acid, thereby reducing the conversion of the fermentation strain to xylose. The ability of alcohol. In addition, such pretreatment techniques also produce fermentation inhibitors such as Acetic Acid, Furfural and Hydroxymethyl Furfural depending on the reaction conditions, so the pretreatment proceeds at this stage. The xylose hydrolysate usually removes furfural and sulfate ions by overbasing, and detoxifies the hydrolyzate produced by the pretreatment to enable subsequent biofermentation to proceed smoothly. However, due to the overbasing process, the xylose is usually lost and will be produced at the same time. Calcium sulphate sludge requires additional funds and equipment to be disposed of and disposed of, thereby increasing the production cost of xylose-converted alcohol.

>> Furthermore, Saccharomyces cerevisiae, which used to be used in the past, does not have the function of converting five-carbon sugar such as xylose into alcohol, and microorganisms with good ability to transform xylose into alcohol in nature are also very limited, only Pichia stipitis , Candida A small number of yeast species such as shehatae have been shown to have the function of converting xylose into alcohol. Moreover, among the relevant literatures collected so far, the types of hydrolyzed fluids studied by the yeast Pichia stipiti for converting xylose into alcohol include corn stalks, corn cobs, hardwoods, and softwoods. ), Water Hyacinth, wheat straw, Sunflower Seed Hull and rice straw, except for a recent paper (Agbogbo, FK and KS Wenger, 2007. Production of ethanol from corn stover hemicellulose) Hydrolyzate using Pichia stipitis . Journal of Industrial Microbiology and Biotechnology 34, 723-727) pointed out that the corn stalk xylose hydrolyzate can be fermented with Pichia stipitis to obtain 85% alcohol yield, and the yields of xylose fermented alcohol obtained in other literatures are roughly Below 75%.

>> On the other hand, since the above literatures all carry out xylose fermentation by hydrolyzing solution of different detoxification conditioning techniques such as alkalization, the highest alcohol yield can be obtained as described above, only Agbogbo et al. The paper published in the journal Biochem.Biotechnol. "Fermentation of acid-pretreated corn stover to ethanol without detoxification using Pichia stipitis " has tested the ability of Pichia stipitis to convert xylose into alcohol without detoxification-conditioned corn stalk hydrolysate. The highest alcohol yield is only about 82%, and the utilization rate of xylose in the hydrolyzate can only reach 35%. Therefore, the above-mentioned conventional techniques cannot reduce the loss of xylose and maintain high alcohol yield to reduce the cost of producing alcohol. Therefore, the average user cannot meet the needs of the user in actual use.

The main object of the present invention is to overcome the above problems encountered in the prior art, and to provide a method for acclimating xylose fermentation strains of a fiber raw material hydrolyzate prepared by high temperature and high pressure reaction treatment by extrusion mixed acid and hot water washing process, and The method for establishing a xylose fermentation method for unhydrogenated fiber raw material hydrolyzate can effectively increase the yield of xylose into alcohol in the fiber raw material hydrolyzate without detoxification conditioning.

>>>> The secondary objective of the present invention is to reduce the xylose loss and the cost of producing alcohol, as well as to have high xylose utilization and high alcohol yield.

For the purpose of the above, the present invention relates to a xylose fermentation method for a non-toxic fiber raw material hydrolyzate, and a domesticated cultured xylose fermentation strain prepared by a high temperature and high pressure reaction process of an extruded mixed acid combined with a hot water washing process. In the xylose hydrolysate, it can be directly grown in the hydrolyzate of the fiber raw material which has not been detoxified and converted into xylose to produce alcohol. First, the domestication and cultivation process is carried out by mixing the undegraded xylose hydrolyzate and the artificial culture solution (Yeast extract and Peptone) in a ratio of 20:80 by volume, and additionally adding xylose (Xylose). and Glucose, the initial xylose and glucose concentrations of the mixed culture solution are controlled at 30 g / liter (g / L) and 10 g / L, respectively, after at least two generations of continuous subculture, according to the above initial xylose and glucose The concentration is maintained at a fixed concentration value, and then the proportion of the xylose hydrolyzate which has not been detoxified is gradually increased, and the ratio of the undegraded xylose hydrolyzate to the artificial culture solution is gradually changed from 20:80. At 35:65, 50:50, 60:40 and 70:30, the yeast exhibits high tolerance to the non-toxically conditioned fiber raw material hydrolysate, and finally directly to the fiber without detoxification conditioning. The raw material hydrolyzate was cultured for at least 60 generations, and it was confirmed that the yeast cultured and cultured by the method of the present invention can directly grow on the undegraded fibrous raw material hydrolyzate, and has good xylose-converting alcohol efficiency.

Therefore, the xylose fermentation method of the undecomposed fiber raw material hydrolyzate of the present invention is not limited to the xylose hydrolyzate obtained by the above pretreatment of rice straw, and can also be applied to bagasse (Bagasse) and Miscanthus. (Silvergrass), Napiergrass, Switchgrass, Corn stover, Wheat straw, Wood, Bamboo, Water hyacinth, and Algae a xylose hydrolyzate obtained by pretreating other fibrous raw materials and pre-catalyzed by dilute acid.

The invention relates to a xylose fermentation method for a non-toxic fiber raw material hydrolyzate, which comprises providing a fiber raw material hydrolyzate domesticated culture prepared by a high temperature and high pressure reaction process by extrusion mixed acid and hot water washing process. The xylose fermentation strain is applied to the fiber raw material hydrolyzate which has not been detoxified and conditioned to increase the yield of xylose into alcohol.

In a preferred embodiment, the present invention is to domestically culture a strain of Pichia stipitis (ATCC 58785; BCRC21777) in a prepared rice straw xylose hydrolysate to enable direct growth without detoxification. The conditioned fiber raw material hydrolyzate and converted xylose to produce alcohol. First, the domestication and cultivation process is carried out by mixing the undegraded xylose hydrolyzate and the artificial culture solution (Yeast extract and Peptone) in a ratio of 20:80 by volume, and additionally adding xylose (Xylose). And glucose (Glucose), the initial xylose and glucose concentration of the mixed culture solution are controlled at 30 g / liter (g / L) and 10 g / L, respectively, after at least two generations of successive passages, according to the initial xylose and The glucose concentration is maintained at a fixed concentration value, and then the proportion of the xylose hydrolyzate which has not been detoxified is gradually increased, and the ratio of the undegraded xylose hydrolyzate to the artificial culture solution is gradually increased from 20:80. Changed to 35:65, 50:50, 60:40 and 70:30, so that yeast showed high tolerance to the fiber raw material hydrolyzate without detoxification conditioning, and finally directly without detoxification conditioning The fiber raw material hydrolyzate is cultured for at least 60 generations, and it is confirmed that the domesticated culture yeast can directly grow on the undegraded fiber raw material hydrolyzate, and has good xylose-converting alcohol efficiency, and the present invention is domesticated and cultured. The novel yeast strain named Pichia stipitis INER 1128.

>>Please refer to "Figure 1" for the hydrolysis of rice straw xylose of the present invention. Schematic diagram of liquid composition. As shown in the figure: when the present invention is actually operated, the xylose hydrolyzate to be used is a liquid solution obtained by using a biaxial screw extruder with a dilute acid catalytic reaction tank as a pretreatment equipment, due to the pretreatment The device is already prior art content, so it will not be repeated here. In the reaction process, firstly, the rice straw which has been cut to the appropriate size is used in the biaxial screw extruder at a dilute acid concentration of 3% (w/w), the screw rotation speed is 40 rpm, and the reaction temperature and time are respectively The stalk is extruded at 145 ° C for 20 minutes and the ratio of the dry weight of the feed rice straw to the aqueous solution is about 50:100. After the structure is destroyed, the dilute acid catalytic reaction is further carried. The tank is filled with an appropriate amount of steam. When the ratio of the dry weight of the rice straw to the aqueous solution is reduced to about 30:100, the reaction temperature is raised to 130 ° C, and after cooking at this high temperature for 15 minutes, the rice after the reaction is immediately The stalk and the aqueous solution are discharged, and the rice straw and the aqueous solution after the reaction are separated by a solid-liquid separation device, and finally the obtained aqueous solution is a xylose hydrolyzate, and the main component thereof comprises glucose, xylose, arabinose (Arabinose), Acetic acid, furfural and Hydroxymethyl Furfural. The xylose hydrolyzate has a xylose concentration of about 20 g/L and a pH of between 1.0 and 1.6. Therefore, the domesticated strain Pichia stipitis INER 1128 of the present invention is not limited to the xylose hydrolyzate obtained by the above pretreatment of rice straw, and can also be applied to bagasse, silvergrass, and pennisetum. (Napiergrass), Switchgrass, Corn stover, Wheat straw, Wood, Bamboo, Bag A xylose hydrolyzate obtained by pretreating other fiber raw materials such as water hyacinth and algae with a dilute acid catalyst.

>> Furthermore, the present invention is not only applicable to a xylose hydrolyzate which has been conventionally detoxified by overbasing, but also has applicability to a xylose hydrolyzate which has not been detoxified.

>> Please refer to the "Fig. 2 and Fig. 3" for the flow chart of the xylose hydrolyzate of the present invention which is subjected to the overbasing method and the process diagram of the xylose hydrolyzate of the present invention without detoxification. As shown in the figure, the xylose hydrolyzate produced by the pretreatment of the rice straw of the above Fig. 1 is taken as an example. When the present invention is applied, wherein the xylose hydrolyzed liquid system by the overbasing method comprises the following conditioning units 101 to 104 in sequence: >> (A) heating 101: heating the xylose hydrolyzate to 50 to 60 ° C; (B) adding excess lime 102: adding excess lime to increase the pH value of the xylose hydrolysate to 9.0~11.0, and producing calcium sulfate precipitate; >> (C) solid-liquid separation 103: using a solid-liquid separation device Removing the calcium sulfate precipitate, wherein the clear liquid is an alkaline xylose hydrolyzate from which furfural and sulfate ions have been removed; and >> (D) adding an acid agent 104: adding an acid agent to adjust the above alkaline xylose hydrolyzate The pH value is weakly acidic.

>> From the above steps, it can be known that conditioning the xylose hydrolyzate by the overbasing method requires heating, adding excess lime, solid-liquid separation and adding an acid agent, and vice versa, and adjusting the xylose hydrolyzate without detoxification. The conditioning unit 201 is relatively simple, and only needs to directly add an alkali agent to the xylose hydrolyzate. The pH value is adjusted from acid to weakly acidic.

>>Please refer to “Fig. 4~6” for the comparison of the highest alcohol yield of the xylose hydrolyzate conditioned by alkalization under different pH values of the present invention, and Fig. 4, hydrolysis of xylose Schematic diagram of the change trend of xylose concentration in liquid fermentation and the alcohol formation of the xylose hydrolyzate conditioned by the alkalization method in a 5 L fermentation tank. As shown in the figure: the alcohol yield obtained by the domesticated strain Pichia stipitis INER 1128 applied to the xylose hydrolyzate subjected to alkalization detoxification conditioning. First, the xylose fermentation was carried out in a 50 ml (mL) shake flask, and the fermentation pH was controlled at 5.0 pH, 6.0 pH and 7.0 pH, respectively, so that the highest alcohol yield was obtained under the pH value during the fermentation. . Among them, the fermentation temperature was controlled at 30 ° C, and the shaking speed of the incubator was maintained at 100 rpm, and the inoculum of the xylose fermentation bacteria was 0.4 to 0.5 g / L.

>> As can be seen from Fig. 4, when the fermentation pH is controlled at pH 5.0, the highest alcohol yield is about 80%; if the fermentation pH is increased to 6.0 and 7.0, the highest alcohol yield is Can be increased to 90%. From this result, it is shown that the domesticated strain Pichia stipitis INER 1128 of the present invention, if applied to the fermentation of the xylose hydrolysate of the rice straw, maintains a high pH value during the fermentation, which will contribute to the improvement of the alcohol yield. In addition, compared with the previous literature, there is a description about the results of the related research on xylose fermentation at present, and the present invention is applied to the fermentation of the xylose hydrolyzate of rice straw, and it is indeed possible to obtain a very desirable alcohol yield.

>> Furthermore, it is further pointed out from Figure 5 that when the pH value of the fermentation is controlled at pH 5.0, pH 6.0 and pH 7.0, respectively, the xylose is concentrated in the hydrolyzate. There is also a significant difference in the trend of the system over time, but the overall xylose utilization rate is close to full utilization. Among them, trend line 1 is when the pH value of the fermentation is controlled at pH 5.0, the xylose concentration of the hydrolyzate can be reduced to negligible in 54 hours; the trend line 2, 3 is increased to pH 6 if the fermentation pH is increased. At .0 and pH 7.0, the xylose concentration in the hydrolyzate can be reduced to a negligible level after a fermentation time of 40 hours. The results show that the domesticated strain of the present invention carries out xylose fermentation at a higher pH value, which not only can obtain a higher alcohol yield, but also can increase the fermentation rate, thereby effectively shortening the reaction time of xylose conversion alcohol.

>> In addition, studies from the 5L fermentation scale have also shown that the domesticated strain Pichia stipitis INER 1128 of the present invention can also obtain good alcohol yield for xylose fermentation. It can be seen from Fig. 6 that when the xylose hydrolyzate is conditioned by alkalization, if the pH value of xylose fermentation is controlled at pH 6.0 and the intake air amount is maintained at 0.02 vvm, the xylose concentration index in the hydrolyzate 4 can be reduced to a negligible level in 40 hours, while obtaining the highest alcohol output concentration index of 5, at this time the alcohol yield of xylose fermentation can reach 92%.

>> From the above description, it can be seen that, under different experimental scales, when the xylose hydrolyzate is detoxified by alkalization, the domesticated strain Pichia stipitis INER 1128 of the present invention can be used for the fermentation of xylose to obtain an ideal alcohol yield. With the utilization of xylose.

>> Please refer to Figure 7 for a comparison of the highest alcohol yields of the xylose hydrolysate treated by alkalization and non-toxicity conditioning under different pH values of the present invention. As shown in the figure: The acclimated strain Pichia stipitis INER 1128 was applied to xylose in the case where the undegraded xylose hydrolysate was previously added with an alkali agent to adjust the pH to pH 5.0, pH 6.0 and pH 7.0. The alcohol yield of the fermentation is very close to the alcohol yield of the xylose hydrolyzate fermented by the overbasing method. When the fermentation pH is controlled at pH 5.0, the highest alcohol yield can still be maintained at 80%; if the fermentation pH is increased to pH 6.0 and pH 7.0, the highest alcohol yield can be maintained. At 90%, it is sufficient to show that if the xylose hydrolysate is not preconditioned to be detoxified, the present invention can still achieve a desired alcohol yield. It can be seen that the domesticated strain of the present invention has a certain degree of tolerance to the fermentation inhibitor in the rice straw hydrolysate, and if applied to xylose fermentation, it will help simplify the conditioning step of the xylose hydrolyzate, thereby reducing the hydrolyzate. The setup cost of the conditioning device.

> The domesticated strain Pichia stipitis INER 1128 of the present invention is not limited to the xylose hydrolyzate obtained by the above pretreatment of rice straw, and can also be applied to bagasse, miscanthus, pennisetum, switchgrass, cornstalk, and straw. The xylose hydrolyzate obtained from the other fiber raw materials such as wood, bamboo, bag lotus and seaweed and pretreated by dilute acid is used for fermentation to produce alcohol.

In summary, the present invention is a xylose fermentation method for a non-toxic fiber raw material hydrolyzate, which can effectively improve various disadvantages of the conventional use, and provides a fiber raw material prepared by pressing mixed acid and hot water to dissolve high temperature and high pressure reaction treatment. The hydrolyzate is used to acclimate and culture the xylose fermentation strain, and can effectively convert the xylose into alcohol in the hydrolyzate of the fiber raw material which is super-alkalized and non-toxically conditioned, and both have high xylose utilization rate and wood The effect of converting sugar into alcohol yield of more than 90% is more traditional than reducing the loss of xylose and the cost of producing alcohol, and also has high alcohol yield, thereby making the invention more progressive and practical. More in line with the needs of the user, it has indeed met the requirements of the invention patent application, and filed a patent application according to law.

The above is only the preferred embodiment of the present invention, and the scope of the present invention is not limited thereto; therefore, the simple equivalent changes made in accordance with the scope of the present invention and the contents of the description of the invention And modifications are still within the scope of the invention.

101~104‧‧‧Over-basic conditioning unit

201‧‧‧Undetoxified conditioning unit

1, 2, 3‧ ‧ trend line

4‧‧‧xylose concentration index

5‧‧‧Alcohol concentration index

Fig. 1 is a schematic view showing the composition of a rice straw xylose hydrolyzate of the present invention.

Fig. 2 is a schematic view showing the flow of the xylose hydrolyzate of the present invention by the overbasing method.

Fig. 3 is a schematic view showing the flow of the xylose hydrolyzate of the present invention without detoxification.

Fig. 4 is a schematic view showing the highest alcohol yield of the xylose hydrolyzate conditioned by alkalization under different pH values of the present invention.

Fig. 5 is a schematic diagram showing the change trend of xylose concentration in the fermentation of xylose hydrolyzate in Fig. 4.

Fig. 6 is a schematic view showing the alcohol formation of the xylose hydrolyzate conditioned by the alkalization method in a 5 L fermentation tank of the present invention.

Fig. 7 is a schematic diagram showing the comparison of the highest alcohol yield of the xylose hydrolyzate which has been subjected to alkalization conditioning and non-toxication conditioning under different pH values of the present invention.

101~104‧‧‧Over-basic conditioning unit

201‧‧‧Undetoxified conditioning unit

1, 2, 3‧ ‧ trend line

4‧‧‧xylose concentration index

5‧‧‧Alcohol concentration index

Claims (7)

The method for fermenting xylose of a non-toxic fiber raw material hydrolysate comprises at least the following steps: (A) mixing a fiber raw material hydrolyzate prepared by detoxification conditioning and a culture medium in a ratio of 20:80 by volume One yeast - Pichia stipitis ; (B) Xylose and Glucose are added to control the initial xylose and glucose concentrations of the above mixed cultures to 30 g / liter (g / L) and 10 g / L, respectively. (C) gradually increasing the proportion of the unfermented fiber raw material hydrolyzate and the artificial culture liquid after continuously subsequent to at least two generations, under the principle of maintaining the concentration of xylose and glucose; and (D) directly The fiber raw material hydrolyzate without detoxification conditioning is cultured for at least 60 generations, and the resulting novel yeast strain Pichia stipitis INER 1128 can be directly grown in the non-toxically conditioned fibrous raw material hydrolyzate by the aforementioned steps and Xylose conversion produces alcohol. A method for fermenting a xylose fermentation of a non-toxic fiber raw material hydrolyzate according to claim 1, wherein the fiber raw material hydrolyzate which has not been detoxified in the step (A) is pre-added with an alkali agent to adjust the acid value Adjust to between 5.0 and 7.0. The method for fermenting a xylose fermentation of a non-toxic fiber raw material hydrolyzate according to claim 1, wherein the fiber raw material hydrolyzate and the artificial culture liquid which are not detoxified in the step (C) are adjusted The ratio is gradually increased from 20:80 to 35:65, 50:50, 60:40 to 70:30. The method for fermenting a non-toxic fiber raw material hydrolyzate according to claim 1, wherein the fiber raw material hydrolyzate is selected from the group consisting of Rice Straw, Bagasse, and Silvergrass. , Napiergrass, Switchgrass, Corn stover, Wheat straw, Wood, Bamboo, Water hyacinth, and Algae The xylose hydrolyzate obtained from the raw material and obtained by the pretreatment. The method for pretreating the fiber raw material hydrolyzate according to the fourth aspect of the patent application, wherein the method for pretreating the fiber raw material is a high temperature and high pressure reaction treatment technology of extrusion mixed acid and hot water washing. The xylose fermentation method of the undecomposed fiber raw material hydrolyzate according to claim 4, wherein the fiber raw material pretreatment device is a biaxial screw extruder combined with a dilute acid catalytic reaction tank. The xylose fermentation method of the undetoxified fiber raw material hydrolyzate according to claim 1, wherein the novel yeast strain can perform xylose decomposition on the hydrolyzate which has not been subjected to detoxification treatment or alkalization treatment .