TWI496890B - The method for improving efficiency of monosaccharide fermentation - Google Patents

Description

Method for improving the efficiency of monosaccharide fermentation

The invention relates to a method for improving the efficiency of monosaccharide fermentation, in particular to a method in which a lignocellulosic material can be appropriately treated and then added to a reactor for cultivating an inoculum, and the cultured inoculum is applied to a fiber raw material hydrolyzate, It will effectively improve the conversion efficiency of sugar fermentation.

According to the increasing price of international crude oil and the increasing global warming, bio-alcohol has been regarded as an alternative fuel to the potential of traditional gasoline. At present, the main raw materials for commercial mass production of raw alcohol are cereals and sugar cane. The raw materials that can be converted into alcohol are mostly starch and sucrose. The raw materials can be fermented by simple fermentation. Produce alcohol.

However, the use of cereals or sugar cane as a raw material for the production of fibrous alcohol has always had doubts about competing for food with humans. Therefore, wood, rice straw, bagasse, corn stover, wheat straw, silvergrass, corncob, and poplar are used. The second-generation bio-alcohol process using Aspen and paper waste as raw materials is considered to have great potential for development in the future due to its diversity of sources, high global storage and no food conflicts. Process technology for the production of raw alcohol.

Generally, fiber raw materials mainly contain 60% to 80% of cellulose and hemicellulose, and 15% to 25% of lignin. Cellulose is mainly composed of glucose monomers, while hemicellulose is made of wood. Sugar is a polymer of the main monomeric sugars. Head In the process of converting fiber into alcohol, it is usually firstly subjected to high temperature and high pressure thermochemical pretreatment technology such as Dilute-acid Hydrolysis or Acid-catalyzed Steam Explosion. Decomposed into xylose.

Basically, in the above-mentioned thermal chemical treatment (Thermal Chemical Pretreatment), a certain proportion of the raw material is mixed with the aqueous solution and then charged into the reactor, and then added under the high temperature and high pressure reaction conditions, 1 to 2%. (w/w) dilute sulfuric acid, so the resulting xylose and glucose fermentation liquid will contain a higher concentration of sulfate ions, which will affect the ability of yeast to convert monosaccharides into alcohol. At the same time, in order to improve the effect of thermochemical pretreatment and increase the concentration of xylose in the hydrolyzate, it is usually necessary to raise the temperature or add the content of organic acid, but also relatively increase the inhibitor in the xylose solution such as acid (Acetic acid). Fermentation inhibitors such as furfural and Hydroxymethyl Furfural affect the yeast fermentation ability.

In the current fiber-to-alcohol process, the fermentation strain is often interfered with by the inhibitor contained in the hydrolysate, which greatly reduces the ability of the strain to ferment alcohol. The most serious situation may cause the strain to have no way to metabolize the sugar to produce alcohol, so there are currently Many studies on the removal of inhibitors, such as the use of overliming, anion exchange resin, active charcoal, etc., but the use of such methods to remove inhibitors, will cause sugars Loss and increase the cost of alcohol production.

In view of this, in order to enable higher fermentation yields for sugar fermentation, thereby increasing the yield of alcohol and reducing the production cost, it is necessary to seek a technique for improving the conversion efficiency of sugar fermentation.

In addition, although there are related and published academic studies (Chandel, AK, et al., Use of Saccharum spontaneum (wild sugarcane) as biomaterial for cell immobilization and modulated ethanol production by thermotolerant Saccharomyces cerevisiae VS3. Bioresource Technology, 2009.100 ( 8): p.2404-2410.), using sweet root grass as the substrate for bacterial adsorption, the raw material needs to be cut finely, and the alkaline chemical is also needed to treat the raw material, and finally it must be made. The procedure of cooling and drying shows that the preparation process of the sweet root grass is complicated, and the waste material produced by using the fibrous alcohol is used as the substrate for the adsorption of the bacteria, and the preparation procedure of the invention is obviously more than that. On the other hand, according to this publication, the strain needs to be centrifuged separately to be cultured with the processed sweetened radish root for 24 hours before fermentation can be carried out, and the invention only needs to be cultured for 24 hours without going through The present invention is superior in efficiency to this publication in comparison with actions such as prior inoculum culture and centrifugation. In addition, the operations such as repeated culture and centrifugation are energy-intensive and time-consuming in the amplification project. In addition to the addition of the lignocellulosic material, the other procedures are the same as those in the general fermentation plant for the cultivation of the inoculum. Has a high industrial applicability.

In view of this, the inventors of this case have intensively discussed the above-mentioned problems of conventional inventions, and actively pursued solutions through years of experience in R&D and manufacturing of related industries. After long-term efforts in research and development, they finally succeeded in developing this book. Invented the "method of improving the efficiency of monosaccharide fermentation" to improve the problems of the conventional use.

The main object of the present invention is that the lignocellulosic material can be appropriately treated and then added to the reactor for cultivating the inoculum. The cultured inoculum is applied to the fiber raw material hydrolyzate, which can effectively improve the sugar fermentation. Conversion effect rate.

In order to achieve the above purpose, the present invention is a method for improving the efficiency of monosaccharide fermentation, which comprises the following steps: Step 1: placing the strain and the lignocellulosic material in an inoculum culture reactor for inoculating culture; Step 2: cultivating the strain After ripening, it can be poured into a fermentation reactor having a liquid to be fermented for fermentation; step 3: after fermentation, the cultured strain can be separated from the fermentation liquid by a solid-liquid separator; and step 4: fermentation liquid is further The purified product was obtained after purification.

In one embodiment of the present invention, the strain used in the first step may be Saccaromyces cerevisiae , Pichia stipitis or other strains which can utilize the cellulose and hemicellulose to be decomposed.

In an embodiment of the present invention, the lignocellulosic material used in the first step may be a rice straw.

In an embodiment of the present invention, the culture time of the inoculum in the step 1 is between 22 hours and 26 hours.

In an embodiment of the present invention, in the second step, Corn stover, Rice straw, Hard wood, Corn cob, and Wheat straw can be used. The liquid produced after the thermochemical reaction is used as the liquid to be fermented.

In one embodiment of the present invention, the thermochemical reaction refers to a pretreatment of dilute acid hydrolysis, and the main components of the desired fermentation liquid are xylose and glucose.

In an embodiment of the present invention, the ratio of the amount of the lignocellulosic material added to the weight of the liquid to be fermented ranges from 0.3% to 3%.

In one embodiment of the present invention, the dilute acid hydrolysis pretreatment has an operating condition of 1 to 3% dilute sulfuric acid concentration, a reaction temperature of 160 to 200 ° C, and a reaction time of 3 to 10 minutes.

In an embodiment of the present invention, the preparation of the lignocellulosic material comprises the following steps: Step 1: mixing the rice straw with a 0.5% to 3% dilute sulfuric acid solution to form a mixed solution, and the rice straw and the dilute sulfuric acid solution The ratio is between 1:5 and 1:10 (w/w); Step 2: The mixed solution is cooked at a temperature of 130 ° C to 200 ° C for 3 minutes to 10 minutes, and then removed by solid-liquid separation. An aqueous solution to obtain a solid matter; Step 3: mixing the solid matter with an aqueous solution containing cellulose hydrolyzing enzyme for 70 hours to 90 hours, and the reaction pH value is controlled between pH 4 and pH 6; and Step 4: After the aqueous solution is separated by means of solid-liquid separation, the obtained residual solid is a lignocellulosic material.

Please refer to FIG. 1 to FIG. 7 , which are schematic diagrams showing the preparation steps of the lignocellulosic material of the present invention, the steps of the steps for adding the lignocellulosic material of the present invention, and the single step of adding the lignocellulosic material when the inoculum of the present invention is cultured. Schematic diagram of the change of the concentration of alcohol in the fermentation of sugar fermentation, the change of the concentration of the monosaccharide-converting alcohol in the fermentation reaction by adding the lignocellulosic material during the cultivation of the inoculum of the present invention, and the conversion of the monosaccharide of the lignocellulosic material and the non-added lignocellulosic material when the inoculum is cultured in the present invention Comparison of efficiency of alcohol, schematic diagram of comparison of monosaccharide conversion alcohol efficiency of adding different weights of lignocellulosic material in the present invention, and alcoholic production of the invention by using Pichia stipitis for monosaccharide fermentation reaction and adding lignocellulosic material and unadded lignocellulosic material A schematic diagram of efficiency comparison. As shown in the figure: the present invention is a method for improving the efficiency of monosaccharide fermentation. Before the present invention, the lignocellulosic material 1 must be prepared by a specific method before the monosaccharide fermentation process, and the preparation of the lignocellulosic material 1 is performed. Contains the following steps (as shown in Figure 1):

Step 1: Mixing the rice straw 10 with 0.5% to 3% of the dilute sulfuric acid solution 11 to form a mixed solution 12, and the ratio of the rice straw 10 to the dilute sulfuric acid solution 11 is between 1:5 and 1:10 (w/w). )between.

Step 2: The mixed solution 12 is subjected to cooking at a temperature of 130 ° C to 200 ° C for 3 minutes to 10 minutes, and then the aqueous solution 122 is removed by solid-liquid separation 121 to obtain a solid 123.

Step 3: The solid matter 123 is mixed with the aqueous solution 13 containing cellulose hydrolyzing enzyme for 70 hours to 90 hours, and the reaction pH value is controlled between pH 4 and pH 6.

Step 4: After separating the aqueous solution 13 by the solid-liquid separation 131, the obtained residual solid is the lignocellulosic material 1.

After the preparation of the above lignocellulosic material 1 is completed, the lignocellulosic material 1 can be applied to the operation steps in the fermentation process, which comprises the following steps (as shown in Fig. 2):

Step 1: The strain 2 and the lignocellulosic material 1 are placed in the inoculum culture reactor 21 for inoculum culture, and the culture time is between 22 hours and 26 hours, and 24 hours is the best time, wherein the strain is The 2 line may be Saccaromyces cerevisiae , Pichia stipitis or other strains which can utilize the cellulose and hemicellulose to decompose the resulting saccharide.

Step 2: After the culture is matured, it can be poured into the liquid with the desired fermentation liquid 3 The fermentation reaction is carried out in the fermentation reactor 31, wherein the corn stover, the rice straw, the hard wood, the corn cob, and the wheat straw are used for heat. The liquid produced after the chemical reaction is used as the fermentation liquid 3, and the thermochemical reaction refers to the pretreatment of the dilute acid hydrolysis, and the main component of the fermentation liquid 3 to be produced is xylose and glucose, and the amount of the lignocellulosic material 1 is added. The weight-to-volume ratio of the liquid to be fermented 3 ranges from 0.3% to 3%, and the dilute acid is pretreated by hydrolysis, and the operating conditions are 1 to 3% dilute sulfuric acid concentration, reaction temperature 160 to 200 ° C, and reaction time 3 to 10 minute.

Step 3: After the fermentation, the cultured strain 2a and the fermented fermentation liquid 3 can be separated by the solid-liquid separator 4.

Step 4: Purification liquid 3 is further purified to obtain purified product 3a.

The effects that can be achieved by the present invention can be verified by the following embodiments:

Implementation case one:

The lignocellulosic material 1 may be added to the nutrient base of the strain 2 cultivated as Saccaromyces cerevisiae , cultured under the culture conditions suitable for the strain 2, and added to the fermentation tank for fermentation of the sugar-converting alcohol after 24 hours; When the inoculum is cultured, the lignocellulosic material 1 is not added, and after 24 hours of cultivation, the alcohol fermentation is similarly performed; while the sugar concentration and the alcohol of the above two methods change with time (as shown in Fig. 3 and Fig. 4) Show), the figure has the added alcohol fiber material 1 for fermentation, the alcohol concentration can reach 8.9g / L, and the highest concentration of alcohol without fermentation of lignocellulosic material 1 is only 6.0g / L. In addition, the addition of lignocellulosic material 1 during the cultivation of the inoculum has an efficiency of about 80% of the sugar-converting alcohol (as shown in Fig. 5), and the efficiency of the sugar-converting alcohol without adding the lignocellulosic material 1 is about 64%. It can be proved that the inoculum culture method of adding lignocellulosic material 1 can effectively improve the efficiency of sugar-converting alcohol by 16%.

Implementation case two:

Different weights of lignocellulosic material 1 (0g, 1g, 2.5g, 5g and 10g) were added to the culture medium, and Saccaromyces cerevisiae was added to fermenting strain 2 for inoculum culture. After 24 hours of cultivation, it was added to the semi-fiber hydrolysis. The fermentation of the monosaccharide-converted alcohol in the liquid is carried out. As shown in Fig. 6, the efficiency of the monosaccharide-converting alcohol without adding the lignocellulosic material 1 is about 0.39 g/g, and the addition of the lignocellulosic material 1 to the cultured inoculum 1 g, 2.5 The ability to convert alcohol of g, 5g and 10g sugars is 0.40, 0.42, 0.43 and 0.42, respectively. Therefore, it is shown that the addition of lignocellulosic material 1 during the cultivation of inoculum can improve the efficiency of alcohol production by monosaccharide fermentation, and according to its transformation. For the comparison of the efficiencies, the optimum additive amount is that the weight ratio of the lignocellulosic material 1 to the fermentation liquid is 1.6%.

Implementation case three:

When the five-carbon sugar fermentation strain Pichia Stipits was tested and cultured, the lignocellulosic material 1 was added to improve the efficiency of the monosaccharide fermentation to convert alcohol. Similarly, 5g of lignocellulosic material 1 was added during the expansion of Pichia Stpitis , and after 10 hours of cultivation, 10 ml was taken. The bacterial liquid was added to 50 mL of the sugar-containing aqueous solution, and during the fermentation reaction, the concentration of the sugar and the alcohol was sampled and analyzed, and it was found that the addition of the lignocellulosic material 1 gave about 51% of the sugar-converting alcohol efficiency, while the inoculum was inoculated. The lignocellulosic material 1 is added during the cultivation, and the alcohol yield can reach 74% (as shown in Fig. 7). Thus, it is proved once again that the culture technique using the material can also be effectively applied to yeast other than the general brewer's yeast. The strain is fermented.

In addition, the present invention can also be applied to a sugar-containing hydrolyzate which has not been detoxified. Fermentation, while applying, can also improve the efficiency of sugar conversion of alcohol, and obtain higher product concentration under the same reaction time. When the pretreatment temperature of dilute acid method is between 130 and 200 °C, the yield is The rate of alcohol production during fermentation with a saccharide hydrolysate can be increased by an average of 1.1 to 3 times.

And after the strain 2 is added to the fermentation tank solution to complete the fermentation, the fermentation strain can be co-sedimented with the added lignocellulosic material 1 under static unstirred conditions, which will facilitate the separation of the strain 2 from the product after the cultivation, and The fermentation reactor was subjected to a batch batch mode to achieve the effect of strain 2 recovery.

In addition, the present invention uses a thermochemical and biochemical method to obtain a lignocellulosic material 1 in a general fiber alcohol factory process, which is originally used only as industrial waste or as a steam for combustion, but In the present invention, the waste can be reused using the lignocellulosic material 1.

In summary, the method for improving the fermentation efficiency of the monosaccharide of the invention can effectively improve various disadvantages of the conventional use, and the lignocellulosic material can be appropriately treated and then added to the reactor for cultivating the inoculum, and the cultured inoculum is applied to the method. The fiber raw material hydrolyzate can effectively improve the conversion efficiency of the sugar fermentation; furthermore, the invention can be more advanced, more practical, and more suitable for the use of the consumer, and has indeed met the requirements of the invention patent application, File a patent application.

However, 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 and modifications made in accordance with the scope of the present invention and the contents of the invention are modified. All should remain within the scope of the invention patent.

1‧‧‧Libre fiber materials

10‧‧‧ straw stalk

11‧‧‧Dilute sulfuric acid solution

12‧‧‧ mixed solution

120‧‧‧cooking

121‧‧‧Solid-liquid separation

122, 13‧‧‧ aqueous solution

123‧‧‧solid objects

131‧‧‧Solid-liquid separation

2, 2a‧‧‧ strain

21‧‧‧ Culture reactor

3‧‧‧desirable liquid

3a‧‧‧purified product

31‧‧‧ Fermentation reactor

4‧‧‧ solid-liquid separator

5‧‧‧purification

Figure 1 is a schematic view showing the preparation steps of the lignocellulosic material of the present invention.

Figure 2 is a schematic view showing the steps of the addition of the lignocellulosic material of the present invention.

Fig. 3 is a schematic diagram showing the concentration change of monosaccharide fermentation-converted alcohol without adding lignocellulosic material when the inoculum of the present invention is cultured.

Fig. 4 is a schematic view showing the change of the concentration of the monosaccharide-converting alcohol by adding the lignocellulosic material to the fermentation reaction in the culture of the present invention.

Fig. 5 is a schematic view showing the comparison of the efficiency of adding the lignocellulosic material and the monosaccharide-converting alcohol not added to the lignocellulosic material when the inoculum is cultured in the present invention.

Fig. 6 is a schematic diagram showing the comparison of the efficiency of monosaccharide conversion of different weights of lignocellulosic materials in the inoculum of the present invention.

Fig. 7 is a schematic diagram showing the comparison of the alcohol production efficiency of the present invention using Pichia stipitis for the monosaccharide fermentation reaction and adding the lignocellulosic material and the non-added lignocellulosic material.

1‧‧‧Libre fiber materials

2, 2a‧‧‧ strain

21‧‧‧ Culture reactor

3‧‧‧desirable liquid

3a‧‧‧purified product

31‧‧‧ Fermentation reactor

4‧‧‧ solid-liquid separator

5‧‧‧purification

Claims (4)

A method for improving the efficiency of monosaccharide fermentation comprises the following steps: Step 1: The strain and the lignocellulosic material are placed in an inoculum culture reactor for inoculum culture for 24 hours; wherein the lignocellulosic material used is prepared as a straw stalk. The strain used is Saccaromyces cerevisiae or Pichia stipitis , and the preparation thereof comprises the following sub-steps: sub-step 1: mixing the rice straw with a 0.5% to 3% dilute sulfuric acid solution to form a mixed solution, and the rice straw and the same The ratio of the dilute sulfuric acid solution is between 1:5 and 1:10 (w/w); sub-step two: the mixed solution is cooked at a temperature of 130 ° C to 200 ° C for 3 minutes to 10 minutes, and then separated by solid and liquid. The aqueous solution is removed to obtain a solid matter; sub-step 3: mixing the solid matter with an aqueous solution containing cellulose hydrolyzing enzyme for 70 hours to 90 hours, and the reaction pH value is controlled between pH 4 and pH 6; Sub-step 4: After separating the aqueous solution by solid-liquid separation, the residual solid obtained is a lignocellulosic material; Step 2: After the culture is matured, the fermentation can be poured into the fermentation liquid with the desired fermentation liquid. Fermentation reaction is carried out, and the weight-to-volume ratio of the amount of the lignocellulosic material added to the liquid to be fermented is in the range of 0.3% to 0.9%; Step 3: after the fermentation, the strain and fermentation after the culture can be passed through the solid-liquid separator Liquid separation; and step 4: the fermentation liquid is purified to obtain a purified product. The method for improving the efficiency of monosaccharide fermentation according to item 1 of the patent application scope, wherein the second step is to use corn stover and rice straw (Rice) The liquid produced by the thermochemical reaction, such as straw), Hard wood, corn cob, and wheat straw, is used as the fermentation liquid. The method for improving the efficiency of monosaccharide fermentation according to item 2 of the patent application scope, wherein the thermochemical reaction refers to pretreatment of dilute acid hydrolysis, and the main components of the fermentation liquid to be produced are xylose and glucose. The method for improving the efficiency of monosaccharide fermentation according to item 3 of the patent application scope, wherein the dilute acid hydrolysis pretreatment has an operating condition of 1 to 3% dilute sulfuric acid concentration, a reaction temperature of 160 to 200 ° C, and a reaction time of 3~ 10 minutes.