CN100339483C - Process for preparing alcohol through utilizing xylose and glucose by microzyme - Google Patents

Abstract

The method for producing alcohol by yeast using xylose and glucose involves the application of yeast, especially the use of one of Pachysomyces tannophilus 2.1622, Pachysomyces tannophilus ATCC 32691, and Candida shohata ATCC 34887 to produce xylose and glucose A method of producing alcohol from a substrate. Based on the existing yeast strains, the present invention can shorten the period for yeast to produce alcohol by fermenting xylose and glucose, and improve the substrate utilization rate by adopting technical means such as domestication, liquid culture, immobilization, and multiplication culture. The method is mainly used in the production of ethanol by fermenting biomass as a raw material.

Description

Method for producing alcohol by yeast using xylose and glucose

technical field

The present invention relates to the application of saccharomycetes, particularly relate to the utilization of one of three strains of yeast species Pachysolen tannophilus (Pachysolen tannophilus) 2.1622, Pachysolen tannophilus ATCC 32691 and Candida shehatae (Candida shehatae) ATCC 34887 Alcohol production from xylose and glucose.

Background technique

Alcohol, also known as ethanol, is usually produced by fermentation of six-carbon sugars by Saccharomyces cerevisiae. The production of fuel ethanol from biomass (crop stalks, wood processing waste, etc.) is an important part of the national energy strategy and national energy security, and it is also an important way to prevent and reduce air pollution caused by vehicle combustion exhaust. An important guarantee for sustainable development. In biomass, cellulose and hemicellulose are the main chemical components. Hemicellulose includes various polypentoses and polyhexoses. The most common hemicellulose is xylan, which accounts for about half of the dry weight of herbaceous plants. , are also present in woody plants. After lignocellulose is pretreated, a hydrolyzed sugar solution mainly containing glucose and xylose can be obtained. In the study of alcoholic fermentation using biomass as raw material, the key is to determine the strains that can efficiently metabolize xylose and glucose into alcohol at the same time, and then improve the alcoholic fermentation process on this basis. Yeasts that can metabolize xylose and glucose into alcohol at the same time include Pachysomyces tannophilus, Candida shohata, Pichia stipitis, Kluyveromyces marxianus and Candida sp XF217) etc., but they utilize xylose and glucose to ferment and produce the actual output of alcohol to have only about 70% of theoretical output, and the fermentation cycle is long, at about 80 hours.

Contents of the invention

The purpose of the present invention is to provide a method for yeast to produce alcohol by fermentation of xylose and glucose. The method is based on existing yeast strains, and can make the alcohol fermentation cycle Shortened, substrate utilization increased.

For realizing the purpose of the present invention, adopted following technical scheme, this method comprises the following steps:

1) inoculating Pachysomyces tannophilus 2.1622 in the acclimation medium for acclimatization;

2) inoculating the acclimated yeast into a liquid medium for liquid culture;

3) immobilizing the yeast obtained by liquid culture;

4) Proliferating and culturing the immobilized yeast;

5) the proliferated yeast carries out alcoholic fermentation;

In the above method of producing alcohol:

When acclimatizing the strain, the formulation of the acclimatization medium is: xylose 10-50g/L, peptone 4-6g/L, yeast juice 2-4g/L, and the acclimation conditions are pH5.0-5.5, 28-35°C, 60-120rpm, the acclimatization time of each batch is 48-96 hours, the inoculation amount is 2%, and more than 5 batches are acclimatized.

When the domesticated yeast is carried out in liquid culture, the formula of the liquid medium is: xylose 10-50g/L, peptone 4-6g/L, yeast juice 2-4g/L, the inoculum size is 1-5%. The conditions are pH5.0-5.5, 28-35° C., 60-120 rpm, and the culture time is 24-48 hours. After the culture is completed, it is concentrated and treated, and the culture solution is collected for use.

When immobilizing the yeast obtained by liquid culture, mix 2ml of concentrated liquid culture with 3-6% sodium _alginate solution containing 1.2% _Al2O3 at room temperature each time, and stir well under sterile conditions After homogeneity, add dropwise to 2% CaCl ₂ aqueous solution, and stand at 4°C for 10-20 hours to obtain gel particles.

When the immobilized yeast cells are propagated and cultured, the formula of the proliferation medium is: xylose 20g/L, glucose 50g/L, peptone 3g/L, yeast juice 2.5g/L, CaCl ₂ 0.25g/L, MgSO ₄ 0.25g /L, KH ₂ PO ₄ 2.5g/L, proliferation culture conditions are 28-35°C, pH 5.0-5.5, 60-120rpm, culture time is 24-48 hours, and fresh culture medium is replaced every 12 hours.

Finally, use xylose and glucose to carry out alcoholic fermentation. The formula of the fermentation medium is: xylose 20g/L, glucose 50g/L, CaCl ₂ 2.5g/L, MgSO ₄ 0.25g/L, KH ₂ PO ₄ 2.5g/L , yeast juice 2.5g/L, peptone 3g/L, urea 0.24g/L, the fermentation conditions are pH5.0-5.5, 28-35°C, 60-120rmp, and the fermentation time is 24-36 hours.

Determination with high pressure liquid chromatography in the laboratory:

Utilization rate of xylose: The utilization rate of xylose in the immobilized cells of three strains of Pachysomyces tannophilus 2.1622, Pachysomyces tannophilus ATCC 32691 and Candida shohata ATCC 34887 were 87.90% and 76.62% respectively and 74.68%, Pachysomyces tannophilus 2.1622 was significantly better than the other two strains.

Glucose utilization rate: The glucose utilization ability of the immobilized cells of the above three strains after domestication has also been significantly improved. Before acclimatization, the immobilized cells were fermented for 36 hours, and the glucose utilization rates of the three strains of Pachysomyces tannophilus 2.1622, Pa. After acclimatization, the immobilized cells were fermented for 24 hours, and the glucose utilization rate of the above three bacterial strains all reached more than 85%. After 36 hours of fermentation, the glucose utilization rates of the three strains of Pachysomyces tannophilus 2.1622, Pachysomyces tannophilus ATCC 32691 and Candida shohata ATCC 34887 were 99.99%, 98.21% and 97.46%, respectively. After 48 hours, the glucose utilization rate of the above three strains all reached 99.99%, close to 100%. For the immobilized cell Pachysolen tannophilus 2.1622, after 24 hours of fermentation, the utilization rate of glucose reached 99.99%, and the fermentation time was shortened by one-third.

Total sugar utilization rate: Before domestication, the total sugar utilization rate of the immobilized cells of the three strains of Pachysomyces tannophilus 2.1622, Pachysomyces tannophilus ATCC 32691 and Candida shehata ATCC 34887 were 85.08%, 79.85%, 77.81%. After domestication, the total sugar utilization rates of the immobilized cells of the above three strains were 93.95%, 88.31%, and 87.34%, respectively.

Ethanol production rates of the three strains after domestication: The ethanol production rates of the three strains were greatly increased after domestication. Overall, among the three strains, the strain Pachysomyces tannophilus 2.1622 was superior to the other two strains.

The yeast of the present invention utilizes xylose and glucose to carry out alcoholic fermentation and has the characteristics of short period, high substrate conversion rate and high alcohol yield.

The present invention will be further described below by specific examples.

Detailed ways

Example 1

Pachysomyces tannophilus 2.1622 A method for the production of alcohol from xylose and glucose.

100ml of acclimatization medium is housed in a 250ml Erlenmeyer flask, the formula of the acclimatization medium is: xylose 20g/L, peptone 5g/L, yeast juice 3g/L. The slant-preserved yeast Pachysolomyces tannophilus 2.1622 was inoculated in the acclimatization medium, and the inoculum amount was 2%. The culture was carried out for 48 hours under the conditions of pH 5.0, 30° C., and 60 rpm, and a total of 7 batches of acclimatization culture were carried out. The number of bacteria in the culture medium was counted by microscope direct counting method, and the number of bacteria increased significantly with the increase of domestication batches. 3.3×10 ⁸ /ml, while the bacterial content of this strain was only 5.0×10 ⁷ /ml after the first domestication, and the bacterial content increased by an order of magnitude after the first domestication. The increase of bacteria content indicated that the ability of metabolizing xylose and glucose was improved.

The domesticated yeast Pachysolen tannophilus 2.1622 was cultured in liquid. 100ml of liquid medium is housed in a 250ml Erlenmeyer flask, and the formula of the liquid medium is: xylose 20g/L, peptone 5g/L, yeast juice 3g/L. The domesticated Pachysomyces tannophilus 2.1622 was inoculated in the liquid medium, and the inoculation amount was 2%. Cultivate for 24 hours under the conditions of pH 5.0, 30° C., and 60 rpm, concentrate after the cultivation, and collect the culture solution for future use.

This culture was then used as an inoculum to immobilize Pachysapsis tannophilus 2.1622 cells. Each time, take 2ml of the culture solution obtained in the previous step and mix it with 68ml of 3% sodium alginate solution containing 1.2% Al ₂ O ₃ at room temperature. After fully stirring under sterile conditions, add dropwise to 2% CaCl ₂ In the aqueous solution, stand at 4°C for 10 hours. Pachysolomyces tannophilus 2.1622 cells were immobilized to make gel particles with a diameter D of about 3 mm. After the immobilized gel particles were washed three times with sterile water, they were transferred to the proliferation medium.

The immobilized Pachysolen tannophilus 2.1622 was propagated and cultured. 100ml of proliferation medium is installed in a 250ml Erlenmeyer flask, the formula of the proliferation medium is: xylose 20g/L, glucose 50g/L, peptone 3g/L, yeast juice 2.5g/L, CaCl ₂ 0.25g/L , MgSO ₄ 0.25g/L, KH ₂ PO ₄ 2.5g/L. Insert the gel particles into 100ml of proliferation medium, culture at 30°C, pH 5.0, 80rpm for 24 hours, and replace with fresh culture medium after 12 hours.

Finally, the alcoholic fermentation was carried out with Pachysomyces tannophilus 2.1622 after propagation. 100ml of fermentation medium is installed in a 250ml Erlenmeyer flask. The formulation of the fermentation medium is: xylose 20g/L, glucose 50g/L, CaCl ₂ 2.5g/L, MgSO ₄ 0.25g/L, KH ₂ PO ₄ 2.5g/L, yeast juice 2.5g/L, peptone 3g/L, urea 0.24g/L. The Pachysomyces tannophilus 2.1622 after multiplication and culture was inserted into 100 ml of fermentation medium, and the fermentation reaction was carried out at 60 rpm, 30° C., and pH value 5.0 for 24 hours.

Measured by high-pressure liquid chromatography: the utilization rate of xylose is 87.90%, the utilization rate of glucose reaches 99.99%, and the utilization rate of total sugar is 93.95%. The alcohol content in the fermentation broth was analyzed by gas chromatography, and the results showed that the ethanol yield reached 97%.

Example 2

Pachysolen tannophilus ATCC 32691 Process for the production of ethanol from xylose and glucose.

100ml of acclimatization medium is housed in a 250ml Erlenmeyer flask, and the formulation of the acclimatization medium is: xylose 10g/L, peptone 4g/L, yeast juice 2g/L. The yeast Pachysolen tannophilus ATCC32691 preserved on the slant was inoculated in the acclimatization medium, and the inoculum amount was 3%. The culture was carried out for 60 hours under the conditions of pH 5.5, 35° C., and 80 rpm, and a total of 6 batches of acclimatization culture were carried out. The bacteria content in the culture solution was counted by microscope direct counting method, and the bacteria content increased obviously with the increase of domestication batches. The increase of bacteria content indicated that the ability of metabolizing xylose and glucose was improved.

The domesticated yeast Pachysolen tannophilus ATCC 32691 was cultured in liquid. 100ml of liquid medium is housed in a 250ml Erlenmeyer flask, and the formula of the liquid medium is: xylose 10g/L, peptone 4g/L, yeast juice 2g/L. The acclimatized Pachysomyces tannophilus ATCC 32691 was inoculated in liquid medium with an inoculum size of 3%. Cultivate for 36 hours under the conditions of pH 5.5, 35° C., and 80 rpm, concentrate after the cultivation, and collect the culture solution for later use.

This culture was then used as an inoculum to immobilize Pachysas tannophilus ATCC 32691 cells. Each time, take 2ml of the culture solution obtained in the previous step and mix it with 68ml of 5% sodium alginate solution containing 1.2% Al ₂ O ₃ at room temperature. After stirring well under sterile conditions, add dropwise to 2% CaCl ₂ In the aqueous solution, stand at 4°C for 15 hours. Pachysolomyces tannophilus ATCC 32691 cells were immobilized to make gel particles with a diameter D of about 3 mm. After the immobilized gel particles were washed three times with sterile water, they were transferred to the proliferation medium.

The immobilized Pachysomyces tannophilus ATCC 32691 was propagated and cultured. 100ml of proliferation medium is placed in a 250ml Erlenmeyer flask, the formula of the proliferation medium is: xylose 20g/L, glucose 50g/L, peptone 3g/L, yeast juice 2.5g/L, CaCl ₂ 0.25g/L , MgSO ₄ 0.25g/L, KH ₂ PO ₄ 2.5g/L. The gel particles were placed in 100ml of proliferation medium, cultured at 35°C, pH 5.5, 70rpm for 36 hours, and replaced with fresh culture medium every 12 hours.

Finally, the alcoholic fermentation was carried out with Pachysomyces tannophilus ATCC 32691 after propagation. 100ml of fermentation medium is installed in a 250ml Erlenmeyer flask. The formula of the fermentation medium is: xylose 20g/L, glucose 50g/L, CaCl ₂ 2.5g/L, MgSO ₄ 0.25g/L, KH ₂ PO ₄ 2.5g/L, yeast juice 2.5g/L, peptone 3g/L, urea 0.24g/L. The Pachysomyces tannophilus ATCC 32691 after multiplication and culture was inserted into 100 ml of fermentation medium, and the fermentation reaction was carried out at 80 rpm, 35° C., and pH 5.5 for 30 hours.

Measured by high-pressure liquid chromatography: the utilization rate of xylose is 76.62%, the utilization rate of glucose reaches 98.08%, and the utilization rate of total sugar is 88.31%. The alcohol content in the fermentation broth was analyzed by gas chromatography, and the results showed that the ethanol yield reached 96%.

Example 3

Candida shohata ATCC 34887 Method for the production of alcohol from xylose and glucose.

100ml of acclimatization medium is housed in a 250ml Erlenmeyer flask, the formulation of the acclimatization medium is: xylose 50g/L, peptone 6g/L, yeast juice 4g/L. The yeast Candida shohata ATCC34887 preserved on the slant was inoculated in the acclimatization medium, and the inoculum amount was 5%. The culture was carried out for 96 hours under the conditions of pH 5.5, 28° C., and 120 rpm, and a total of 5 batches of acclimatization culture were carried out. The bacteria content in the culture solution was counted by microscope direct counting method, and the bacteria content increased obviously with the increase of domestication batches. The increase of bacteria content indicated that the ability of metabolizing xylose and glucose was improved.

The domesticated yeast Candida shohata ATCC 34887 was cultured in liquid. 100ml of liquid medium is housed in a 250ml Erlenmeyer flask, and the formula of the liquid medium is: xylose 50g/L, peptone 6g/L, yeast juice 4g/L. The acclimatized Pachysomyces tannophilus ATCC 34887 was inoculated in the liquid medium, and the inoculation amount was 5%. Cultivate for 48 hours under the conditions of pH 5.5, 28° C., and 120 rpm, concentrate after the cultivation, and collect the culture solution for later use.

This culture was then used as an inoculum to immobilize Candida shehata ATCC 34887 cells. Each time, take 2ml of the culture solution obtained in the previous step and mix it with 68ml of 6% sodium alginate solution containing 1.2% Al ₂ O ₃ at room temperature. After stirring well under sterile conditions, add dropwise to 2% CaCl ₂ In the aqueous solution, it was left to stand at 4°C for 20 hours. Candida shohata ATCC 34887 cells were immobilized and made into gel particles with a diameter D of about 3 mm. After the immobilized gel particles were washed three times with sterile water, they were transferred to the proliferation medium.

The immobilized Candida shohata ATCC 34887 was propagated and cultured. 100ml of proliferation medium is installed in a 250ml Erlenmeyer flask, the formula of the proliferation medium is: xylose 20g/L, glucose 50g/L, peptone 3g/L, yeast juice 2.5g/L, CaCl ₂ 0.25g/L , MgSO ₄ 0.25g/L, KH ₂ PO ₄ 2.5g/L. The gel particles were placed in 100ml of proliferation medium, cultured at 28°C, pH 5.5, 120rpm for 48 hours, and replaced with fresh culture medium every 12 hours.

Finally, alcoholic fermentation was carried out with Candida shohata ATCC 34887 after multiplication and cultivation. 100ml of fermentation medium is installed in a 250ml Erlenmeyer flask. The formulation of the fermentation medium is: xylose 20g/L, glucose 50g/L, CaCl ₂ 2.5g/L, MgSO ₄ 0.25g/L, KH ₂ PO ₄ 2.5g/L, yeast juice 2.5g/L, peptone 3g/L, urea 0.24g/L. The proliferated Candida shohata ATCC 34887 was inserted into 100 ml of fermentation medium, and the fermentation reaction was carried out at 120 rpm, 28° C., and pH 5.5 for 36 hours.

Measured by high-pressure liquid chromatography: the utilization rate of xylose is 74.68%, the utilization rate of glucose reaches 97.46%, and the utilization rate of total sugar is 87.34%. The alcohol content in the fermentation broth was analyzed by gas chromatography, and the results showed that the ethanol yield reached 95%.

Claims (6)

1. a yeast utilizes the method for wood sugar and glucose production alcohol, it is characterized in that this method may further comprise the steps:

1) pachysolen tannophilus 2.1622 be inoculated in the domestication substratum tame;

2) yeast after will taming is inoculated in and carries out liquid culture in the liquid nutrient medium;

3) yeast that liquid culture is obtained carries out immobilization;

4) immobilized yeast is carried out multiplication culture;

5) yeast after the propagation carries out zymamsis;

In the method for above-mentioned production alcohol:

When bacterial strain was tamed, the prescription of domestication substratum was: wood sugar 10-50g/L, and peptone 4-6g/L, yeast water 2-4g/L, the domestication condition is pH5.0-5.5,28-35 ℃, 60-120rpm, each batch domestication time is 48-96 hour.

2. the method for a production alcohol as claimed in claim 1, when it is characterized in that bacterial strain tamed, inoculum size is 2%, domestication is more than 5 batches.

3. the method for a production alcohol as claimed in claim 2, it is characterized in that with the yeast after the domestication prescription of substratum is: wood sugar 10-50g/L, peptone 4-6g/L when carrying out liquid culture, yeast water 2-4g/L, inoculum size is 1-5%, and culture condition is pH5.0-5.5,28-35 ℃, 60-120rpm, incubation time is 24-48 hour, cultivates and finishes the back concentration, and it is standby to collect nutrient solution.

4. the method for a production alcohol as claimed in claim 3, when it is characterized in that the yeast that liquid culture obtains carried out immobilization, at every turn with the concentrated liquid culture of 2ml with contain 1.2%Al ₂O ₃The 3-6% sodium alginate soln mix at normal temperatures, after under aseptic condition, stirring, be added drop-wise to 2% CaCl ₂In the aqueous solution, after leaving standstill 10-20 hour under 4 ℃, make gel particles.

5. the method for a production alcohol as claimed in claim 4, when it is characterized in that fixed yeast cell carried out multiplication culture, the prescription of proliferated culture medium is: wood sugar 20g/L, glucose 50g/L, peptone 3g/L, yeast water 2.5g/L, CaCl ₂0.25g/L, MgSO ₄0.25g/L, KH ₂PO ₄2.5g/L the multiplication culture condition is 28-35 ℃, pH5.0-5.5, and 60-120rpm, incubation time were 24-48 hour, changed a fresh medium every 12 hours.

6. the method for a production alcohol as claimed in claim 5, when it is characterized in that yeast behind the multiplication culture carries out zymamsis, the prescription of fermention medium is: wood sugar 20g/L, glucose 50g/L, CaCl ₂2.5g/L, MgSO ₄0.25g/L, KH ₂PO ₄2.5g/L, yeast water 2.5g/L, peptone 3g/L, urea 0.24g/L, fermentation condition are pH5.0-5.5,28-35 ℃, 60-120rmp, fermentation time are 24-36 hour.