CN108203721A - A kind of method that zymomonas mobilis utilizes corn producing and ethanol - Google Patents

Abstract

The invention discloses a method for producing ethanol by Zymomonas mobilis using corn, and belongs to the technical field of microbial fermentation. The method of the present invention comprises the following steps: (1) degerming the corn embryos under the beating of a degerminator to obtain corn grits, and crushing the corn grits through a pulverizer to obtain corn flour, adding water and stirring to obtain corn steep liquor; (2) adding water to the corn steep liquor and mixing Homogenize, cook and liquefy, then add glucoamylase for saccharification to obtain corn syrup saccharification liquid; (3) Trichoderma reesei fermentation liquid and Aspergillus niger fermentation liquid are mixed and centrifuged according to the volume ratio of 1:1, and the supernatant is taken for Centrifuge for the second time, and take the supernatant to obtain the crude cellulase enzyme liquid; (4) Add corn syrup saccharification liquid to the crude cellulase enzyme liquid, enzymatically hydrolyze at 45-55°C for 3-5 days, and then inoculate motile zymounit cells Bacteria, anaerobic fermentation at 30-35°C for 2-3 days. The conversion rate of converting Zymomonas mobilis into ethanol by corn is 35% through the method of the present invention.

Description

A kind of method that Zymomonas mobilis utilizes corn to produce ethanol

technical field

The invention belongs to the technical field of microbial fermentation, and in particular relates to a method for producing ethanol by Zymomonas mobilis using corn.

Background technique

Zymomonas mobilis ( Z. mobilis ) is a Gram-negative bacterium, a species of Zymomonas, named for its strong motility. Most of them are straight rods with round or oval tails, often in pairs, but rarely assembled into short chains. It grows under facultative anaerobic conditions, grows best under anaerobic conditions, and can survive under aerobic conditions. It is highly acid-resistant and can grow between pH 3.5 and 7.5. Zymomonas mobilis uses the Entner-Doudomff pathway to metabolize ethanol, which requires less energy and produces higher yields. As an ethanol producing strain, Zymomonas mobilis has certain excellent characteristics: such as high ethanol yield, strong ethanol tolerance, and high specificity of fermentation substrate. Compared with yeast, the ethanol yield of Zymomonas mobilis is 5% to 10% higher than that of yeast, which can reach 97% of the theoretical value, and the ethanol conversion rate is 5 times that of yeast. However, Zymomonas mobilis also has its own limitations, and its substrate utilization range is narrow, and it can only utilize glucose, fructose and sucrose. Recently, some people have used genetic engineering methods to modify its unfavorable factors, so that it can use a variety of substrates.

Due to the disadvantages of insufficient mining of traditional energy sources and environmental pollution, fuel ethanol is considered to be the most likely energy source to replace fossil fuels.

Contents of the invention

The present invention provides a method for producing ethanol by Zymomonas mobilis using corn steep liquor. The method takes corn steep liquor as a substrate, processes corn into sugar solution suitable for fermentation, and then uses Zymomonas mobilis to ferment to produce ethanol.

The object of the present invention is achieved through the following technical solutions:

A method for producing ethanol by Zymomonas mobilis using corn steep liquor, comprising the steps of:

(1) Degerming, crushing and pulping of corn: The corn is beaten by the degerminator to remove the corn germ to obtain corn grits, and the corn grits are crushed by a grinder to obtain corn flour, which is then stirred with water to obtain corn steep liquor.

(2) Liquefaction and saccharification of corn steep liquor: Mix corn steep liquor and water evenly at a mass ratio of 1:3.5-4, heat to 100±5°C and cook until liquefaction is complete to obtain liquefied liquid. Add glucoamylase when the temperature of the liquefied solution is lowered to 55°C-65°C, and add hydrochloric acid to adjust the pH to 4.1-4.3 for saccharification to obtain corn syrup saccharified liquid. The water is preferably water at 45°C-55°C.

(3) Preparation of crude cellulase enzyme liquid: Trichoderma reesei fermentation liquid and Aspergillus niger fermentation liquid were mixed according to the volume ratio of 1:1, the mixture of the two was centrifuged, and the supernatant was taken for the second The second centrifugation, the supernatant of the second centrifugation is the cellulase crude enzyme liquid.

(4) Enzymatic hydrolysis of corn starch: Add the corn syrup saccharification solution obtained in step (2) to the crude cellulase solution obtained in step (3), and inoculate Zymomonas mobilis after enzymatic hydrolysis at 45-55°C for 3-5 days , 30-35 ℃ anaerobic, static fermentation for 2-3 days.

Step (1) is preferably: the corn is beaten by a degerminator to remove the corn germ to obtain corn grits, and the corn grits are crushed into 200-mesh fine powder by a pulverizer. Add 50g of corn flour to 20g of water into a mixing tank, heat to 63-68°C and stir to obtain corn steep liquor.

The final concentration (concentration in the system) of the saccharification enzyme described in step (2) is preferably 1 μg/L, and the saccharification time is preferably 20 minutes.

The Trichoderma reesei and Aspergillus niger culture, activation and fermentation methods described in step (3) are the same, and the Trichoderma reesei fermentation liquid or Aspergillus niger fermentation liquid is preferably prepared by a method comprising the following steps:

1) Inoculate Trichoderma reesei or Aspergillus niger in PDA solid medium, culture in a sealed container at a constant temperature of 28°C for 3-5 days, then wash the generated spores from the medium with sterilized physiological saline , and use a pipette gun to break up the spores existing in agglomerates to completely disperse them, and dilute to a concentration of ¹⁰⁷ /mL to obtain Trichoderma reesei spore suspension or Aspergillus niger spore suspension.

2) Inoculate the spore suspension of Trichoderma reesei or Aspergillus niger in the seed medium at an inoculation amount of 10% (V/V), and culture it in a shaker at 28°C and 150rpm for 48 hours to obtain activated Lisler Trichoderma liquid or activated Aspergillus niger liquid. Described seed culture medium comprises the raw material of following concentration: corn grits 10g/L, Tween-80 is 5mL/L, Mandles trace element salt solution 1mL/L, Mandles nutrient salt concentrated solution 100mL/L, pH is 4.8 Concentration of 1mol/L citrate buffer 50mL/L.

3) Inoculate the above-mentioned activated Trichoderma reesei liquid or activated Aspergillus niger liquid into the fermentation medium at 10% inoculum amount and culture at 28° C. and 150 rpm for 7 days to obtain Trichoderma reesei fermentation liquid or Aspergillus niger fermentation liquid. The fermentation medium contains raw materials of the following concentrations: corn grits 30g/L, Tween-80 5mL/L, Mandles trace element salt solution 1mL/L, Mandles nutrient salt concentrated solution 100mL/L, pH 4.8 Concentration of 1mol/L citrate buffer 50mL/L.

The centrifugation conditions described in step (3) are preferably 4° C., 8000 rpm for 10 min.

The Zymomonas mobilis described in step (4) is preferably an activated bacterial liquid, preferably prepared by a method comprising the following steps: activating Zymomonas mobilis with ZM seed culture medium, at 10% (v/v ) into the ZM seed medium, placed in a constant temperature shaker at 32°C, and cultured statically for 10 hours. Take the seed solution grown for 10 hours and inoculate it into the ZM seed medium at an inoculation amount of 10%, place it in a constant temperature shaker at 32°C, and let it stand for the second time to activate. When the OD ₆₀₀ of the bacterial solution reaches about 10, the final activation will be obtained. The bacterium solution is used to inoculate into the corn syrup saccharification solution enzymatically hydrolyzed by the above crude enzyme solution.

The relationship between the amount of the crude enzyme solution and the corn syrup saccharification solution in step (4) is preferably: add 1 g of corn flour to every 12 mL of crude enzyme solution and obtain the corn syrup saccharification solution in steps (1) and (2).

Step (4) is preferably: add 1g of corn flour per 12mL of crude enzyme solution, add corn syrup saccharification solution to the crude enzyme solution in proportion to the corn syrup saccharification solution obtained in steps (1) and (2), and enzymatically hydrolyze at 50°C After 4 days, Zymomonas mobilis was inoculated, anaerobically and statically fermented in a shaker at 32° C. for 60 h.

The action mechanism of the present invention is as follows: Zymomonas mobilis has certain excellent characteristics as an ethanol production strain, such as higher ethanol yield and stronger ethanol tolerance. However, Zymomonas mobilis also has its own limitations, the substrate utilization range is narrow, and it cannot directly act on starch. In the invention, aspergillus niger and wood enzyme are used to hydrolyze mixed fermented corn grits to produce crude cellulase enzyme liquid, and enzymatically hydrolyze corn steep liquor to produce reducing sugar. By improving the traditional mixed fermentation of Aspergillus niger and Trichoderma to produce cellulase by separate fermentation and then mixing the crude enzyme solution, the product feedback inhibition phenomenon caused by the accumulation of five-carbon sugars is effectively improved, and the efficiency of starch saccharification is improved. . Then, the Zymomonas mobilis acts on the reducing sugar to produce ethanol, thereby improving the ethanol production efficiency of the Zymomonas mobilis using corn steep liquor. Solve the problem of single narrow substrate utilization by Zymomonas mobilis.

The beneficial technical effects of the present invention are: (1) In the present invention, the crude cellulase enzyme liquids fermented by Aspergillus niger and Trichoderma respectively are mixed at 1:1, which effectively controls the feedback inhibition of the product, thereby obtaining higher activity and yield. High cellulase crude enzyme solution and can target starch saccharification. (2) Inoculating Zymomonas mobilis in the fermentation broth containing reducing sugar after enzymolysis greatly improved the efficiency of Zymomonas mobilis in producing absolute ethanol from corn steep liquor, and solved the problem of Zymomonas mobilis substrates The utilization scope is narrow, and the drawback of cornstarch can not be effectively utilized. By the method of the present invention, Zymomonas mobilis converts corn into ethanol with a conversion rate of 35%, that is, 100g of corn produces 35g of ethanol.

Detailed ways

The following examples are used to further illustrate the content of the present invention, but should not be interpreted as limiting the present invention, without departing from the spirit and essence of the present invention, the modifications or replacements made to the methods, steps or conditions of the present invention all belong to the present invention range. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.

Example 1

1. Materials

(1) Strains: Trichoderma reesei ( T. reesei ) RUT C30, Aspergillus niger ( Aspergillus niger ), Zymomonas mobilis ( Z. mobilis ).

(2) PDA solid medium: PDA is potato dextrose agar medium. Wash and peel the potatoes, weigh 200g of potatoes and cut them into small pieces, add them to a beaker, add water and cook until the potatoes can be gently pierced by a glass, and cook About 30min. Filter with gauze, add 20g of glucose and 20g of agar to the filtrate, and dilute to 1L. Autoclave at 115°C for 30 minutes, pour solid plate.

(3) Seed medium: corn grits 10g/L, Tween-80 5mL/L, Mandles trace element salt solution 1mL/L, Mandles nutrient salt concentrated solution 100mL/L, pH 4.8 concentration 1mol/L Citric acid buffer 50mL/L. Autoclave at 115°C for 30 minutes and set aside.

(4) Fermentation medium: corn grits 30g/L, Tween-80 5mL/L, Mandles trace element salt solution 1mL/L, Mandles nutrient salt concentrated solution 100mL/L, pH 4.8 concentration 1mol/L Citric acid buffer 50mL/L. Autoclave at 115°C for 30 minutes and set aside.

(5) ZM seed medium: glucose 100g/L, urea 0.05g/L, yeast extract 1g/L, potassium dihydrogen phosphate 0.05g/L, magnesium sulfate 0.05g/L, add deionized water to 1000mL , adjusted the pH to 6.0 with HCL, and sterilized at 115°C for 30min.

(6) ZM seed medium without glucose: Except for glucose, the other components and contents are the same as ZM seed medium.

2. Specific steps

(1) Degerming, crushing and pulping of corn: The corn germs are degerminated by the degerming machine to obtain corn grits, and the corn grits are crushed into 200-mesh fine powder by a pulverizer, and the crushing particle size is generally ≥88%. Add 50g of corn flour to 20g of water into the mixing tank, heat to 63-68°C and stir to obtain corn steep liquor with a pH value of 5.8-6.0.

(2) Corn syrup liquefaction and saccharification: Mix corn steep liquor with water at 45°C-55°C in a mass ratio of 1:3.5-4, the pH value is 6.5-7.0, heat to 100±5°C and cook for about 100 minutes, iodine The liquid test is pink, indicating that the liquefaction is completed and the liquefied liquid is obtained. When the temperature of the liquefied solution is lowered to about 60°C, add glucoamylase (final concentration 1 μg/L), and add hydrochloric acid to adjust the pH to 4.1-4.3, and the saccharification time is controlled at 20 minutes to obtain corn syrup saccharification solution.

(3) Preparation of cellulase crude enzyme solution:

1) Pick a small amount of preserved spores of Trichoderma reesei and Aspergillus niger with an inoculation loop, streak on different PDA solid plates, seal with a parafilm, and culture in a 28°C incubator for 3-5 days.

2) After 3-5 days of cultivation in the incubator, Trichoderma reesei and Aspergillus niger will produce a layer of spores, wash the spores from the solid plate with sterilized saline, and use a pipette gun to form clumps The spores were crushed, dispersed completely, and diluted to a concentration of ¹⁰⁷ /mL to obtain Trichoderma reesei spore suspensions and Aspergillus niger spore suspensions, respectively.

3) The above-mentioned Trichoderma reesei spore suspension and Aspergillus niger spore suspension were respectively used as strains, inoculated into different seed media at an inoculation amount of 10% (V/V), and cultured in a shaker at 28°C and 150rpm After 48 hours, the activated Trichoderma reesei liquid and the activated Aspergillus niger liquid were obtained.

4) Inoculate the above-mentioned activated Trichoderma reesei liquid and the activated Aspergillus niger liquid into 250 mL shake flasks containing 50 mL of different fermentation media according to the inoculum amount of 10%, and cultivate them at 28°C and 150 rpm for 7 days to obtain Li Trichoderma fermented liquid or Aspergillus niger fermented liquid.

5) Mix Trichoderma reesei fermentation broth and Aspergillus niger fermentation broth at a volume ratio of 1:1, centrifuge the mixture at 4°C and 8000rpm for 10min; take the supernatant and transfer it Centrifuge for the second time under the same conditions in another centrifuge tube (the centrifugation conditions are the same as above), and take the supernatant to obtain the crude cellulase enzyme solution.

(4) Preparation of Zymomonas mobilis:

Zymomonas mobilis was activated with ZM seed culture medium, inoculated into ZM seed culture medium at an inoculation amount of 10% (v/v), placed in a constant temperature shaker at 32°C, and cultured statically for 10 h. Take 1.4 mL of the seed solution grown for 10 hours and inoculate it into the ZM seed medium at a 10% inoculation volume (v/v), place it in a constant temperature shaker at 32°C, and culture it for a second time, during which the OD ₆₀₀ of the bacterial solution is measured. When the OD ₆₀₀ reaches about 10, it is the activated Zymomonas mobilis, which can be used as a strain for ethanol production.

Take 1.4 mL of activated Zymomonas mobilis strains, remove the glucose and ethanol contained in them by centrifugation and water washing, and at the same time use 1.4 mL of glucose-free ZM seed medium to suspend and mix the strains Homogenize Zymomonas mobilis suspension.

(5) Enzymatic hydrolysis of corn steep liquor: add the corn syrup saccharification liquid obtained in step (2) to the Into 50mL of crude enzyme solution, that is, every 12mL of crude enzyme solution, add 1g of corn flour saccharification solution obtained by steps (1) and (2). Under the condition of 50°C, after 4 days of enzymatic hydrolysis, 1.4 mL of Zymomonas mobilis suspension was inoculated and fermented anaerobically and statically in a shaker at 32°C for 60 hours. After the fermentation finished, the ethanol content in the fermented liquid measured by gas chromatography was 35g/L. The conversion rate of corn into ethanol is 35%, that is, 100g of corn produces 35g of ethanol.

Claims (8)

1. a kind of method that zymomonas mobilis utilizes corn pulp producing and ethanol, it is characterised in that：Include the following steps：

（1）The de- embryo of corn is crushed and is sized mixing：Maize is made to deviate to obtain hominy grits under the impact of germ separator corn, it is beautiful Rice crushed grain crushes to obtain corn flour by pulverizer, and water is added to stir to get corn pulp；

（2）The liquefaction and saccharification of corn pulp：By corn pulp and water in mass ratio 1:3.5-4 is uniformly mixed, and is heated to 100 ± 5 DEG C Boiling is completed to obtain liquefier to liquefying；Liquefier adds in carbohydrase when being cooled to 55 DEG C -65 DEG C, and adds hydrochloric acid tune pH to 4.1- 4.3, it is saccharified to obtain corn pulp saccharified liquid；

（3）The preparation of cellulase crude enzyme liquid：Trichoderma reesei zymotic fluid and black-koji mould zymotic fluid are 1 according to volume ratio:1 item Part is mixed, and the mixed liquor of the two is centrifuged, and supernatant is taken to carry out second and is centrifuged, takes supernatant up to the thick enzyme of cellulase Liquid；

（4）Digest cornstarch：Toward step（3）Step is added in obtained cellulase crude enzyme liquid（2）Obtained corn pulp sugar Change liquid, 45-55 DEG C of enzymolysis is inoculated with zymomonas mobilis, 30-35 DEG C of anaerobism, standing for fermentation 2-3 days after 3-5 days.

2. according to the method described in claim 1, it is characterized in that：Step（1）For：Corn is made into jade under the impact of germ separator Rice embryo deviates to obtain hominy grits, and hominy grits is ground into 200 mesh fine-powders by pulverizer；Add 20g water according to 50g corn flour Ratio is added to mixing tank, is heated to 63-68 DEG C and stirs to get corn pulp.

3. according to the method described in claim 1, it is characterized in that：Step（2）Described in carbohydrase final concentration of 1 μ g/ L, the time of saccharification is 20 minutes.

4. according to the method described in claim 1, it is characterized in that：Step（3）Described in trichoderma reesei zymotic fluid or black song Mold fermentation liquid is prepared by a method comprising the following steps to obtain：

1）Li's Trichoderma or black-koji mould are seeded in PDA solid mediums, the sealing culture 3-5 under 28 DEG C of constant temperatures After it, the spore of generation is washed from culture medium with the physiological saline that sterilization treatment is crossed, and will be into bulk with liquid-transfering gun Existing spore is smashed, and is allowed to be completely dispersed, and is diluted to 10⁷The concentration of a/mL, obtain trichoderma reesei spore suspension or Aspergillus niger spore suspension；

2）Trichoderma reesei spore suspension or aspergillus niger spore suspension are inoculated in 10% inoculum concentration in seed culture medium, The trichoderma reesei bacterium solution or the aspergillus niger bacterium solution of activation activated 28 DEG C in shaking table, after 150rpm cultures 48h；The kind Sub- culture medium includes the raw material of following concentration：Hominy grits 10g/L, Tween-80 5mL/L, Mandles trace element salting liquid 1mL/L, Mandles nutritive salt concentrated solution 100mL/L, pH are the citrate buffer solution 50mL/L of 4.8 a concentration of 1mol/L；

3）The aspergillus niger bacterium solution of the trichoderma reesei bacterium solution of activation or activation is inoculated by 10% inoculum concentration 28 in fermentation medium DEG C, 150rpm culture 7d obtain trichoderma reesei zymotic fluid or black-koji mould zymotic fluid；The fermentation medium includes following dense The raw material of degree：Hominy grits 30g/L, Tween-80 5mL/L, Mandles trace element salting liquid 1mL/L, Mandles nutritive salt Concentrated solution 100mL/L, pH are the citrate buffer solution 50mL/L of 4.8 a concentration of 1mol/L.

5. according to the method described in claim 1, it is characterized in that：Step（3）Described in centrifugation condition for 4 DEG C, 8000rpm centrifuges 10min.

6. according to the method described in claim 1, it is characterized in that：Step（4）Described in zymomonas mobilis pass through packet The method for including following steps is prepared：Zymomonas mobilis is activated with ZM seed culture mediums, ZM is accessed by 10% inoculum concentration Seed culture medium is placed in 32 DEG C of constant-temperature table, quiescent culture 10h；Take growth 10h after seed liquor by 10% inoculum concentration It is inoculated in ZM seed culture mediums, is placed in 32 DEG C of constant-temperature table, it is secondary to stand activation to bacterium solution OD₆₀₀Reach 10.

7. according to the method described in claim 1, it is characterized in that：Step（4）The area of a room of middle crude enzyme liquid and corn pulp saccharified liquid Relationship be：Step is pressed per 12mL crude enzyme liquids addition 1g corn flour（1）、（2）Obtained corn pulp saccharified liquid.

8. according to the method described in claim 1, it is characterized in that：Step（4）For：1g corn flour is added by every 12mL crude enzyme liquids By step（1）、（2）Corn pulp saccharified liquid is added in crude enzyme liquid by the obtained ratio of corn pulp saccharified liquid, and 50 DEG C digest 4 days After be inoculated with zymomonas mobilis, in 32 DEG C of shaking tables anaerobism, standing for fermentation 60h.