CN117947122A - Method for producing L-lysine by fermentation - Google Patents
Method for producing L-lysine by fermentation Download PDFInfo
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- CN117947122A CN117947122A CN202211279778.1A CN202211279778A CN117947122A CN 117947122 A CN117947122 A CN 117947122A CN 202211279778 A CN202211279778 A CN 202211279778A CN 117947122 A CN117947122 A CN 117947122A
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Abstract
The invention provides a method for producing L-lysine by fermentation, which comprises the following steps: (1) Pretreating and hydrolyzing lignocellulose raw materials to obtain hydrolysate; (2) And (3) fermenting and producing L-lysine by using a fermentation medium containing hydrolysate, wherein the glucose content in a fermentation system is maintained to be more than 1g/L by feeding in the fermentation process. According to the invention, the lignocellulose raw material is used for hydrolysis and fermentation to produce lysine, and meanwhile, the fed-batch fermentation process of the lysine is realized by feeding the hydrolysate concentrated solution of the lignocellulose raw material, so that the yield of the lysine is improved, and the method has a wide industrial application prospect.
Description
Technical Field
The invention relates to a novel method for producing L-lysine by taking lignocellulose as a raw material through fermentation.
Background
L-lysine (abbreviated as lysine) is a large amount of biotechnology products, is mainly used as a feed additive, and is widely applied to industries such as livestock breeding and the like. At present, raw materials used for producing the L-lysine are mainly starchy raw materials such as corn, wheat, cassava and the like, along with the rising of the price of the raw materials, the production cost of the L-lysine is higher and higher, and the limitation of using grain raw materials for industrial production of the L-lysine is increased. The lignocellulose raw material such as straw raw material is used for replacing starchiness raw material to produce the L-lysine, so that the raw material cost can be obviously reduced, the yield can be improved, and the method has important significance for market expansion and chemical application of the L-lysine.
The main components of lignocellulose include cellulose, hemicellulose, and lignin. The inherent structure of lignocellulose can be broken through by a pretreatment means, the package of lignin and hemicellulose on cellulose is eliminated, and the contact area of cellulose and cellulose is increased. Common pretreatment modes comprise dilute acid pretreatment, steam explosion pretreatment, ammonia fiber explosion pretreatment, alkali pretreatment, ionic liquid pretreatment and the like. The pretreatment process inevitably produces a series of toxic inhibitors that inhibit the subsequent cellulase activity, cell growth and metabolic activity of the fermenting microorganism. These toxicity inhibitors mainly include furfural, 5-hydroxymethylfurfural, acetic acid, 4-p-hydroxybenzaldehyde, vanillin, syringaldehyde, and the like. The pretreated material is often detoxified to remove these toxic inhibitors. The currently adopted toxic inhibitor removal means mainly comprise water washing, excessive lime treatment, ion exchange adsorption, activated carbon adsorption, microbial detoxification (biological detoxification for short) and the like.
The fed-batch fermentation process of lysine by using straw raw materials reported in the currently disclosed patent is complex, a detoxification process is needed, toxic inhibitors in a pretreatment process are removed, and a supernatant fluid obtained after the detoxification liquid is centrifuged is sterilized and can be used as a fed-batch carbon source, so that the production cost is increased, the application prospect of lignocellulose raw materials in lysine fermentation is limited, and the requirement of industrial production cannot be met.
Disclosure of Invention
In order to solve the above problems, the present invention provides a method for producing L-lysine by fermentation, comprising the steps of:
(1) Pretreating and hydrolyzing lignocellulose raw materials to obtain hydrolysate;
(2) And (3) fermenting and producing L-lysine by using a fermentation medium containing hydrolysate, wherein the glucose content in a fermentation system is maintained to be more than 1g/L by feeding in the fermentation process.
In one embodiment, in step (1), the pretreatment is an acid pretreatment comprising: the lignocellulose raw material and sulfuric acid solution are added into a reaction container, and saturated water vapor is introduced to heat reactants.
In one embodiment, the lignocellulosic feedstock is present in a solids to solids ratio of (1-8) to sulfuric acid solution of 1.
In one embodiment, the saturated water vapor has a temperature of 150 to 200deg.C, preferably 165 to 175 deg.C.
In one embodiment, the heating time is 1-10min, preferably 2-5min.
In one embodiment, the amount of H 2SO4 is 500 to 5000g per 100kg of the mixed system of lignocellulosic feedstock and sulfuric acid solution. Preferably, the content of H 2SO4 in the mixed system of the lignocellulose raw material and the sulfuric acid solution is 500-2000g per 100 kg.
In one embodiment, in step (1), the lignocellulosic feedstock is subjected to hydrolysis treatment with a cellulase comprising a neutral cellulase, an acid cellulase, preferably an acid cellulase.
In one embodiment, the cellulase is used in an amount of 1 to 20U/g of lignocellulosic feedstock, further 5 to 10U/g of lignocellulosic feedstock.
In one embodiment, the temperature of the hydrolysis treatment is 40-60 ℃, further 45-55 ℃, further 47-53 ℃.
In one embodiment, the pH of the hydrolysis treatment is from 4.5 to 7.0, further from 4.5 to 5.5. The pH can be adjusted during the hydrolysis treatment according to the invention, for example by means of ammonia, the concentration of which is preferably 25% to 30%, said percentages being by volume.
In one embodiment, the glucose content of the hydrolysate in step (1) is 60-120g/L, and further 100-120g/L.
As is known in the art, the pretreatment process inevitably produces a series of inhibitors for inhibiting the activity of subsequent cellulase and the cell growth and metabolic activity of fermenting microorganisms, and the pretreated materials are often detoxified to remove the inhibitors.
In one embodiment, the fermentation temperature is changed every 1-30 hours in the fermentation production of L-lysine in the step (2), and the fermentation temperature is generally in an upward trend.
In one embodiment, the fermentation temperature is controlled to be 32-38.5 ℃ and further 35-38 ℃ every 1-30 h. Specific examples of fermentation temperatures may be: 33 ℃, 34 ℃, 35 ℃, 36 ℃, 37 ℃, etc.
In one embodiment, the difference in fermentation temperatures between two adjacent time periods is controlled to be 0-2 ℃, and further 0.5-1 ℃. For example, the fermentation temperature is controlled to be 0.5℃and 1 ℃.
In a preferred embodiment of the invention, the fermentation time is controlled to 48 hours, and the fermentation temperature is controlled to 32-34 ℃, for example 34 ℃ in 1-8 hours; controlling the fermentation temperature to 34-35 ℃ for 8-16h, such as 35 ℃; controlling the fermentation temperature to 35-36 ℃ at 16-34h, such as 36 ℃; controlling the fermentation temperature at 36-37deg.C, such as 37deg.C, for 34-46 hr; the fermentation temperature is controlled at 37.5-38.5 deg.C, such as 38 deg.C, for 46-48 h.
In one embodiment, the pH is controlled to be 6.2-7.5, further 6.5-7 during the fermentation production of L-lysine in step (2).
In one embodiment, the ventilation rate is controlled to be 0.5-5vvm in the fermentation production of L-lysine in the step (2).
In one embodiment, the fermentation medium of step (2) contains 0.01% -0.2% KH2PO4、0.15％-0.45％MgSO4·7H2O、0.01％-0.06％MnSO4·H2O、0.5％-2.5％% ammonium sulfate, said percentages being mass volume percentages (w/v), mass units are g, volume units are ml).
In one embodiment, the total nitrogen content in the fermentation medium of step (2) is 1% -5%, and the percentages are mass percentages.
In one embodiment, the fermentation medium of step (2) contains a nitrogen source, which includes corn steep liquor, yeast extract, and the like.
In one embodiment, the fermentation medium of step (2) contains 0.05% -0.5% corn steep liquor, wherein the percentage is mass volume percentage (w/v), mass unit is g, volume unit is ml).
In one embodiment, the corn steep liquor has a total nitrogen content of 1% to 5% by weight.
In one embodiment, the yeast extract has a total nitrogen content of 5% to 15% by weight.
In one embodiment, the fermentation medium in the step (2) further contains 0.005% -0.5% of a growth factor, wherein the percentage is mass volume percentage (w/v%, mass unit is g, volume unit is ml), and the growth factor comprises any one or more than two of vitamin B 2, vitamin D, glutamine, lysine, methionine, leucine and phenylalanine.
In one embodiment, in the step (2), before fermentation, seed liquid is inoculated into the fermentation medium, wherein the inoculation amount is 10-40%, and the percentage is volume percentage.
In one embodiment, the concentration OD562 of the seed solution is 0.6-1.0 after 40-fold dilution.
In one embodiment, the seed solution contains bacterial cells selected from the group consisting of Corynebacterium glutamicum, escherichia coli, brevibacterium, and Bacillus, preferably Corynebacterium glutamicum.
In one embodiment, the corynebacterium glutamicum is Corynebacterium glutamicum CathS141 (Corynebacterium glutamicum CathS 141), and the corynebacterium glutamicum is CathS which is now deposited with the China center for type culture Collection, address: chinese university of Wuhan, post code 430072, preservation number CCTCC M20211495, and preservation date 2021, 11 months and 29 days.
In one embodiment, in step (2), the feeding is started when the glucose concentration in the fermentation system falls below 5-20g/L, further below 5-15g/L, further below 5-10 g/L. Feeding is started when the concentration is reduced to below 15g/L, below 10g/L or below 5 g/L.
In one embodiment, in step (2), the feed is a glucose-containing solution, preferably a hydrolysate concentrate of a lignocellulosic feedstock, more preferably a straw hydrolysate concentrate. Preferably, the concentration of the glucose-containing solution is 60-800g/L, and further 200-400g/L.
In the invention, the hydrolysate concentrate of the lignocellulose raw material used for material supplementing is prepared by pretreating and hydrolyzing the lignocellulose raw material, and then the hydrolysate concentrate is obtained by concentrating. Preferably, the preparation method of the hydrolysate is the same as that of the hydrolysate in the step (1), and the concentration may be performed by a conventional process in the art, so long as the obtained concentrate satisfies the above glucose concentration.
In one embodiment, in step (2), the feed is further supplemented with an ammonium sulfate solution having a concentration of 5% -40%, further 10% -35%, and the ammonium sulfate concentration in the fermentation system is maintained at a level of 0.5-8g/L by the feed. The percentage is mass volume percentage (w/v), the mass unit is g, and the volume unit is ml).
In one embodiment, the glucose content in the fermentation system is maintained at 5-20g/L, further at 5-15g/L, by feeding during the fermentation in step (2).
In one embodiment, in step (2), the volume of the fermentation broth in the post-feed fermenter is controlled to be 0.7 to 3 times, further 0.7 to 2.0 times, further 0.7 to 1.5 times the volume of the fermentation broth before feed. It is well known to those skilled in the art that the volume of the fed fermentation broth may be controlled within the above-defined limits by controlling the feed rate and/or the discharge rate, or otherwise.
In one embodiment, in step (1), the pretreated lignocellulosic feedstock has a solids content of from 5 to 45% (w/w), and further from 10 to 40% (w/w), the percentages being by mass.
In one embodiment, the lignocellulosic feedstock of step (1) comprises one or more of corn stover, wheat straw, cotton stalk, sesame stalk, canola stalk, sweet sorghum stalk, corn cob, rice hull, chaff, bagasse, straw, wood chips, hardwood, and softwood.
Compared with the prior art, the invention has the following positive effects:
According to the invention, the lignocellulose raw material is used for hydrolysis and fermentation to produce lysine, and meanwhile, the fed-batch fermentation process of the lysine is realized by feeding the hydrolysate concentrated solution of the lignocellulose raw material, so that the yield of the lysine is improved, and the method has a wide industrial application prospect;
In addition, the fermentation medium and the feed supplement medium for producing lysine by fermentation directly use the hydrolysate or the hydrolysate concentrate of the lignocellulose raw material for fermentation and feed supplement, the hydrolysate does not need to be detoxified, and meanwhile, the lignocellulose raw material contains nutrients such as trace elements, nitrogen, phosphorus and the like, so that the addition proportion of the nutrients in the feed supplement medium can be reduced, and the production cost is reduced.
Detailed Description
The following detailed description of the invention is provided in detail, with the understanding that the embodiments described herein are merely illustrative and explanatory of the invention and are not intended to limit the invention.
The reagents, raw materials and test methods used are described as follows:
The total nitrogen content of the corn steep liquor in the examples was 2.7wt%.
Culture of seed solution of fermentation strain
The strain for fermentation is Corynebacterium glutamicum CathS141 (Corynebacterium glutamicum CathS 141), which is now deposited in China center for type culture Collection, address: chinese university of Wuhan, post code 430072, preservation number CCTCC M20211495, and preservation date 2021, 11 months and 29 days.
The composition of the seed culture medium is as follows: KH 2PO4, mgSO 4·7H2 O, mnSO 4·H2 O, ammonium sulfate, glucose, and growth factor vitamin B 2 in amounts of 0.45, 0.5, 10, 15, 0.15, 0.035w/v, respectively.
C.glutamicum CathS141 is inoculated into a seed culture medium, the inoculation amount is 0.5% (v/v), and the culture is carried out for 14 hours under the conditions that the ventilation amount is 0.4vvm, the temperature is 37 ℃, the pressure is 0.05MPa and the pH value is 6.5 (the culture is regulated by ammonia water with the concentration of 25 (v/v)%, so that the concentration of the thallus OD562 reaches 0.75 after the culture is diluted 40 times, and the mature seed liquid is obtained.
Example 1
1 Preparation of straw hydrolysate
Adding corn stalk material and sulfuric acid solution into a reaction container according to a solid-to-liquid ratio (g/g) of 2:1, wherein the H 2SO4 content in a mixed system of each 100kg corn stalk and sulfuric acid solution is 1100g, and introducing 172 ℃ saturated steam into the reaction container to heat the material in the reaction container for 5min. Then adjusting the pH value of the material to 5.0, adding the pretreated straw raw material (solid content of 30 wt%) into a saccharification tank, adding acid cellulase into the saccharification tank according to the proportion of 8U/g straw to carry out enzymolysis saccharification treatment, controlling the pH value to 5.0 by using ammonia water during the period of 50 ℃ to obtain straw hydrolysate. The content of glucose in the straw hydrolysate is 80g/L, the content of furfural is 0.8g/L, the content of hydroxymethylfurfural is 1.6g/L, and the content of acetic acid is 8g/L.
Concentrating the straw hydrolysate to obtain straw hydrolysate concentrate serving as a feed solution, wherein the glucose content in the straw hydrolysate concentrate is 300g/L.
Fermentation of 2L-lysine
Adding a fermentation medium into a fermentation tank filled with straw hydrolysate, wherein the content of KH 2PO4 in the fermentation medium is 0.04w/v%, the content of MgSO 4·7H2 O is 0.32w/v%, the content of MnSO 4·H2 O is 0.028w/v%, the content of ammonium sulfate is 1.0w/v%, the content of corn steep liquor is 0.26w/v%, and the content of growth factor vitamin B 2 is 0.017w/v%.
Inoculating seed solution into fermentation medium, inoculating seed solution with inoculum size of 20% (v/v), pH of 6.5 (controlled by 30% (v/v) ammonia water), and air volume of 1vvm, and starting fermentation process. In the whole fermentation process, the fermentation temperature is controlled to be 34 ℃ for 1-8h, 35 ℃ for 8-16h, 36 ℃ for 16-34h, 37 ℃ for 34-46h and 38 ℃ for 46-48 h.
When the glucose concentration in the fermentation liquor is reduced to below 5g/L, the concentrated solution of straw hydrolysate (with the glucose content of 300g/L and the preparation method as above) and the ammonium sulfate solution (with the ammonium sulfate content of 20 w/v%) are fed and fermented, and the glucose concentration in the fermentation liquor is maintained at the level of 5-10 g/L and the ammonium sulfate concentration is maintained at the level of 2-5 g/L through feeding. The volume of the fermentation liquor in the fermentation tank after material supplementing is maintained to be 1.1-1.5 times of the volume of the fermentation liquor before material supplementing by controlling the discharging speed. The fermentation period was 48 hours.
Example 2
1 Preparation of straw hydrolysate
Adding corn stalk material and sulfuric acid solution into a reaction container according to a solid-to-liquid ratio (g/g) of 2:1, wherein the H 2SO4 content in a mixed system of each 100kg corn stalk and sulfuric acid solution is 1100g, and introducing saturated water vapor at 180 ℃ into the reaction container to heat the material in the reaction container for 10min. Then adjusting the pH value of the material to 5.0, adding the pretreated straw raw material (solid content of 30 wt%) into a saccharification tank, adding acid cellulase into the saccharification tank according to the proportion of 8U/g straw to carry out enzymolysis saccharification treatment, controlling the pH value to 5.0 by using ammonia water during the period of 50 ℃ to obtain straw hydrolysate. The content of glucose in the straw hydrolysate is 80g/L, the content of furfural is 1.5g/L, the content of hydroxymethylfurfural is 2.8g/L, and the content of acetic acid is 15g/L.
Concentrating the straw hydrolysate to obtain straw hydrolysate concentrate serving as a feed solution, wherein the glucose content in the straw hydrolysate concentrate is 300g/L.
Fermentation of 2L-lysine
Adding a fermentation medium into a fermentation tank filled with straw hydrolysate, wherein the content of KH 2PO4 in the fermentation medium is 0.04w/v%, the content of MgSO 4·7H2 O is 0.32w/v%, the content of MnSO 4·H2 O is 0.028w/v%, the content of ammonium sulfate is 1.0w/v%, the content of corn steep liquor is 0.26w/v%, and the content of growth factor vitamin B 2 is 0.017w/v%.
Inoculating seed solution into fermentation medium, inoculating seed solution with 20% (v/v), and starting fermentation under pH 6.5 (controlled by 30% (v/v) ammonia water) and air volume of 1 vvm. In the whole fermentation process, the fermentation temperature is controlled to be 32 ℃ for 1-6h, 34 ℃ for 6-16h, 35 ℃ for 16-34h, 37 ℃ for 34-46h and 37.5 ℃ for 46-48 h.
When the glucose concentration in the fermentation liquor is reduced to below 5g/L, the concentrated solution of straw hydrolysate (with the glucose content of 300g/L and the preparation method as above) and the ammonium sulfate solution (with the ammonium sulfate content of 20 w/v%) are fed and fermented, and the glucose concentration in the fermentation liquor is maintained at the level of 5-10 g/L and the ammonium sulfate concentration is maintained at the level of 2-5 g/L through feeding. The volume of the fermentation liquor in the fermentation tank after material supplementing is maintained to be 1.1-1.5 times of the volume of the fermentation liquor before material supplementing by controlling the discharging speed. The fermentation period was 48 hours.
Example 3
1 Preparation of straw hydrolysate
Adding corn stalk material and sulfuric acid solution into a reaction container according to a solid-to-liquid ratio (g/g) of 2:1, wherein the H 2SO4 content in a mixed system of each 100kg corn stalk and sulfuric acid solution is 1100g, and introducing 170 ℃ saturated steam into the reaction container to heat the material in the reaction container for 2.5min. Then adjusting the pH value of the material to 5.0, adding the pretreated straw raw material (solid content of 30 wt%) into a saccharification tank, adding acid cellulase into the saccharification tank according to the proportion of 8U/g straw to carry out enzymolysis saccharification treatment, controlling the pH value to 5.0 by using ammonia water during the period of 50 ℃ to obtain straw hydrolysate. The content of glucose in the straw hydrolysate is 80g/L, the content of furfural is 0.5g/L, the content of hydroxymethylfurfural is 0.8g/L, and the content of acetic acid is 4g/L.
Concentrating the straw hydrolysate to obtain straw hydrolysate concentrate serving as a feed solution, wherein the glucose content in the straw hydrolysate concentrate is 300g/L.
Fermentation of 2L-lysine
Adding a fermentation medium into a fermentation tank filled with straw hydrolysate, wherein the content of KH 2PO4 in the fermentation medium is 0.04w/v%, the content of MgSO 4·7H2 O is 0.32w/v%, the content of MnSO 4·H2 O is 0.028w/v%, the content of ammonium sulfate is 1.0w/v%, the content of corn steep liquor is 0.26w/v%, and the content of growth factor vitamin B 2 is 0.017w/v%.
Inoculating seed solution into fermentation medium, inoculating seed solution with 20% (v/v), and starting fermentation under pH 6.5 (controlled by 30% (v/v) ammonia water) and air volume of 1 vvm. In the whole fermentation process, the fermentation temperature is controlled to be 34 ℃ for 1-8h, 35 ℃ for 8-16h, 36.6 ℃ for 16-36h, 37 ℃ for 36-47h and 38 ℃ for 47-48 h.
When the glucose concentration in the fermentation liquor is reduced to below 5g/L, the concentrated solution of straw hydrolysate (with the glucose content of 300g/L and the preparation method as above) and the ammonium sulfate solution (with the ammonium sulfate content of 20 w/v%) are fed and fermented, and the glucose concentration in the fermentation liquor is maintained at the level of 5-10 g/L and the ammonium sulfate concentration is maintained at the level of 2-5 g/L through feeding. The volume of the fermentation liquor in the fermentation tank after material supplementing is maintained to be 1.1-1.5 times of the volume of the fermentation liquor before material supplementing by controlling the discharging speed. The fermentation period was 48 hours.
Example 4
1 Preparation of straw hydrolysate
Adding corn stalk material and sulfuric acid solution into a reaction container according to a solid-to-liquid ratio (g/g) of 2:1, wherein the H 2SO4 content in a mixed system of each 100kg corn stalk and sulfuric acid solution is 1100g, and introducing saturated water vapor at 200 ℃ into the reaction container to heat the material in the reaction container for 10min. Then adjusting the pH value of the material to 5.0, adding the pretreated straw raw material (solid content of 30 wt%) into a saccharification tank, adding acid cellulase into the saccharification tank according to the proportion of 8U/g straw to carry out enzymolysis saccharification treatment, controlling the pH value to 5.0 by using ammonia water during the period of 50 ℃ to obtain straw hydrolysate. The content of glucose in the straw hydrolysate is 80g/L, the content of furfural is 3g/L, the content of hydroxymethylfurfural is 5g/L, and the content of acetic acid is 20g/L.
Concentrating the straw hydrolysate to obtain straw hydrolysate concentrate serving as a feed solution, wherein the glucose content in the straw hydrolysate concentrate is 300g/L.
Fermentation of 2L-lysine
Adding a fermentation medium into a fermentation tank filled with straw hydrolysate, wherein the content of KH 2PO4 in the fermentation medium is 0.04w/v%, the content of MgSO 4·7H2 O is 0.32w/v%, the content of MnSO 4·H2 O is 0.028w/v%, the content of ammonium sulfate is 1.0w/v%, the content of corn steep liquor is 0.26w/v%, and the content of growth factor vitamin B 2 is 0.017w/v%.
Inoculating seed solution into fermentation medium, inoculating seed solution with 20% (v/v), and starting fermentation under pH 6.5 (controlled by 30% (v/v) ammonia water) and air volume of 1 vvm. In the whole fermentation process, the fermentation temperature is controlled to be 34 ℃ for 1-8h, 35 ℃ for 8-16h, 36 ℃ for 16-34h, 37 ℃ for 34-46h and 38.5 ℃ for 46-48 h.
When the glucose concentration in the fermentation liquor is reduced to below 5g/L, the concentrated solution of straw hydrolysate (with the glucose content of 300g/L and the preparation method as above) and the ammonium sulfate solution (with the ammonium sulfate content of 20 w/v%) are fed and fermented, and the glucose concentration in the fermentation liquor is maintained at the level of 5-10 g/L and the ammonium sulfate concentration is maintained at the level of 2-5 g/L through feeding. The volume of the fermentation liquor in the fermentation tank after material supplementing is maintained to be 1.1-1.5 times of the volume of the fermentation liquor before material supplementing by controlling the discharging speed. The fermentation period was 48 hours.
Example 5
1 Preparation of straw hydrolysate
Adding corn stalk material and sulfuric acid solution into a reaction container according to a solid-to-liquid ratio (g/g) of 2:1, wherein the H 2SO4 content in a mixed system of each 100kg corn stalk and sulfuric acid solution is 1100g, and introducing saturated water vapor at 168 ℃ into the reaction container to heat the material in the reaction container for 2min. Then adjusting the pH value of the material to 5.0, adding the pretreated straw raw material (solid content of 30 wt%) into a saccharification tank, adding acid cellulase into the saccharification tank according to the proportion of 8U/g straw to carry out enzymolysis saccharification treatment, controlling the pH value to 5.0 by using ammonia water during the period of 50 ℃ to obtain straw hydrolysate. The content of glucose in the straw hydrolysate is 80g/L, the content of furfural is 0.4g/L, the content of hydroxymethylfurfural is 0.7g/L, and the content of acetic acid is 3.5g/L.
Concentrating the straw hydrolysate to obtain straw hydrolysate concentrate serving as a feed solution, wherein the glucose content in the straw hydrolysate concentrate is 300g/L.
Fermentation of 2L-lysine
Adding a fermentation medium into a fermentation tank filled with straw hydrolysate, wherein the content of KH 2PO4 in the fermentation medium is 0.04w/v%, the content of MgSO 4·7H2 O is 0.32w/v%, the content of MnSO 4·H2 O is 0.028w/v%, the content of ammonium sulfate is 1.0w/v%, the content of corn steep liquor is 0.26w/v%, and the content of growth factor vitamin B 2 is 0.017w/v%.
Inoculating seed solution into fermentation medium, inoculating seed solution with 20% (v/v), and starting fermentation under pH 6.5 (controlled by 30% (v/v) ammonia water) and air volume of 1 vvm. In the whole fermentation process, the fermentation temperature is controlled to be 33 ℃ for 1-10h, 35 ℃ for 10-20h, 37 ℃ for 20-46h and 38 ℃ for 46-48 h.
When the glucose concentration in the fermentation liquor is reduced to below 5g/L, the concentrated solution of straw hydrolysate (with the glucose content of 300g/L and the preparation method as above) and the ammonium sulfate solution (with the ammonium sulfate content of 20 w/v%) are fed and fermented, and the glucose concentration in the fermentation liquor is maintained at the level of 5-10 g/L and the ammonium sulfate concentration is maintained at the level of 2-5 g/L through feeding. The volume of the fermentation liquor in the fermentation tank after material supplementing is maintained to be 1.1-1.5 times of the volume of the fermentation liquor before material supplementing by controlling the discharging speed. The fermentation period was 48 hours.
Example 6
1 Preparation of straw hydrolysate
Adding corn stalk material and sulfuric acid solution into a reaction container according to a solid-to-liquid ratio (g/g) of 2:1, wherein the H 2SO4 content in a mixed system of each 100kg corn stalk and sulfuric acid solution is 1100g, and introducing 172 ℃ saturated steam into the reaction container to heat the material in the reaction container for 5min. Then adjusting the pH value of the material to 5.0, adding the pretreated straw raw material (solid content of 30 wt%) into a saccharification tank, adding acid cellulase into the saccharification tank according to the proportion of 8U/g straw to carry out enzymolysis saccharification treatment, controlling the pH value to 5.0 by using ammonia water during the period of 50 ℃ to obtain straw hydrolysate. The content of glucose in the straw hydrolysate is 80g/L, the content of furfural is 0.8g/L, the content of hydroxymethylfurfural is 1.6g/L, and the content of acetic acid is 8g/L.
Concentrating the straw hydrolysate to obtain straw hydrolysate concentrate serving as a feed solution, wherein the glucose content in the straw hydrolysate concentrate is 300g/L.
Fermentation of 2L-lysine
Adding a fermentation medium into a fermentation tank filled with straw hydrolysate, wherein the content of KH 2PO4 in the fermentation medium is 0.02w/v%, the content of MgSO 4·7H2 O is 0.16w/v%, the content of MnSO 4·H2 O is 0.014w/v%, the content of ammonium sulfate is 1.0w/v%, the content of corn steep liquor is 0.26w/v%, and the content of growth factor vitamin B 2 is 0.008w/v%.
Inoculating seed solution into fermentation medium, inoculating seed solution with 20% (v/v), and starting fermentation under pH 6.5 (controlled by 30% (v/v) ammonia water) and air volume of 1 vvm. In the whole fermentation process, the fermentation temperature is controlled to be 34 ℃ for 1-8h, 35 ℃ for 8-16h, 36 ℃ for 16-34h, 37 ℃ for 34-46h and 38 ℃ for 46-48 h.
When the glucose concentration in the fermentation liquor is reduced to below 5g/L, the concentrated solution of straw hydrolysate (with the glucose content of 300g/L and the preparation method as above) and the ammonium sulfate solution (with the ammonium sulfate content of 20 w/v%) are fed and fermented, and the glucose concentration in the fermentation liquor is maintained at the level of 5-10 g/L and the ammonium sulfate concentration is maintained at the level of 2-5 g/L through feeding. The volume of the fermentation liquor in the fermentation tank after material supplementing is maintained to be 1.1-1.5 times of the volume of the fermentation liquor before material supplementing by controlling the discharging speed. The fermentation period was 48 hours.
Example 7
1 Preparation of straw hydrolysate
Adding corn stalk material and sulfuric acid solution into a reaction container according to a solid-to-liquid ratio (g/g) of 2:1, wherein the H 2SO4 content in a mixed system of each 100kg corn stalk and sulfuric acid solution is 1100g, and introducing 172 ℃ saturated steam into the reaction container to heat the material in the reaction container for 5min. Then adjusting the pH value of the material to 5.0, adding the pretreated straw raw material (solid content of 30 wt%) into a saccharification tank, adding acid cellulase into the saccharification tank according to the proportion of 8U/g straw to carry out enzymolysis saccharification treatment, controlling the pH value to 5.0 by using ammonia water during the period of 50 ℃ to obtain straw hydrolysate. The content of glucose in the straw hydrolysate is 80g/L, the content of furfural is 0.8g/L, the content of hydroxymethylfurfural is 1.6g/L, and the content of acetic acid is 8g/L.
Concentrating the straw hydrolysate to obtain straw hydrolysate concentrate serving as a feed solution, wherein the glucose content in the straw hydrolysate concentrate is 300g/L.
Fermentation of 2L-lysine
Adding a fermentation medium into a fermentation tank filled with straw hydrolysate, wherein the content of KH 2PO4 in the fermentation medium is 0.01w/v%, the content of MgSO 4·7H2 O is 0.08w/v%, the content of MnSO 4·H2 O is 0.007w/v%, the content of ammonium sulfate is 1.0w/v%, the content of corn steep liquor is 0.26w/v%, and the content of growth factor vitamin B 2 is 0.004w/v%.
Inoculating seed solution into fermentation medium, inoculating seed solution with 20% (v/v), and starting fermentation under pH 6.5 (controlled by 30% (v/v) ammonia water) and air volume of 1 vvm. In the whole fermentation process, the fermentation temperature is controlled to be 34 ℃ for 1-8h, 35 ℃ for 8-16h, 36 ℃ for 16-34h, 37 ℃ for 34-46h and 38 ℃ for 46-48 h.
When the glucose concentration in the fermentation liquor is reduced to below 5g/L, the concentrated solution of straw hydrolysate (with the glucose content of 300g/L and the preparation method as above) and the ammonium sulfate solution (with the ammonium sulfate content of 20 w/v%) are fed and fermented, and the glucose concentration in the fermentation liquor is maintained at the level of 5-10 g/L and the ammonium sulfate concentration is maintained at the level of 2-5 g/L through feeding. The volume of the fermentation liquor in the fermentation tank after material supplementing is maintained to be 1.1-1.5 times of the volume of the fermentation liquor before material supplementing by controlling the discharging speed. The fermentation period was 48 hours.
Example 8
1 Preparation of straw hydrolysate
Adding corn stalk material and sulfuric acid solution into a reaction container according to a solid-to-liquid ratio (g/g) of 2:1, wherein the H 2SO4 content in a mixed system of each 100kg corn stalk and sulfuric acid solution is 1100g, and introducing 172 ℃ saturated steam into the reaction container to heat the material in the reaction container for 5min. Then adjusting the pH value of the material to 5.0, adding the pretreated straw raw material (solid content of 30 wt%) into a saccharification tank, adding acid cellulase into the saccharification tank according to the proportion of 8U/g straw to carry out enzymolysis saccharification treatment, controlling the pH value to 5.0 by using ammonia water during the period of 50 ℃ to obtain straw hydrolysate. The content of glucose in the straw hydrolysate is 80g/L, the content of furfural is 0.8g/L, the content of hydroxymethylfurfural is 1.6g/L, and the content of acetic acid is 8g/L.
Concentrating the straw hydrolysate to obtain straw hydrolysate concentrate serving as a feed solution, wherein the glucose content in the straw hydrolysate concentrate is 300g/L.
Fermentation of 2L-lysine
Adding a fermentation medium into a fermentation tank filled with straw hydrolysate, wherein the content of ammonium sulfate in the fermentation medium is 1.0w/v%, and the content of corn steep liquor is 0.26w/v%.
Inoculating seed solution into fermentation medium, inoculating seed solution with 20% (v/v), and starting fermentation under pH 6.5 (controlled by 30% (v/v) ammonia water) and air volume of 1 vvm. In the whole fermentation process, the fermentation temperature is controlled to be 34 ℃ for 1-8h, 35 ℃ for 8-16h, 36 ℃ for 16-34h, 37 ℃ for 34-46h and 38 ℃ for 46-48 h.
When the glucose concentration in the fermentation liquor is reduced to below 5g/L, the concentrated solution of straw hydrolysate (with the glucose content of 300g/L and the preparation method as above) and the ammonium sulfate solution (with the ammonium sulfate content of 20 w/v%) are fed and fermented, and the glucose concentration in the fermentation liquor is maintained at the level of 5-10 g/L and the ammonium sulfate concentration is maintained at the level of 2-5 g/L through feeding. The volume of the fermentation liquor in the fermentation tank after material supplementing is maintained to be 1.1-1.5 times of the volume of the fermentation liquor before material supplementing by controlling the discharging speed. The fermentation period was 48 hours.
Comparative example 1
Fermentation of L-lysine
Glucose solution (glucose content in glucose solution is 80 g/L) is added into a fermentation tank, and a fermentation medium is added into the fermentation tank, wherein the content of KH 2PO4 in the fermentation medium is 0.04w/v%, the content of MgSO 4·7H2 O in the fermentation medium is 0.32w/v%, the content of MnSO 4·H2 O in the fermentation medium is 0.028w/v%, the content of ammonium sulfate in the fermentation medium is 1.0w/v%, the content of corn steep liquor is 0.26w/v%, and the content of growth factor vitamin B 2 is 0.017w/v%.
Inoculating seed solution into fermentation medium, inoculating seed solution with 20% (v/v), and starting fermentation under pH 6.5 (controlled by 30% (v/v) ammonia water) and air volume of 1 vvm. In the whole fermentation process, the fermentation temperature is controlled to be 34 ℃ for 1-8h, 35 ℃ for 8-16h, 36 ℃ for 16-34h, 37 ℃ for 34-46h and 38 ℃ for 46-48 h.
When the glucose concentration in the fermentation broth is reduced below 5g/L, feeding glucose solution (glucose content is 300 g/L) and ammonium sulfate solution (ammonium sulfate content in ammonium sulfate solution is 20 w/v%) to perform a fed-batch fermentation process, wherein the glucose concentration in the fermentation broth is maintained at a level of 5-10 g/L and the ammonium sulfate concentration is maintained at a level of 2-5 g/L through feeding. The volume of the fermentation liquor in the fermentation tank after material supplementing is maintained to be 1.1-1.5 times of the volume of the fermentation liquor before material supplementing by controlling the discharging speed. The fermentation period was 48 hours.
Comparative example 2
Fermentation of L-lysine
Glucose solution (glucose content in the glucose solution is 80 g/L) is added into a fermentation tank, and a fermentation medium is added into the fermentation tank, wherein the ammonium sulfate content in the fermentation medium is 1.0w/v%, and the corn steep liquor content is 0.26w/v%.
Inoculating seed solution into fermentation medium, inoculating seed solution with 20% (v/v), and starting fermentation under pH 6.5 (controlled by 30% (v/v) ammonia water) and air volume of 1 vvm. In the whole fermentation process, the fermentation temperature is controlled to be 34 ℃ for 1-8h, 35 ℃ for 8-16h, 36 ℃ for 16-34h, 37 ℃ for 34-46h and 38 ℃ for 46-48 h.
When the glucose concentration in the fermentation broth is reduced below 5g/L, feeding glucose broth (glucose content is 300 g/L) and ammonium sulfate solution (ammonium sulfate content is 20 w/v%) are started to perform a fed-batch fermentation process, the glucose concentration in the fermentation broth is maintained at a level of 5-10 g/L and the ammonium sulfate concentration is maintained at a level of 2-5 g/L through feeding. The volume of the fermentation liquor in the fermentation tank after material supplementing is maintained to be 1.1-1.5 times of the volume of the fermentation liquor before material supplementing by controlling the discharging speed. The fermentation period was 48 hours.
Comparative example 3
1 Preparation of straw hydrolysate
Adding corn stalk material and sulfuric acid solution into a reaction container according to a solid-to-liquid ratio (g/g) of 2:1, wherein the H 2SO4 content in a mixed system of each 100kg corn stalk and sulfuric acid solution is 1100g, and introducing 172 ℃ saturated steam into the reaction container to heat the material in the reaction container for 5min. Then adjusting the pH value of the material to 5.0, adding the pretreated straw raw material (solid content of 30 wt%) into a saccharification tank, adding acid cellulase into the saccharification tank according to the proportion of 8U/g straw to carry out enzymolysis saccharification treatment, controlling the pH value to 5.0 by using ammonia water during the period of 50 ℃ to obtain straw hydrolysate. The content of glucose in the straw hydrolysate is 80g/L, the content of furfural is 0.8g/L, the content of hydroxymethylfurfural is 1.6g/L, and the content of acetic acid is 8g/L.
Concentrating the straw hydrolysate to obtain straw hydrolysate concentrate serving as a feed solution, wherein the glucose content in the straw hydrolysate concentrate is 300g/L.
Fermentation of 2L-lysine
Adding a fermentation medium into a fermentation tank filled with straw hydrolysate, wherein the content of KH 2PO4 in the fermentation medium is 0.04w/v%, the content of MgSO 4·7H2 O is 0.32w/v%, the content of MnSO 4·H2 O is 0.028w/v%, the content of ammonium sulfate is 1.0w/v%, the content of corn steep liquor is 0.26w/v%, and the content of growth factor vitamin B 2 is 0.017w/v%.
Inoculating seed solution into fermentation medium, inoculating seed solution with inoculum size of 20% (v/v), and starting fermentation process at 37deg.C and pH of 6.5 (controlled by 30% (v/v) ammonia water) and air volume of 1 vvm.
When the glucose concentration in the fermentation liquor is reduced to below 5g/L, the concentrated solution of straw hydrolysate (with the glucose content of 300g/L and the preparation method as above) and the ammonium sulfate solution (with the ammonium sulfate content of 20 w/v%) are fed and fermented, and the glucose concentration in the fermentation liquor is maintained at the level of 5-10 g/L and the ammonium sulfate concentration is maintained at the level of 2-5 g/L through feeding. The volume of the fermentation liquor in the fermentation tank after material supplementing is maintained to be 1.1-1.5 times of the volume of the fermentation liquor before material supplementing by controlling the discharging speed. The fermentation period was 48 hours.
TABLE 1
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Claims (10)
1. A method for the fermentative production of L-lysine, characterized in that it comprises the following steps:
(1) Pretreating and hydrolyzing lignocellulose raw materials to obtain hydrolysate;
(2) And (3) fermenting and producing L-lysine by using a fermentation medium containing hydrolysate, wherein the glucose content in a fermentation system is maintained to be more than 1g/L by feeding in the fermentation process.
2. The method of claim 1, wherein in step (1), the pretreatment is an acid pretreatment comprising: adding lignocellulose raw material and sulfuric acid solution into a reaction container, and introducing saturated water vapor to heat reactants;
Preferably, the solid-to-liquid ratio of the lignocellulose raw material to the sulfuric acid solution is (1-8): 1; and/or the number of the groups of groups,
Preferably, the temperature of the saturated water vapor is 150-200 ℃, and more preferably 165-175 ℃; and/or the number of the groups of groups,
Preferably, the heating time is 1-10min, more preferably 2-5min.
3. The method according to claim 1, wherein in step (1) the lignocellulosic feedstock is subjected to a hydrolysis treatment with a cellulase comprising a neutral cellulase, an acid cellulase, preferably an acid cellulase; and/or the number of the groups of groups,
The dosage of the cellulase is 1-20U/g of lignocellulose raw material, and further 5-10U/g of lignocellulose raw material; and/or the number of the groups of groups,
The temperature of the hydrolysis treatment is 40-60 ℃, further 45-55 ℃, further 47-53 ℃; and/or the number of the groups of groups,
The pH of the hydrolysis treatment is 4.5-7.0, and further 4.5-5.5; and/or the number of the groups of groups,
The content of glucose in the hydrolysate in the step (1) is 60-120g/L, and further 100-120g/L.
4. The method according to claim 1, wherein the fermentation temperature is changed every 1 to 30 hours in the fermentation production of L-lysine in the step (2), and the fermentation temperature is overall increased;
Preferably, the fermentation temperature is controlled to be 32-38.5 ℃ and further 35-38 ℃ every 1-30 h;
preferably, the difference between the fermentation temperatures of the adjacent two time periods is controlled to be 0-2 ℃, and further 0.5-1 ℃.
5. The method according to claim 1, wherein the pH is controlled to be 6.2 to 7.5, further 6.5 to 7, and/or the ventilation is controlled to be 0.5 to 5vvm in the fermentation production of L-lysine in step (2).
6. The method of claim 1, wherein the fermentation medium of step (2) comprises 0.01% -0.2% KH 2PO4, 0.15% -0.45% MgSO 4·7H2 O, 0.01% -0.06% MnSO 4·H2 O, and 0.5% -2.5% ammonium sulfate; and/or the number of the groups of groups,
The fermentation medium in the step (2) contains a nitrogen source, wherein the nitrogen source comprises corn steep liquor and yeast extract; and/or the number of the groups of groups,
The fermentation medium in the step (2) contains 0.005% -0.5% of growth factors, wherein the growth factors comprise any one or more than two of vitamin B 2, vitamin D, glutamine, lysine, methionine, leucine and phenylalanine.
7. The method according to claim 1, wherein in step (2), seed liquid is inoculated into the fermentation medium before fermentation in an amount of 10% to 40%; and/or the number of the groups of groups,
Further, the concentration OD 562 of the seed liquid is 0.6-1.0 after 40 times dilution; and/or the number of the groups of groups,
Further, the seed solution contains a bacterial cell selected from the group consisting of Corynebacterium glutamicum, escherichia coli, brevibacterium, and Bacillus, preferably Corynebacterium glutamicum.
8. The method according to claim 1, wherein in step (2), feeding is started when the glucose concentration in the fermentation system falls below 5-20 g/L; and/or the number of the groups of groups,
The feed uses a solution containing glucose, preferably a hydrolysate concentrate of a lignocellulose raw material; preferably, the concentration of the glucose-containing solution is 60-800g/L, and further 200-400g/L; and/or the number of the groups of groups,
The feed is also supplemented with an ammonium sulfate solution, the concentration of which is 5% -40%, and further 10% -35%.
9. The process according to claim 1 or 8, wherein the glucose content in the fermentation system is maintained at 5-20g/L, further at 5-15g/L, by feeding during the fermentation in step (2); and/or the number of the groups of groups,
In the step (2), the volume of the fermentation liquor in the fermentation tank after feeding is controlled to be 0.7-3 times, further 0.7-2.0 times and further 0.7-1.5 times of the volume of the fermentation liquor before feeding.
10. The method according to claim 1, wherein in step (1), the solid content of the pretreated lignocellulosic feedstock is 5-45% (w/w), further 10-40% (w/w); and/or the number of the groups of groups,
The wood fiber raw material in the step (1) comprises one or more of corn stalk, wheat stalk, cotton stalk, sesame stalk, rape stalk, sweet sorghum stalk, corncob, rice hull, chaff, bagasse, straw, wood dust, hardwood and cork.
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