CN108424896B - Method for producing cellulase by mixed fermentation of corn straw furfural residues - Google Patents

Method for producing cellulase by mixed fermentation of corn straw furfural residues Download PDF

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CN108424896B
CN108424896B CN201710077685.3A CN201710077685A CN108424896B CN 108424896 B CN108424896 B CN 108424896B CN 201710077685 A CN201710077685 A CN 201710077685A CN 108424896 B CN108424896 B CN 108424896B
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cellulase
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trichoderma reesei
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CN108424896A (en
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张宗超
刘会芳
刘秀梅
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Dalian Institute of Chemical Physics of CAS
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Abstract

The invention provides a method for producing cellulase by mixed fermentation of corn straw furfural residues, which produces cellulase by mixed fermentation of trichoderma reesei CICC13052 and CICC40360 in an enzyme production culture medium taking the corn straw furfural residues as an induced carbon source. The method has the advantages of cheap and easily-obtained raw materials, overcomes the defect of low enzyme production of a single strain, improves the production efficiency, ensures that the two strains in the mixed strain belong to the trichoderma reesei, has the same growth conditions and no antagonistic action, and is beneficial to mixed culture. The method has simple and easy operation and low cost, and is expected to provide a simple and economic fermentation process for the industrialization of producing the cellulase by a microbial fermentation method.

Description

Method for producing cellulase by mixed fermentation of corn straw furfural residues
Technical Field
The invention belongs to the field of biological energy utilization, and particularly relates to a method for producing cellulase by fermenting corn straw furfural residues through mixed bacteria.
Background
The cellulase has good application prospect in the aspects of textile industry, feed industry, brewing, food processing, new energy development and the like. In recent years, with the increasing severity of energy problems, the proposition of producing novel energy such as fiber fuel ethanol by using cellulase to degrade straw raw materials is more on schedule, but the wide application of the novel energy is hindered due to the higher price. Therefore, the production of the high-efficiency and low-price cellulase has epoch-making significance for finally solving the problem of energy shortage faced by human beings.
The cellulase has wide sources, and can produce cellulase in bacteria, actinomycetes, fungi, animal bodies and the like. The cellulase synthesized by different microorganisms has obvious difference in composition and greatly different cellulose degrading capability. The cellulase yield of the actinomycetes is extremely low, and the research is very little. The bacterial yield is also not high, and is mainly endoglucanase, and in addition, the produced enzyme is intracellular enzyme or adsorbed on cell walls, which increases the purification difficulty and is rarely used industrially. Filamentous fungi have many advantages for enzyme production: the produced cellulase is extracellular enzyme, and is convenient to extract; high enzyme production efficiency, reasonable enzyme system and the like. At present, most microbial strains for producing cellulase are filamentous fungi, which typically comprise Trichoderma (Trichoderma), Aspergillus (Aspergillus) and Penicillium (Penicillium), wherein Trichoderma reesei is the cellulase producing strain with the most industrial application value due to the characteristics of high enzyme production activity, complete enzyme system, extensive growth environment, easy enzyme extraction, safe and nontoxic strains and the like.
Cellulases are multicomponent complex enzymes, typically comprising endo-beta-glucanases (Cx enzymes), exo-beta-glucanases (C1 enzymes) and beta-glucosidases (CB enzymes). When hydrolyzing natural cellulose into glucose, the synergistic effect of the above 3 components must be relied on to complete: exonucleases (cellobiohydrolases) can hydrolyze crystalline regions of cellulose, (CBH I) sustained hydrolysis starting from the reducing end or (CBH II) non-reducing end of the cellulose chain, releasing cellobiose; the endonuclease mainly acts on an amorphous area of cellulose, randomly hydrolyzes glycosidic bonds in a cellulose chain, cuts off a long cellulose chain, and converts the long cellulose chain into a large number of short cellulose chains with different polymerization degrees, so that the polymerization degree of cellulose molecules is reduced, and the number of tail ends of the cellulose chain for the action of the exonuclease is increased; beta-glucosidase then primarily hydrolyzes cellobiose and soluble cellooligosaccharides, ultimately converting cellulose to available glucose.
The single strain fermentation has the defects of low enzyme activity and incomplete enzyme system components, which can seriously affect the enzymolysis efficiency, improve the cellulase activity and improve the enzyme system composition, and the research is very active at present, and a plurality of methods are proposed: constructing a multigene strain through genetic engineering and expressing a plurality of enzyme components at the same time, but the cost is high and the time consumption is long; in comparison, mixed fermentation is the simplest and most effective method. In the process of mixed fermentation, the mixed bacteria are mutually beneficial and inhabit, so that the proportion of enzyme systems is coordinated, feedback inhibition caused by the mass accumulation of a certain intermediate product in the fermentation process is avoided, the enzyme production capacity of the mixed fermentation is greatly higher than that of a single bacterial strain, and the yield is improved. CN 101100660A proposes a method for producing cellulase by mixed fermentation of trichoderma koningii and rhizopus oryzae, but the growth conditions of the two strains are different, the rhizopus oryzae has higher growth speed than that of the trichoderma koningii, the advantage growth of the rhizopus oryzae in the mixed culture process is easily caused, the enzyme activity of filter paper is reduced on the contrary under the condition, the culture mode and the conditions need to be strictly controlled, the two strains need to be inoculated stage by stage, the operation is complex, and the problems that the nutrition competition cannot be avoided when the two strains exist at the same time in the later stage of fermentation and the like are solved; CN 102154243A utilizes the mixed fermentation of trichoderma viride and aspergillus niger to improve the activity of beta-glucosidase, but has the same problem; the homostella and the like are fermented by mixing trichoderma reesei and aspergillus niger, and besides a certain antagonistic action exists due to different growth conditions, the addition of the aspergillus niger changes the pH value of a fermentation system, so that the trichoderma reesei is not beneficial to producing cellulase, and the enzyme activity of filter paper obtained by mixed fermentation is lower than that of single trichoderma reesei (food science, 2012,33(19): 193-198). Aiming at the defects, the invention provides a novel mixed fermentation method, two strains of trichoderma reesei are mixed and fermented for enzyme production, the growth conditions of the two strains are the same, and the operation is simple and convenient.
In the furfural production process, a large amount of furfural waste residues are generated, more than 10 tons of residues are discharged from each ton of furfural products, and about thousands of tons of furfural waste residues are discharged every year in China, so that great pressure is brought to the environment and enterprises. The furfural residues contain a large amount of cellulose with high added value, and if the furfural residues are subjected to enzymolysis saccharification treatment, the saccharification can change waste into valuable, so that great economic benefit is generated. The production of the furfural mostly adopts a weak acid hydrolysis method, so that the original cellulose of the raw material and the complex network structures among the hemicellulose and the lignin are damaged to a certain degree while most of the hemicellulose is separated, the complex pretreatment process of the plant fiber raw material can be omitted, and favorable conditions are provided for utilizing furfural residue cellulose for biotransformation. The invention selects furfural residue with rich resources and special structure as the cellulose raw material to produce cellulase by fermentation, and has double benefits of economy and environmental protection.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a method for producing cellulase by fermenting corn straw furfural residues by using mixed bacteria.
The production strains are Trichoderma reesei (Trichoderma reesei) CICC13052 and CICC40360, which are purchased from China Center of Industrial Culture Collection (CICC).
The purpose of the invention is achieved by the following technical scheme:
a method for producing cellulase by fermenting corn straw furfural residues with mixed bacteria is characterized in that Trichoderma reesei (Trichoderma reesei) CICC13052 and CICC40360 are subjected to mixed fermentation in an enzyme production culture medium taking the furfural residues as an induction carbon source to produce cellulase.
The method for producing the cellulase by fermenting the furfural residues by using mixed bacteria preferably adopts the technical scheme that the method comprises the following steps:
(1) the purchased freeze-dried powder of Trichoderma reesei (Trichoderma reesei) CICC13052 and CICC40360 was activated with slant activation medium: the slant tube coated with the above strain is subjected to static culture at 28 deg.C for 5-7 days until spores grow, and then subculture activation is carried out by the same method (since the strain is in dormant state after freeze-drying preservation, the first generation strain needs to be cultured for a proper time and transferred to 2-3 generations to recover activity).
(2) Respectively adding appropriate amount of sterile physiological saline into activated Trichoderma reesei (Trichoderma reesei) CICC13052 and CICC40360 slant, mixing for 2min, and preparing into 1 × 106-7Spore suspension per ml. Inoculating the spore suspension into a fresh seed culture medium in an inoculation amount of 5%, and performing shake culture at 28 ℃ and 180rpm for 24-48h to respectively obtain seed culture solutions of the two bacteria;
(3) inoculating the mixed seed culture solution of the two bacteria into an enzyme production culture medium according to the volume ratio of 1-10%, and fermenting under the conditions of 28 ℃ and 180rpm to produce the enzyme.
The inoculation ratio of the CICC40360 to the CICC13052 in the mixed seed culture solution is 1-10: 10-1.
Further, the preferred ratio is 1: 3.
Furthermore, the initial concentration of the furfural residue obtained by mixed fermentation is 20-200 g/L, and preferably 100 g/L.
The culture medium adopted in the method for producing the cellulase by fermenting the furfural residues by using the mixed bacteria is as follows:
activating a culture medium: 1.0L of potato extract, 20.0g of glucose and 15.0g of agar, and the pH is natural.
Seed culture medium: 10g of microcrystalline cellulose, 10g of glucose, 0.3g of urea and KH2PO4 2.0g，(NH4)2SO41.4g，MgSO4-7H20.3g of O, 0.75g of peptone, 0.25g of yeast powder and CaCl2-2H2O0.4g, water 1L, trace elements CoCl20.002g，ZnSO4-7H2O 0.0014g，MnS04-7H2O 0.0016g，FeS04-7H2O 0.005g。
Enzyme production culture medium: 20-200g of corn straw furfural residue, 0.3g of urea and KH2PO4 2.0g，(NH4)2SO41.4g，MgSO4-7H20.3g of O, 0.75g of peptone, 0.25g of yeast powder and CaCl2-2H20.4g of O, 1L of water, and the trace element CoCl20.002g，ZnSO4-7H2O 0.0014g，MnS04-7H2O 0.0016g，FeS04-7H2O 0.005g。
Compared with the prior art, the invention has the following beneficial effects:
(1) the industrial furfural waste residue has large resource amount, low price, rich cellulose, low degree of polymerization of the cellulose and loose structure, and is a preferred raw material for preparing cellulose for bioconversion. The invention takes the furfural residues of the corn straws as the raw material, thereby not only saving the cost, but also solving the environmental problem, and having no environmental pollution while developing and utilizing natural cellulose resources such as the straws, and the like, and being an effective means for promoting the industrialization of the straw ethanol.
(2) The mixed fermentation has higher cellulose utilization rate and enzyme production efficiency than single strain fermentation.
(3) The concentration of the substrate tolerant to mixed fermentation is high, and the mixed fermentation can tolerate 200g/L of corn straw furfural residues.
(4) The single bacteria contained in the mixed bacteria are all aerobic fungi-trichoderma reesei, and the growth conditions are the same, so that the co-culture is convenient.
The method solves the problem of furfural residue utilization, improves the production efficiency of the cellulase, has low cost, simple process and good economic and social benefits, is environment-friendly, is suitable for industrial application, and has good industrial application prospect.
Drawings
FIG. 1 shows the situation of enzyme production by furfural residue fermentation with mixed bacteria;
FIG. 2 shows the condition of producing enzyme by fermentation of furfural residues in Trichoderma reesei CICC 40360;
FIG. 3 shows the condition of producing enzyme by fermentation of Trichoderma reesei CICC13052 by using furfural residues;
FIG. 4 effect of substrate concentration on enzyme production in mixed fermentation;
FIG. 5 influence of different initial pH values on the enzyme production in mixed fermentation;
FIG. 6 shows the effect of different inoculum sizes on the enzyme production in mixed fermentation;
FIG. 7 shows the effect of different inoculation ratios on the enzyme production in mixed fermentation;
FIG. 8 effect of different co-inducers on enzyme production in mixed fermentation.
Detailed Description
The following non-limiting examples are presented to enable those of ordinary skill in the art to more fully understand the present invention and are not intended to limit the invention in any way. In the following examples, unless otherwise specified, the experimental methods used were all conventional methods, and the reagents used were all available from chemical or biological reagents companies.
1. And (3) microbial culture and fermentation:
activating strains: activating the purchased strain by using a slant activation medium, performing static culture on a slant test tube coated with the bacterial liquid at 28 ℃ for 5-7 days until spores grow out, and performing subculture for 2-3 generations by using the same method to obtain the activated strain.
Seed culture: sealing a 100mL triangular flask filled with 20mL seed culture medium by using a cotton plug and a bottle opening membrane, sterilizing at 115 ℃ for 20 minutes, cooling, inoculating the spore suspension of the slant microorganism, and culturing in a shaker at 28 ℃ and 180rpm for 24-48 hours to obtain a seed culture solution.
And (3) fermenting to produce enzyme: transferring the cultured seed culture solution into a 100mL shaking bottle containing 20mL of enzyme-producing medium at an inoculum size of 1-10%, culturing in a shaking table at 28 ℃ and 180rpm for 3-11d, and sampling at regular time to determine enzyme activity.
2. The analysis method comprises the following steps: the enzyme activity of the fermentation liquor is determined by adopting an international standard method recommended by the International Union of theory and applied chemistry (IUPAC).
Example 1
Mixed-strain fermentation for producing cellulase
The slant preserved trichoderma reesei CICC13052 and CICC40360 are respectively activated, and the spore suspension is respectively transferred to a seed culture solution by 5 percent of inoculation amount to shake culture on a shaking table at 28 ℃ and 180rpm for 24-48 h. Then transferring the mixture into an enzyme production culture medium containing 50g/L furfural residue according to the inoculation ratio of 1:1 and the total inoculation amount of 5%, and sampling at regular time to determine the enzyme activity. The result is shown in figure 1, when the mixed strain takes 50g/L corn straw furfural residue as an induced carbon source, the cellulase activity of the mixed strain can reach 0.82 FPU/mL.
Comparative example 1
Single-bacterium CICC40360 fermentation cellulase
The Trichoderma reesei CICC40360 preserved on the activated slant is transferred from the spore suspension into a seed culture solution by 5 percent of inoculation amount and is subjected to shake culture on a shaking table at 28 ℃ and 180rpm for 24-48 h. And then transferring the strain with the inoculation amount of 5% to an enzyme production culture medium containing 50g/L of furfural residues, and sampling at regular time to determine the enzyme activity, wherein the result is shown in figure 2, when 50g/L of corn straw furfural residues are used as an induced carbon source, the cellulase enzyme activity of the Trichoderma reesei CICC40360 is only 0.47FPU/mL and is 42.68% lower than that of mixed fermentation.
Comparative example 2
Single-bacterium CICC13052 fermentation production cellulase
According to the method of the comparative example 1, the Trichoderma reesei CICC13052 is inoculated to an enzyme production culture medium containing 50g/L furfural residue for fermentation, and the enzyme activity is measured by sampling at regular time. The result is shown in FIG. 3, and it can be seen from the figure that when 50g/L corn straw furfural residue is used as an induced carbon source, the cellulase activity of the CICC13052 can reach 0.67FPU/mL, which is higher than that of CICC40360, and is 18.29% lower than that of mixed fermentation.
Example 2
Influence of different substrate concentrations on enzyme production by mixed fermentation
According to the method described in the embodiment 1, the mixed bacteria are transferred into enzyme production culture media with different furfural residue concentrations according to the inoculation ratio of 1:1 and the total inoculation amount of 5%, the enzyme activity is measured by sampling at regular time, and the optimal carbon source content and the enzyme production condition are explored. As shown in FIG. 4, it can be seen that the mixed bacteria have better substrate tolerance, can ferment and produce enzyme even at a furfural residue concentration of 200g/L, and have a cellulase activity of 0.87FPU/mL at maximum when 100g/L of furfural residue is used as an induced carbon source, so that the substrate concentration in subsequent tests is preferably 100 g/L.
Example 3
Influence of different initial pH values on enzyme production in mixed fermentation
The pH of the culture medium is closely related to the growth and the propagation of cells and the enzyme production by fermentation, and the pH of the culture medium is often changed along with the growth and the propagation of the cells and the accumulation of metabolic products in the fermentation process, so that the experiment only controls the initial pH.
According to the method described in the embodiment 2, the mixed bacteria are transferred into an enzyme production culture medium containing 100g/L furfural residue and having initial pH of 2.80, 4.32, 4.80, 6.00, 7.00 and 8.00 according to the inoculation ratio of 1:1 and the total inoculation amount of 5% for fermentation, the enzyme activity is measured by sampling at regular time, the influence of the initial pH on the enzyme production of the mixed bacteria fermentation is observed, and the optimal initial pH and the enzyme production condition are searched. As shown in fig. 5, the difference between the cases of producing cellulase by fermentation of mixed trichoderma reesei under different initial pH conditions is large, the cellulase is formed under a meta-acid environment, but the formation of cellulase is not favored by too low pH (pH 2.8), while the formation of cellulase is also disadvantageous under neutral meta-base conditions (pH 7, 8), and the production of cellulase is affected by too low pH or too high pH. According to experimental data, the pH value of the initial pH formed by the cellulase is preferably 4.8, the enzyme production time is shortened compared with that before optimization, the enzyme production peak can reach 0.91FPU/mL at 7d, the single-bacterium fermentation is 3 days earlier than that of CICC13052, the single-bacterium fermentation is 2 days earlier than that of CICC40360, and the enzyme activity is improved.
Example 4
Influence of different inoculation amounts on enzyme production by mixed fermentation
According to the method described in example 3, the mixture is transferred from the seed liquid into an enzyme production medium with the initial pH of 4.8 for enzyme production by fermentation according to the inoculation ratio of 1:1 and the total inoculation amount of 1%, 2.5%, 5%, 8% and 10%, and the enzyme activity is measured by sampling at regular time and the optimal inoculation amount is searched.
The influence of different inoculum sizes on the enzyme production by mixed fermentation is shown in FIG. 6, and the difference of the conditions of producing cellulase by mixed fermentation under different inoculum sizes is large. The inoculation amount is too small, the thallus concentration is insufficient, and the production of cellulase is hindered; the inoculation amount is too large, the cell concentration is too high, the dissolved oxygen is insufficient, the substrate nutrition is deficient, and the formation of cellulase is also inhibited, and the inoculation amount of 5% is preferable from experimental data.
Example 5
Influence of different inoculation ratios on enzyme production by mixed fermentation
The inoculation ratio is an important parameter in mixed fermentation. According to the method described in example 4, the total inoculation amount is 5%, and the inoculation ratios are respectively CICC40360: the method of CICC13052 is 1:1, 1:2, 2:1, 1:3, 3:1, 1:4, 4:1, 1:5 and 5:1, inoculating the seed liquid into an enzyme-producing culture medium with the initial pH of 4.8 for fermentation, sampling at regular time to measure the enzyme activity, and searching the optimal inoculation ratio. The influence of different inoculation ratios on the enzyme production of mixed fermentation is shown in FIG. 7, the enzyme activity produced by the mixed fermentation is lower along with the increase of the proportion of the CICC40360, and the enzyme activity is increased along with the increase of the proportion of the CICC 13052. Wherein, the enzyme yield of the CICC40360: CICC 13052: 1:3 is high (0.98FPU/mL), the speed is high, the enzyme activity is stable, and the industrial production is facilitated. Therefore, the preferred inoculation ratio is CICC40360: CICC 13052-1: 3.
Example 6
Influence of different auxiliary inducers on cellulase production by mixed fermentation
The cellulase is a composite induction enzyme, so an experiment that different auxiliary inducers are added on the basis of taking furfural residue as an induction carbon source is designed.
According to the method described in the example 5, the seed liquids of the cultured CICC40360 and CICC13052 are respectively inoculated into enzyme-producing culture media containing different inducers and having the initial pH of 4.8 according to the inoculation proportion of CICC40360: CICC 13052: 1:3 and the inoculation amount of 5% to carry out fermentation enzyme-producing culture, sampling is carried out at regular intervals to detect the enzyme activity of filter paper, and the influence of different inducers on the fermentation enzyme production of the strains is observed.
The CK group was a control in which the carbon source to which the auxiliary inducer was not added was 100g/L furfural residue, and the test groups were prepared by adding 0.5% of glucose, lactose, xylose, cellobiose, microcrystalline cellulose (MCC), and sodium carboxymethylcellulose (CMC) to the CK group.
The test result is shown in fig. 8, compared with CK, each auxiliary inducer does not play a positive inducing role, which shows that the furfural residue of the corn straw can independently induce mixed bacteria to produce cellulase without adding other auxiliary inducers, thereby saving the cost of producing the cellulase and being beneficial to industrial production.

Claims (4)

1. A method for producing cellulase by mixed fermentation of corn straw furfural residues is characterized in that two strains of Trichoderma reesei (Trichoderma reesei) (Trichoderma reesei)) (Trichoderma reesei ) Mixing and fermenting in an enzyme production culture medium which takes corn straw furfural residues as an induced carbon source to produce cellulase; the trichoderma reesei is CICC13052 and CICC 40360.
2. The method for producing the cellulase by fermenting the corn straw furfural residues through the mixed strain according to claim 1, wherein the inoculation ratio of the trichoderma reesei CICC13052 to the Trichoderma reesei CICC40360 is 1-10: 10-1.
3. The method for producing the cellulase by mixed fermentation of the corn straw furfural residues as claimed in claim 1, wherein the induced carbon source of the enzyme production medium is the corn straw furfural residues; the mixed bacteria has high substrate tolerant concentration and can tolerate 200g/L corn straw furfural residue.
4. The method for producing the cellulase by mixed fermentation of the corn straw furfural residues according to claim 1, wherein the initial pH of the mixed fermentation is 2-8; the inoculation amount is 1-10%.
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