WO2020034821A1 - 用于木质纤维素的全菌糖化方法 - Google Patents

用于木质纤维素的全菌糖化方法 Download PDF

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WO2020034821A1
WO2020034821A1 PCT/CN2019/097813 CN2019097813W WO2020034821A1 WO 2020034821 A1 WO2020034821 A1 WO 2020034821A1 CN 2019097813 W CN2019097813 W CN 2019097813W WO 2020034821 A1 WO2020034821 A1 WO 2020034821A1
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saccharification
lignocellulose
glucose
bacterial
seed
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French (fr)
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崔球
刘亚君
祁宽
刘世岳
李仁民
冯银刚
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中国科学院青岛生物能源与过程研究所
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/02—Monosaccharides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P2201/00—Pretreatment of cellulosic or lignocellulosic material for subsequent enzymatic treatment or hydrolysis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P2203/00—Fermentation products obtained from optionally pretreated or hydrolyzed cellulosic or lignocellulosic material as the carbon source

Definitions

  • the invention belongs to the field of biotechnology, and relates to a method for biotransformation of lignocellulose, in particular to a method for bacterial saccharification of lignocellulose.
  • Lignocellulosic biomass is the most abundant renewable biomass resource on the earth. It has great application and development prospects to convert it into energy, chemicals or materials. In particular, China is a large agricultural country, with annual crop straw output exceeding 900 million tons. On the other hand, environmental pollution and traffic safety accidents caused by straw burning have also prompted the country to launch a series of plans, programs and measures for comprehensive utilization of straw. Therefore, promoting the industrialized development of comprehensive utilization of agricultural waste biomass is of great significance to the establishment of China's green circular economy and the sustainable development of society.
  • the "one-pot” integrated bacteria-catalyzed biological saccharification strategy integrated with bioprocessing technology has the advantages of simplifying processes and reducing equipment requirements, and is the most suitable for the conversion and utilization of cellulose-based biomass. Routing.
  • the core of the "one-pot method” is the use of an efficient whole-bacterial catalyst, a microorganism that has the ability to degrade lignocellulosic substrates and can efficiently hydrolyze cellulose substrates into fermentable sugars.
  • the catalytic saccharification of lignocellulosic whole bacteria mainly uses high-temperature anaerobic bacteria based on Clostridium thermocellum, a fiber-producing body, as a whole-bacterial catalyst. Fibrosomes are extracellular multi-enzyme complexes with complex structures and components, and are one of the most efficient cellulose degradation systems known in nature.
  • Catalytic saccharification of whole cellulosic lignocellulose includes a two-step semi-continuous production mode of whole cell catalyst pre-culture (seed culture stage) and whole cell saccharification.
  • Glucose is a commonly used carbon source for microbial culture in the fermentation industry. It is relatively inexpensive and is a product of cellulose saccharification. However, at present, glucose cannot be used for the pre-culture of whole bacteria catalysts. This is because when C. thermocellum is cultured with glucose as the sole carbon source, not only will there be a growth delay of tens of hours, but the production of fibrosomes will be significantly reduced, which will lead to a long production cycle. In addition, The cellulose degradation activity of the obtained whole bacteria catalyst was low. Yoav et al.
  • the present invention provides a bacterial saccharification method for lignocellulose, which not only greatly shortens the seed culture stage.
  • the cycle reduces the cost of seed liquid and shortens the cycle of whole saccharification, thus laying a foundation for the industrialization process.
  • a bacterial saccharification method for lignocellulose including the following steps:
  • the glucose medium contains glucose at a concentration of 1-20 g / L; the passage and acclimatization are performed 2-5 times, and each passage is inoculated to 0.2-2% (volume fraction) after culturing the fibrosomal strain.
  • the added amount of the lignocellulose raw material is 0.1-2.0 g / L; the temperature condition for inducing domestication is 34-65 ° C; and the time for inducing domestication is 12-48 hours.
  • the pretreated lignocellulose raw material that is, the pretreated substrate, is added to the glucose medium in order to induce matching fibrosomal synthesis.
  • the specific activity of the fiber bodies of the seed liquid obtained by the present invention is consistent with the fiber bodies produced from cellulose as a carbon source, and is higher than that produced from cellobiose as a carbon source.
  • the fiber bodies produced for the carbon source are consistent, and the seed growth rate is consistent with the seeds grown with cellobiose as the carbon source, higher than the seeds grown with cellulose as the carbon source, and much faster than the pretreated lignocellulose as the carbon source. Seed.
  • Pre-impregnation of raw materials According to the solid-liquid weight-volume ratio of 1: 3-1: 50, the pretreated lignocellulose raw material and the culture medium solution are mixed uniformly in a container at a speed of 0.5-100 r / min. And last for 1-48h; the pretreated lignocellulose raw material is the same as in step (1), the purpose is to achieve sufficient mixing of the substrate and the culture medium before the saccharification starts, which is beneficial to the growth and vitality of the whole bacteria catalyst To achieve faster saccharification.
  • the container is an anaerobic fermentation device with a stirring paddle or an anaerobic rotary mixer; the anaerobic fermentation device with a stirring paddle achieves uniform mixing by stirring, and the anaerobic rotary mixer passes through the entirety of the container Roll and flip for even blending.
  • step (3) Whole saccharification: the optimized whole saccharified seed solution of step (1) is inoculated into the pre-impregnated reaction system obtained in step (2), and the saccharification reaction is performed at a speed of 0.5-100 r / min A sugar solution containing glucose was obtained.
  • the inoculum amount of the whole bacteria saccharified seed liquid is 0.1-10% volume fraction.
  • the pH can be controlled to 5.8-6.2 by adding sodium hydroxide.
  • the fibrosomal producing strain is Clostridium thermocellum, Clostridium yellow fibrinolyticus, Clostridium fibrophilus, Clostridium fibrinolyticus, Vibrio fibrolyticus, Pseudomonas fibrinolyticus, Rumenococcus albus, or yellow Rumen cocci.
  • the pretreatment is a combination of one or more of the pretreatment technologies of alkali method, acid method, hydrothermal method, steam explosion method and sulfonation method.
  • the lignocellulose raw material is a combination of one or more of corn straw, wheat straw, shrub branches, wood chips, corn cob, straw, and waste paper.
  • the glucose medium described in step (1) is 2.9 g / L of dipotassium hydrogen phosphate, 1.5 g / L of potassium dihydrogen phosphate, 0.8 g / L of urea, 0.1 g / L of calcium chloride, and magnesium chloride per liter of water.
  • the bacterial saccharification method for lignocellulose according to the present invention which uses relatively inexpensive glucose and adds a low concentration of pretreated lignocellulosic biomass as a carbon source, is used for cultivating small fibers such as Clostridium thermocellum Compared with the prior art, the whole-saccharified saccharified seed liquid achieves the effects of cost reduction and cycle shortening, and overcomes the problems that cannot be obtained in the prior art.
  • the method for bacterial saccharification of lignocellulose according to the present invention adopts a technique of pre-impregnation of raw materials, which significantly shortens the adaptation buffer period of bacterial saccharification and improves the efficiency of bacterial saccharification, thereby reducing the cycle and cost of the entire saccharification process. .
  • a bacterial saccharification method for lignocellulose including the following steps:
  • the Clostridium thermocellum was subcultured 3 times in a medium containing 5 g / L of glucose as the sole carbon source, that is, a glucose medium, according to an inoculation ratio of 1% by volume; Adding 0.1g / L dry weight of pretreated corn stalk to glucose medium, induced domestication at 55 ° C for 48h, so as to obtain whole bacteria with high growth rate, high fiber body production and high fiber body specific activity Saccharified seed fluid.
  • the glucose culture medium also has dipotassium hydrogen phosphate 2.9g / L, potassium dihydrogen phosphate 1.5g / L, urea 0.8g / L, and calcium chloride 0.1g / L per liter of water. , 1.8g / L of magnesium chloride, 0.0005g / L of ferrous sulfate, 2g / L of sodium sulfide, 4g / L of corn slurry, 2g / L of trisodium citrate, pH 6.5-7.5.
  • the pretreated lignocellulose raw material that is, the pretreated substrate, is added to the glucose medium in order to induce matching fibrosomal synthesis.
  • the pretreatment method is a sulfonation method pretreatment used in the literature (China Paper, 2015, 34, 1-6).
  • the fibrosomal specific activity, yield and cell biomass of the seed liquid are detected.
  • the detection of fiber body production is: extracting the fiber bodies from the seed liquid by cellulose adsorption method and quantifying the protein by the Bradford method to obtain the fiber body production (the fiber bodies per unit volume of the seed liquid) Milligrams).
  • the specific activity of fiber bodies was measured by using the pretreated corn stalk as a substrate to detect the hydrolysis activity of the extracted fiber bodies. The hydrolysis experiment was continued at 55 ° C for 24 hours, and then the concentration of reducing sugars released by hydrolysis was measured by DNS method.
  • fibrosomal specific activity U / mg based on reducing sugar and fibrosomal production, where the enzyme activity unit (U) is defined as the amount of protein required to hydrolyze cellulose substrate per hour to produce 1mg of reducing sugar under measurement conditions. (mg).
  • U enzyme activity unit
  • the detection of cell biomass is as follows: the precipitate after centrifuging the seed solution is hydrolyzed with sodium hydroxide, and then the Bradford method is used to detect the concentration of cellular protein in the precipitate (the number of milligrams of precipitated protein per unit volume of seed solution). The growth curve and the concentration of precipitated protein were analyzed to analyze the time (hours) required to grow to the middle logarithm, and the growth rate was judged.
  • Pre-impregnation of raw materials According to the solid-liquid weight-volume ratio of 1: 6.5, 1 kg of the pretreated lignocellulosic raw material and 6.5 L of the culture medium solution are mixed uniformly at a speed of 50 r / min for 4 h;
  • the pretreated lignocellulose raw material is the same as in step (1), the purpose is to achieve sufficient mixing of the substrate and the culture medium before saccharification starts, which is beneficial to the growth and vitality of the whole bacteria catalyst, thereby achieving a faster Saccharification.
  • the uniform mixing is to achieve uniform mixing by stirring by a stirring device in a container.
  • Whole bacteria saccharification Inoculate the optimized whole bacteria saccharified seed solution in step (1) according to the inoculation amount of 10% by volume into the pre-impregnated reaction system obtained in step (2), and continue at 50 r / min.
  • a saccharification reaction is performed under a rotation speed condition to obtain a sugar liquid containing glucose.
  • the content of reducing sugar in the fermentation broth was sampled every 12 hours, and the conversion rate of cellulose to soluble sugar in the substrate was calculated.
  • the time when the conversion rate is lower than 10% is defined as the lag period (days) of hydrolysis of the lignocellulose substrate.
  • the time until the conversion reached 80% (mass ratio) was defined as the hydrolysis cycle (days) of the lignocellulose substrate.
  • the method for bacterial saccharification of lignocellulose without the steps of domestication and pre-impregnation, includes the following steps:
  • Seed liquid culture Clostridium thermocellum is cultured in a medium (components other than glucose in glucose medium) of glucose pretreated corn stover as a carbon source for 5 hours. After the end of step (1), the method of Example 1 was used to detect the specific activity of fibrosomes, yield and cell biomass of the seed solution.
  • the method for bacterial saccharification of lignocellulose includes the following steps:
  • C. thermocellum was subcultured 3 times in a medium with 10 g / L of glucose as the sole carbon source, that is, a glucose medium, according to an inoculation ratio of 1% by volume; 0.5g / L dry weight of pretreated wheat straw was added to the glucose medium and induced domestication at a temperature of 58 ° C for 36h, so as to obtain whole bacteria with high growth rate, high fiber body production, and high fiber body specific activity. Saccharified seed fluid.
  • the pretreatment method is a combined hydrothermal and sulfonation pretreatment method used in CN201610133959.
  • step (1) The optimized whole saccharified seed solution of step (1) is inoculated into the pre-soaked reaction system obtained in step (2) at an inoculation amount of 10% by volume, and continued at 25 r / min.
  • a saccharification reaction is performed under a rotation speed condition to obtain a sugar liquid containing glucose.
  • the content of reducing sugar in the fermentation broth was sampled every 12 hours, and the conversion rate of cellulose to soluble sugar in the substrate was calculated.
  • the method for bacterial saccharification of lignocellulose includes the following steps:
  • C. thermocellum was subcultured twice in a medium with 20 g / L of glucose as the sole carbon source, that is, a glucose medium, according to an inoculation ratio of 1% by volume; Add 2g / L dry weight of pretreated shrub branches to glucose medium and induce domestication at 60 °C for 24h, in order to obtain high growth rate, high fiber body production, and high fiber body specific vigor glycosylation Seed liquid.
  • the pretreatment method is the alkali method in the literature (Bin Li, et al. Recent progress on the pretreatment and fragmentation of Bioignorery at Biosciences, Journal of Bioresources, 2017, 2 (1), 4-9) Pretreatment technology. After the end of step (1), the method of Example 1 was used to detect the specific activity of fibrosomes, yield and cell biomass of the seed solution.
  • Pre-impregnation of raw materials According to the solid-liquid weight-volume ratio of 1: 8, 1 kg of the pretreated lignocellulose raw material and 8 L of the culture medium solution are mixed uniformly at a speed of 45 r / min for 28 h; The pretreated lignocellulose raw material is the same as in step (1).
  • Whole saccharification The optimized whole saccharified seed solution in step (1) is inoculated into the pre-impregnated reaction system obtained in step (2) at an inoculation amount of 6% by volume, and continued at 45 r / min. A saccharification reaction is performed under a rotation speed condition to obtain a sugar liquid containing glucose. During this process, samples of reducing sugar in the fermentation broth were sampled every 12 hours.
  • the method for bacterial saccharification of lignocellulose without the steps of domestication and pre-impregnation, includes the following steps:
  • Seed liquid culture Clostridium thermocellum was cultured in a medium (a component of glucose medium other than glucose) containing 20 g / L microcrystalline cellulose as a carbon source for 24 hours. After the end of step (1), the method of Example 1 was used to detect the specific activity of fibrosomes, yield and cell biomass of the seed solution.
  • the method for bacterial saccharification of lignocellulose includes the following steps:
  • the pre-treatment method is a pre-treatment technology combining the alkali method and hydrothermal in the literature (Biotechnology for Biofuels, 2014, 7: 116).
  • Pre-impregnation of raw materials According to the solid-liquid weight-volume ratio of 1: 8, 100 kg of the pretreated lignocellulose raw material and 800 L of the culture medium solution are mixed uniformly at a speed of 1.5 r / min for 24 hours; The pretreated lignocellulose raw material is the same as in step (1).
  • the mixing uniformity is achieved by the overall rolling and turning of the container.
  • the method for bacterial saccharification of lignocellulose includes the following steps:
  • Clostridium thermocellum that is, a glucose medium
  • Clostridium thermocellum that is, a glucose medium
  • subcultured at 2% by volume inoculation ratio then inoculated to the 1g / L dry weight of pretreated straw glucose medium, induced domestication at 60 °C for 48h, so as to obtain high growth rate, high fiber body production, high fiber body specific vigor saccharified seed liquid .
  • the pretreatment method is the steam explosion pretreatment technology in the literature (Cellulose Science and Technology, 2002, 3, 47-52).
  • Pre-impregnation of raw materials According to a solid-liquid weight-volume ratio of 1:11, 100 kg of the pretreated lignocellulosic raw material and 1100 L of the culture medium solution are mixed uniformly at a speed of 0.5 r / min for 48 h;
  • the pretreated lignocellulose raw material is the same as in step (1).
  • the mixing uniformity is achieved by the overall rolling and turning of the container.
  • step (1) The optimized whole saccharified seed solution of step (1) is inoculated into the pre-impregnated reaction system obtained in step (2) at an inoculation amount of 2% by volume, and at a rate of 1.6 r / min.
  • a saccharification reaction is performed under a rotation speed condition to obtain a sugar liquid containing glucose.
  • the content of reducing sugar in the fermentation broth was sampled every 12 hours, and the conversion rate of cellulose to soluble sugar in the substrate was calculated.
  • the method for bacterial saccharification of lignocellulose includes the following steps:
  • the culture medium of Clostridium thermocellum with 5g / L glucose as the sole carbon source that is, glucose medium
  • 5g / L glucose as the sole carbon source
  • glucose medium was subcultured 4 times according to the inoculation ratio of 0.5% by volume; then inoculated to the 2g / L dry weight pretreated waste paper in glucose medium, induced domestication for 12h at a temperature of 60 ° C, to obtain high-growth, high-fibrosomal yield, and high-fibrosomal specific vigor glycosylated seeds liquid.
  • the pretreatment method is a hydrothermal pretreatment technique in the literature (Bioresource Technology, 2004, 91, 93-100). After the end of step (1), the method of Example 1 was used to detect the specific activity of fibrosomes, yield and cell biomass of the seed solution.
  • Pre-impregnation of raw materials According to the solid-liquid weight-volume ratio of 1:10, 0.2 kg of the pretreated lignocellulose raw material and 2 L of the culture medium solution are mixed uniformly at a speed of 5.0 r / min for 40 h.
  • the pretreated lignocellulose raw material is the same as in step (1).
  • the mixing uniformity is achieved by the overall rolling and turning of the container.
  • the method for bacterial saccharification of lignocellulose without the steps of domestication and pre-impregnation, includes the following steps:
  • Seed liquid culture Clostridium thermocellum is cultured in a medium (a component of glucose medium other than glucose) containing 5 g / L cellobiose as a carbon source for 12 hours. After the end of step (1), the method of Example 1 was used to detect the specific activity of fibrosomes, yield and cell biomass of the seed solution.
  • the method for bacterial saccharification of lignocellulose includes the following steps:
  • the culture medium of Clostridium yellow fibrinolyticus with 10 g / L glucose as the sole carbon source, that is, glucose medium was subcultured 3 times according to the inoculation ratio of 1% by volume; Add 0.5g / L dry weight of pretreated wheat straw to glucose medium and induce domestication at 65 °C for 24h, in order to obtain high growth rate, high fiber body production, high fiber body specific vigor glycosylation Seed liquid.
  • the pretreatment method is the same as the sulfonation pretreatment technique in Example 1. After the end of step (1), the method of Example 1 was used to detect the specific activity of fibrosomes, yield and cell biomass of the seed solution.
  • Pre-impregnation of raw materials According to the solid-liquid weight-volume ratio of 1:15, 0.2 kg of the pretreated lignocellulose raw material and 3 L of the culture medium solution are mixed uniformly at a speed of 20 r / min for 12 h;
  • the pretreated lignocellulose raw material is the same as in step (1).
  • the uniform mixing is to achieve uniform mixing by stirring by a stirring device in a container.
  • the method for bacterial saccharification of lignocellulose without the acclimation and pre-impregnation steps includes the following steps:
  • Seed liquid culture C. yellow cellulolytic is cultured in a medium (a component of glucose medium other than glucose) containing 10 g / L microcrystalline cellulose as a carbon source for 24 hours. After the end of step (1), the method of Example 1 was used to detect the specific activity of fibrosomes, yield and cell biomass of the seed solution.
  • a medium a component of glucose medium other than glucose
  • the method for bacterial saccharification of lignocellulose includes the following steps:
  • the culture medium of Clostridium fibrosus with 10 g / L of glucose as the sole carbon source was subcultured 3 times according to the inoculation ratio of 1% by volume; 0.5g / L dry weight of pretreated wheat straw glucose medium, induced domestication for 36h at 37 ° C, so as to obtain high-growth, high-fibrosomal yield, and high-fibrosomal specific vigor glycosylated seeds liquid.
  • the pretreatment method is the same as the sulfonation pretreatment technique in Example 1. After the end of step (1), the method of Example 1 was used to detect the specific activity of fibrosomes, yield and cell biomass of the seed solution.
  • Pre-impregnation of raw materials According to the solid-liquid weight-volume ratio of 1:20, 0.2 kg of the pretreated lignocellulose raw material and 4 L of the culture medium solution are mixed uniformly at a speed of 20 r / min for 10 h; The pretreated lignocellulose raw material is the same as in step (1).
  • the uniform mixing is to achieve uniform mixing by stirring by a stirring device in a container.
  • step (1) The optimized whole saccharified seed solution of step (1) is inoculated into the pre-impregnated reaction system obtained in step (2) at an inoculum volume of 1% by volume, and continued at 20 r / min.
  • a saccharification reaction is performed under a rotation speed condition to obtain a sugar liquid containing glucose.
  • the content of reducing sugar in the fermentation broth was sampled every 12 hours, and the conversion rate of cellulose to soluble sugar in the substrate was calculated.
  • the method for bacterial saccharification of lignocellulose without the domestication and pre-impregnation steps includes the following steps:
  • Seed liquid culture Clostridium fibrosus is cultured in a medium (a component of glucose medium other than glucose) containing 10 g / L microcrystalline cellulose as a carbon source for 36 h. After the end of step (1), the method of Example 1 was used to detect the specific activity of fibrosomes, yield and cell biomass of the seed solution.
  • the method for bacterial saccharification of lignocellulose includes the following steps:
  • the culture medium of Clostridium cellulolyticum with 10 g / L glucose as the sole carbon source was subcultured 3 times according to the inoculation ratio of 1% by volume; 0.5g / L dry weight of pretreated wheat straw glucose medium, induced domestication for 36h at 34 °C, so as to obtain the whole bacteria saccharified seeds with high growth rate, high fiber body production and high fiber body specific activity liquid.
  • the pre-processing method is the same as the pre-processing technology in Embodiment 2. After the end of step (1), the method of Example 1 was used to detect the specific activity of fibrosomes, yield and cell biomass of the seed solution.
  • Pre-impregnation of raw materials According to the solid-liquid weight-volume ratio of 1:40, 0.1 kg of the pretreated lignocellulose raw material and 4 L of the culture medium solution are mixed uniformly at a rotation speed of 75 r / min for 5 h; The pretreated lignocellulose raw material is the same as in step (1).
  • the uniform mixing is to achieve uniform mixing by stirring by a stirring device in a container.
  • the method for bacterial saccharification of lignocellulose without the domestication and pre-impregnation steps includes the following steps:
  • Seed liquid culture Clostridium cellulolyticus was cultured in a medium (a component of glucose medium other than glucose) containing 10 g / L cellobiose as a carbon source for 36 hours. After the end of step (1), the method of Example 1 was used to detect the specific activity of fibrosomes, yield and cell biomass of the seed solution.
  • Embodiment 16 is a diagrammatic representation of Embodiment 16:
  • the method for bacterial saccharification of lignocellulose includes the following steps:
  • Vibrio cellulolyticus was cultured with 10 g / L of glucose as the sole carbon source, that is, glucose medium, and subcultured 3 times according to the inoculation ratio of 1% by volume; 0.5g / L dry weight pre-treated corn cob glucose medium was induced to acclimate for 36h at 37 ° C to obtain whole bacteria with high growth rate, high fiber body production, and high fiber body specific activity. Saccharified seed fluid.
  • the pretreatment method is the same as the steam explosion pretreatment technology in Embodiment 5. After the end of step (1), the method of Example 1 was used to detect the specific activity of fibrosomes, yield and cell biomass of the seed solution.
  • Pre-impregnation of raw materials According to a solid-liquid weight-volume ratio of 1:50, 0.1 kg of the pretreated lignocellulose raw material and 5 L of the culture medium solution are mixed uniformly at a speed of 100 r / min for 1 h; The pretreated lignocellulose raw material is the same as in step (1).
  • the uniform mixing is to achieve uniform mixing by stirring by a stirring device in a container.
  • step (1) The optimized whole saccharified seed solution of step (1) is inoculated into the pre-impregnated reaction system obtained in step (2) at an inoculation amount of 0.1% by volume, and continued at 100 r / min.
  • a saccharification reaction is performed under a rotation speed condition to obtain a sugar liquid containing glucose.
  • the content of reducing sugar in the fermentation broth was sampled every 12 hours, and the conversion rate of cellulose to soluble sugar in the substrate was calculated.
  • the method for bacterial saccharification of lignocellulose without the acclimation and pre-impregnation steps includes the following steps:
  • Seed liquid culture Vibrio acetolyticum was cultured in a medium (a component of glucose medium other than glucose) containing 10 g / L cellobiose as a carbon source for 36 hours. After the end of step (1), the method of Example 1 was used to detect the specific activity of fibrosomes, yield and cell biomass of the seed solution.
  • a medium a component of glucose medium other than glucose
  • Embodiment 18 is a diagrammatic representation of Embodiment 18:
  • the method for bacterial saccharification of lignocellulose includes the following steps:
  • Pseudomonas fibrinolyticus was cultured with 5g / L of glucose as the sole carbon source medium, that is, glucose medium, and subcultured at 2% by volume inoculation ratio; then inoculated to Add 1g / L dry weight of pretreated waste paper to glucose medium and induce domestication at 42 ° C for 40h to obtain full bacterial saccharification with high growth rate, high fiber body production, and high fiber body specific activity. Seed liquid.
  • the pretreatment method is the same as the steam explosion pretreatment technology in Embodiment 5. After the end of step (1), the method of Example 1 was used to detect the specific activity of fibrosomes, yield and cell biomass of the seed solution.
  • Pre-impregnation of raw materials According to the solid-liquid weight-volume ratio of 1:25, 0.2 kg of the pretreated lignocellulose raw material and 5 L of the culture medium solution are mixed uniformly at a speed of 1.6 r / min for 12 h.
  • the pretreated lignocellulose raw material is the same as in step (1).
  • the mixing uniformity is achieved by the overall rolling and turning of the container.
  • the bacterial saccharification method for lignocellulose without the steps of domestication and pre-impregnation, includes the following steps:
  • Seed liquid culture Pseudomonas fibrinolyticus was cultured in a medium (a component of glucose medium other than glucose) containing 10 g / L of pretreated waste paper as a carbon source for 40 hours. After the end of step (1), the method of Example 1 was used to detect the specific activity of fibrosomes, yield and cell biomass of the seed solution.
  • the method for bacterial saccharification of lignocellulose includes the following steps:
  • rumen coccus albicans cultured with 20 g / L glucose as the sole carbon source, that is, glucose medium was subcultured twice according to a 1% volume fraction inoculation ratio; then inoculated to 2 g / L dry weight pre-treated corn cob glucose medium, induced domestication for 40 h at 37 ° C, to obtain a whole bacterial saccharified seed solution with high growth rate, high fiber body production, and high fiber body specific activity .
  • the pretreatment method is the same as the steam explosion pretreatment technology in Embodiment 5. After the end of step (1), the method of Example 1 was used to detect the specific activity of fibrosomes, yield and cell biomass of the seed solution.
  • Pre-impregnation of raw materials According to the solid-liquid weight-volume ratio of 1: 3, 0.2 kg of the pretreated lignocellulose raw material and 0.6 L of the culture medium solution are mixed uniformly at a speed of 2 r / min for 48 h.
  • the pretreated lignocellulose raw material is the same as in step (1).
  • the mixing uniformity is achieved by the overall rolling and turning of the container.
  • Whole bacteria saccharification Inoculate the optimized whole bacteria saccharified seed solution in step (1) with a 10% volume fraction inoculation amount into the pre-impregnated reaction system obtained in step (2), and continue at 2r / min.
  • a saccharification reaction is performed under a rotation speed condition to obtain a sugar liquid containing glucose.
  • the content of reducing sugar in the fermentation broth was sampled every 12 hours, and the conversion rate of cellulose to soluble sugar in the substrate was calculated.
  • the method for bacterial saccharification of lignocellulose without the acclimation and pre-impregnation steps includes the following steps:
  • Seed liquid culture Rumenococcus albicans was cultured in a medium (a component of glucose medium other than glucose) of 20 g / L pretreated corn cob as a carbon source for 40 hours. After the end of step (1), the method of Example 1 was used to detect the specific activity of fibrosomes, yield and cell biomass of the seed solution.
  • the method for bacterial saccharification of lignocellulose includes the following steps:
  • rumen coccus luteus is cultured with 20g / L glucose as the sole carbon source, that is, glucose medium, and then subcultured twice according to a 1% volume fraction inoculation ratio; then inoculated to the 2g / L dry weight pre-treated corn cob glucose media, induced domestication at 40 ° C for 48h, to obtain high-growth, high-fibre body yield, and high-fibre body specific vigor saccharified seeds liquid.
  • the pretreatment method is a dilute acid hydrolysis pretreatment technique in the literature (bioprocessing process, 2010, 3, 66-72). After the end of step (1), the method of Example 1 was used to detect the specific activity of fibrosomes, yield and cell biomass of the seed solution.
  • Pre-impregnation of raw materials According to a solid-liquid weight-volume ratio of 1: 5, 0.4 kg of the pretreated lignocellulose raw material and 2 L of the culture medium solution are mixed uniformly at a speed of 10 r / min for 36 h; The pretreated lignocellulose raw material is the same as in step (1).
  • the mixing uniformity is achieved by the overall rolling and turning of the container.
  • Whole bacteria saccharification Inoculate the optimized whole bacteria saccharified seed solution in step (1) with a 10% volume fraction inoculation amount into the pre-impregnated reaction system obtained in step (2), and continue at 10 r / min.
  • a saccharification reaction is performed under a rotation speed condition to obtain a sugar liquid containing glucose.
  • the content of reducing sugar in the fermentation broth was sampled every 12 hours, and the conversion rate of cellulose to soluble sugar in the substrate was calculated.
  • the bacterial saccharification method used for lignocellulose without the steps of domestication and pre-impregnation, includes the following steps:
  • Seed liquid culture Rumenococcus luteans was cultured in a medium (components other than glucose of glucose medium) of glucose pretreated corn cob as a carbon source at 20 g / L for 48 h. After the end of step (1), the method of Example 1 was used to detect the specific activity of fibrosomes, yield and cell biomass of the seed solution.
  • Growth rate refers to the time required to grow to mid-log phase.
  • the lag period refers to the time during which the conversion of cellulose to soluble sugar in the substrate is less than 10% (mass ratio) during the whole saccharification process.
  • the hydrolysis cycle refers to the time when the conversion rate of cellulose to soluble sugar in the substrate reaches 80% (mass ratio) during the whole saccharification process.
  • the specific activity of fibrosomes is 1.3-4.1U / mg; the yield of fibrosomes is 1.4-2.1mg / mL; and the seed growth rate is 12-36 hours.
  • the yield of glucose in the sugar solution is 10.7-50.3 g / L; the lag period is 2-3 days; and the saccharification cycle is 10-17.5 days.
  • the fiber body of the seed liquid obtained by using the bacterial saccharification method for lignocellulose described in the present application obtained by using the bacterial saccharification method for lignocellulose described in the present application.
  • the specific activity is 4.1-17.7 U / mg; the yield of fibrosomes is 13.9-25.5 mg / mL; the seed growth rate is 16-32 hours.
  • the yield of glucose in the sugar solution is 26.6-62.5g / L; the lag period is 0.5-1.5 days; and the saccharification cycle is 7.5-13.5 days.
  • the specific activity of the fibrous bodies is 3.3-17.1 U / mg; the yield of the fibrous bodies is 15.0-21.3 mg / mL; and the seed growth rate is 24-40 hours.
  • the yield of glucose in the sugar solution is 23.9-63.1g / L; the lag period is 2.5-4.5 days; and the saccharification cycle is 12-18 days.
  • the specific activity of the fibrous bodies is 5.1-15.3 U / mg; the yield of the fibrous bodies is 8.1-14.8 mg / mL; and the seed growth rate is 66-72 hours.
  • the yield of glucose in the sugar solution is 24.7-103.1 g / L; the lag period is 2.5-5.0 days; and the saccharification cycle is 15-26.5 days.
  • the specific activity of the fibrous bodies of the seed liquid obtained by the present invention is consistent with the fibrous bodies produced by microcrystalline cellulose as a carbon source, and higher than the fibrous bodies produced by using cellobiose as a carbon source
  • the fiber body production of the seed liquid obtained by the present invention is the same as that produced by cellulose as a carbon source; higher than that produced by cellobiose as a carbon source;
  • the present invention The seed growth rate of the obtained seed liquid is consistent with that of cellobiose as a carbon source, which is higher than that of cellulose as a carbon source, and much faster than that of pretreated lignocellulose as a carbon source;
  • the lag period and saccharification period in the invention are greatly shortened.
  • the bacterial saccharification method for lignocellulose according to the present invention uses relatively inexpensive glucose and low-concentration pretreated lignocellulosic biomass as a carbon source, and is used for culturing small fibers such as Clostridium thermocellum.
  • the whole-saccharified saccharified seed liquid achieves the effects of cost reduction and cycle shortening, and overcomes the problems that cannot be obtained in the prior art.
  • the method for bacterial saccharification of lignocellulose according to the present invention adopts a technique of pre-impregnation of raw materials, which significantly shortens the adaptation buffer period (delay period) of bacterial saccharification, especially under the condition of high solid content. The efficiency of whole bacteria saccharification, thereby reducing the cycle and cost of the entire saccharification process.

Abstract

提供了用于木质纤维素的全菌糖化方法，包括以下步骤：(1)种子液优化：在厌氧条件下，将产纤维小体菌株在以葡萄糖为唯一碳源的培养基，进行传代驯化；然后接种到添加了预处理后的木质纤维素原料的葡萄糖培养基中进行诱导驯化。(2)原料预浸渍：将预处理后的木质纤维素原料和培养基溶液混合均匀。(3)全菌糖化：将步骤(1)优化的全菌糖化种子液接种到步骤(2)得到的预浸渍后的反应体系中，进行糖化反应，得到含有葡萄糖的糖液。该方法不但大大缩短了种子培养阶段的周期，降低了种子液的成本，也缩短了全菌糖化的周期，从而为工业化的进程奠定了基础。

Description

用于木质纤维素的全菌糖化方法 技术领域
本发明属于生物技术领域，涉及一种木质纤维素的生物转化方法，具体涉及一种用于木质纤维素的全菌糖化方法。
背景技术
近年来，从生物质中获取可再生的能源、材料或化学品得到世界各国的普遍关注。利用木质纤维素类生物质为原料生产第二代生物基燃料或化学品，成为能否大规模替代石油基产品的关键。
木质纤维素类生物质是地球上蕴藏最为丰富的可再生生物质资源，将其转化为能源、化学品或材料具有巨大的应用和发展前景。特别的，我国是农业大国，每年的农作物秸秆产量超过9亿吨。另一方面，秸秆焚烧引起的环境污染与交通安全事故等问题，也促使国家推出一系列秸秆综合利用的规划、方案与措施。所以，推动农业废弃生物质综合利用的产业化发展，对我国绿色循环经济的建立和社会的可持续发展意义重大。
然而，生物质糖化所用的酶制剂技术目前被发达国家公司垄断，由此而造成的酶的高成本成为制约木质纤维素工业化应用的关键问题。此外，技术上的困难也阻碍了木质纤维素生物转化技术的发展和应用。因此，开发低成本、高效率的糖化技术，对发展中国家尤为必要。
与依赖于酶制剂的糖化工艺相比，整合生物加工技术的“一锅法”全菌催化的生物糖化策略具有简化流程、降低设备要求等优势，是最适合纤维素类生物质生物转化利用的工艺路线。“一锅法”技术的核心在于采用高效的全菌催化剂，即一种具有木质纤维素底物降解能力、可以高效将纤维素底物水解转化为可发酵糖的微生物。目前，木质纤维素全菌催化糖化主要利用基于热纤梭菌这一产纤维小体的高温厌氧菌作为全菌催化剂。纤维小体是一种具有复杂结构和组分的胞外多酶复合体，是自然界中已知的最高效的纤维素降解体系之一。木质纤维素全菌催化糖化包括全菌催化剂预培养(种子培养阶段)和全菌糖化的两步半连续生产模式。在种子培养阶段，目前通常有两种方法：(1)以市售纤维二糖或者纤维素作为碳源进行培养，种子生长较快，但价格昂贵；(2)以预处理生物质原料为碳源进行种子培养，这一方法虽然可以降低成本，但种子培养周期较长。
葡萄糖是发酵行业常用的微生物培养碳源，价格较低，而且是纤维素糖化的产物。然而，目前，葡萄糖还不能应用于全菌催化剂的预培养。这是由于，当以葡萄糖为唯一碳源对热纤梭菌进行培养时，不但会有几十小时的生长延滞期，而且会导致纤维小体的产量显著降低， 从而导致生产周期长，此外，所获得的全菌催化剂的纤维素降解活力较低。Yoav等人将热纤梭菌菌株在以葡萄糖为唯一碳源的培养基里进行驯化(Yoav,S.,Barak,Y.,Shamshoum,M.,et al.,Biotechnology for biofuels 2017,10:222)，解决了葡萄糖作为单一碳源所导致的生长延滞期的问题。然而，该报道只是为了更快的得到菌体和纤维小体，并没有解决纤维小体产量显著降低以及所获得的全菌催化剂的纤维素降解活力较低的问题。此外，在全菌糖化阶段，将种子液接种进具有高固含量预处理木质纤维素底物的大规模糖化体系后，一般需要2-5天的缓冲期才开始酶解糖化过程，也会导致生产周期长，从而增加生产过程的人工、能耗成本。
因此，木质纤维素全菌催化糖化中种子培养阶段的种子液制备的技术难题，以及全菌糖化步骤中存在的周期长、成本高的问题，是基于全菌催化的木质纤维素生物糖化技术难以大规模应用于工业生产的主要瓶颈之一。
发明内容
针对现有技术中木质纤维素全菌糖化中所存在周期长、种子液制备成本高的问题，本发明提供了用于木质纤维素的全菌糖化方法，该方法不但大大缩短了种子培养阶段的周期，降低了种子液的成本，也缩短了全菌糖化的周期，从而为工业化的进程奠定了基础。
本发明的技术方案：
用于木质纤维素的全菌糖化方法，包括以下步骤：
(1)种子液优化：在厌氧条件下，将产纤维小体菌株在以葡萄糖为唯一碳源的培养基，即葡萄糖培养基中，进行传代驯化；然后接种到添加了预处理后的木质纤维素原料的葡萄糖培养基中进行诱导驯化。从而获得高生长速度、高纤维小体产量、高纤维小体比活力的全菌糖化种子液。
所述的葡萄糖培养基含有葡萄糖的浓度为1-20g/L；所述传代驯化进行2-5次，每次传代为将产纤维小体菌株培养后按照0.2-2％(体积分数)接种至新鲜培养基中进行培养。所述木质纤维素原料的添加量为0.1-2.0g/L；所述诱导驯化的温度条件为34-65℃；所述诱导驯化的时间为12-48小时。向葡萄糖培养基中添加预处理后的木质纤维素原料，即预处理底物，是为了诱导匹配的纤维小体合成。
本发明所获得的种子液的纤维小体的比活力与纤维素为碳源生产的纤维小体一致、高于以纤维二糖为碳源生产的纤维小体，其纤维小体产量与纤维素为碳源生产的纤维小体一致，其种子生长速度与以纤维二糖为碳源生长的种子一致，高于纤维素为碳源生长的种子，远高于预处理木质纤维素为碳源生长的种子。
(2)原料预浸渍：按照1:3-1:50的固液重量体积比，将预处理后的木质纤维素原料和培养基溶液在容器中在0.5-100r/min的转速条件下混合均匀并持续1-48h；所述预处理后的木质 纤维素原料与步骤(1)中相同，目的是在糖化开始前实现底物与培养基进行充分的混合，有利于全菌催化剂的生长和活力，从而实现更快速的糖化。所述容器为带有搅拌桨的厌氧发酵设备或者厌氧旋转混匀器；所述带有搅拌桨的厌氧发酵设备通过搅拌实现混合均匀，而厌氧旋转混匀器则通过容器的整体滚动翻转实现混合均匀。
(3)全菌糖化：将步骤(1)优化的全菌糖化种子液接种到步骤(2)得到的预浸渍后的反应体系中，并在0.5-100r/min的转速条件下进行糖化反应，得到含有葡萄糖的糖液。所述的全菌糖化种子液的接种量为0.1-10％的体积分数。糖化过程中可通过流加氢氧化钠的方式使pH控制在5.8-6.2。
其中，所述的产纤维小体菌株为热纤梭菌、黄色溶纤梭菌、嗜纤维梭菌、解纤维梭菌、解纤维醋弧菌、溶纤维假拟杆菌、白色瘤胃球菌或黄化瘤胃球菌。所述的预处理为碱法、酸法、水热法、汽爆法和磺化法预处理技术中的一种或多种的组合。所述木质纤维素原料为玉米秸秆、麦秸、灌木条枝、木片、玉米芯、稻草和废纸中的一种或多种的组合。
其中，步骤(1)所述的葡萄糖培养基为每升水中添加磷酸氢二钾2.9g/L、磷酸二氢钾1.5g/L、尿素0.8g/L、氯化钙0.1g/L、氯化镁1.8g/L、硫酸亚铁0.0005g/L、硫化钠2g/L、玉米浆4g/L、柠檬酸三钠2g/L、葡萄糖1-20g/L、pH 6.5-7.5。
本发明的有益效果：
(1)本发明所述的用于木质纤维素的全菌糖化方法，以相对廉价的葡萄糖添加低浓度的预处理木质纤维素生物质为碳源，用于培养热纤梭菌等产纤维小体菌株，作为全菌糖化的种子液；与现有技术相比，所述全菌糖化的种子液实现了成本降低和周期缩短的效果，克服了现有技术中二者不可兼得的难题。
(2)本发明所述的木质纤维素的全菌糖化方法，采用原料预浸渍的技术，显著缩短全菌糖化的适应缓冲期，提高全菌糖化的效率，从而降低整个糖化过程的周期和成本。
具体实施方式
下面结合实施例对本发明做进一步的说明。
实施例1：
用于木质纤维素的全菌糖化方法，包括以下步骤：
(1)种子液优化：
在厌氧条件下，将热纤梭菌在以5g/L的葡萄糖为唯一碳源的培养基，即葡萄糖培养基中，按照1％体积分数的接种比例，进行3次传代培养；然后接种到添加0.1g/L干重的预处理玉米秸秆的葡萄糖培养基中，在55℃的温度条件下诱导驯化48h，从而获得高生长速度、高纤维小体产量、高纤维小体比活力的全菌糖化种子液。所述的葡萄糖培养基除含有5g/L葡萄糖 外，每升水中还具有磷酸氢二钾2.9g/L、磷酸二氢钾1.5g/L、尿素0.8g/L、氯化钙0.1g/L、氯化镁1.8g/L、硫酸亚铁0.0005g/L、硫化钠2g/L、玉米浆4g/L、柠檬酸三钠2g/L、pH 6.5-7.5。向葡萄糖培养基中添加预处理后的木质纤维素原料，即预处理底物，是为了诱导匹配的纤维小体合成。所述的预处理方法为文献(中国造纸，2015,34,1-6)中采用的磺化法预处理。
在步骤(1)结束后，检测种子液的纤维小体比活力、产量和细胞生物量。其中：(1)纤维小体产量的检测为：利用纤维素吸附法提取种子液中的纤维小体，以Bradford法进行蛋白质定量，从而获得纤维小体产量(每单位体积种子液中纤维小体的毫克数)。(2)纤维小体比活力的检测为：以预处理玉米秸秆为底物，检测提取的纤维小体的水解活力。水解实验在55℃条件下持续24小时，然后用DNS法检测水解释放的还原糖的浓度。根据还原糖和纤维小体产量计算纤维小体比活力(U/mg)，其中酶活单位(U)定义为在测定条件下，每小时水解纤维素底物生成1mg还原糖所需要的蛋白量(mg)。(3)细胞生物量的检测为：将种子液离心后的沉淀用氢氧化钠进行水解，然后利用Bradford法检测沉淀中细胞蛋白的浓度(每单位体积种子液中沉淀蛋白的毫克数)，根据生长曲线以及沉淀蛋白浓度分析生长至对数中期所需时间(小时)，判断生长速度。
(2)原料预浸渍：按照1:6.5的固液重量体积比，将1kg预处理后的木质纤维素原料和6.5L的培养基溶液在50r/min的转速条件下混合均匀，并持续4h；所述预处理后的木质纤维素原料与步骤(1)中相同，目的是在糖化开始前实现底物与培养基进行充分的混合，有利于全菌催化剂的生长和活力，从而实现更快速的糖化。所述混合均匀为通过容器中搅拌装置的搅拌实现混合均匀。
(3)全菌糖化：将步骤(1)优化的全菌糖化种子液按照10％体积分数的接种量接种到步骤(2)得到的预浸渍后的反应体系中，并继续在50r/min的转速条件下进行糖化反应，得到含有葡萄糖的糖液。在此过程中，每12小时取样检测发酵液中还原糖的含量，计算底物中纤维素到可溶性糖的转化率。
其中，转化率低于10％(质量比)的时间定义为木质纤维素底物的水解的延滞期(天)。转化率达到80％(质量比)的时间定义为木质纤维素底物的水解周期(天)。
实施例2：
与实施例1不同的是，用于木质纤维素的全菌糖化方法，不经驯化及预浸渍步骤，包括以下步骤：
(1)种子液培养：将热纤梭菌在以5g/L预处理玉米秸秆为碳源的培养基(葡萄糖培养基除葡萄糖以外的成分)中培养48h。在步骤(1)结束后，采用实施例1的方法检测种子液的纤维小体比活力、产量和细胞生物量。
(2)全菌糖化：按照10％体积分数的接种量，将种子液直接接种到含有1kg预处理后的玉米秸秆和6.5L的培养基溶液的反应体系中进行全菌糖化，预处理原料及全菌糖化的条件与实施例1相同。在此过程中，每12小时取样检测发酵液中还原糖的含量，计算底物中纤维素到可溶性糖的转化率。
实施例3：
与实施例1不同的是，用于木质纤维素的全菌糖化方法，包括以下步骤：
(1)种子液优化：
在厌氧条件下，将热纤梭菌在以10g/L的葡萄糖为唯一碳源的培养基，即葡萄糖培养基中，按照1％体积分数的接种比例，进行3次传代培养；然后接种到添加0.5g/L干重的预处理小麦秸秆的葡萄糖培养基中，在58℃的温度条件下诱导驯化36h，从而获得高生长速度、高纤维小体产量、高纤维小体比活力的全菌糖化种子液。所述的预处理方法为CN201610133959中采用的水热与磺化法联合预处理法。
(2)原料预浸渍：按照1:5的固液重量体积比，将1kg预处理后的木质纤维素原料和5L的培养基溶液在25r/min的转速条件下混合均匀，并持续36h；所述预处理后的木质纤维素原料与步骤(1)中相同。
(3)全菌糖化：将步骤(1)优化的全菌糖化种子液以10％体积分数的接种量接种到步骤(2)得到的预浸渍后的反应体系中，并继续在25r/min的转速条件下进行糖化反应，得到含有葡萄糖的糖液。在此过程中，每12小时取样检测发酵液中还原糖的含量，计算底物中纤维素到可溶性糖的转化率。
实施例4：
与实施例1不同的是，用于木质纤维素的全菌糖化方法，包括以下步骤：
(1)种子液优化：
在厌氧条件下，将热纤梭菌在以20g/L的葡萄糖为唯一碳源的培养基，即葡萄糖培养基中，按照1％体积分数的接种比例，进行2次传代培养；然后接种到添加2g/L干重的预处理灌木枝条的葡萄糖培养基中，在60℃的温度条件下诱导驯化24h，从而获得高生长速度、高纤维小体产量、高纤维小体比活力的全菌糖化种子液。所述的预处理方法为文献(Bin Li,et  al.Recent progress on the pretreatment and fractionation of lignocelluloses for Biorefinery at QIBEBT.Journal of Bioresources and Bioproducts,2017,2(1),4-9)中的碱法预处理技术。在步骤(1)结束后，采用实施例1的方法检测种子液的纤维小体比活力、产量和细胞生物量。
(2)原料预浸渍：按照1:8的固液重量体积比，将1kg预处理后的木质纤维素原料和8L的培养基溶液在45r/min的转速条件下混合均匀，并持续28h；所述预处理后的木质纤维素原料与步骤(1)中相同。(3)全菌糖化：将步骤(1)优化的全菌糖化种子液以6％体积分数的接种量接种到步骤(2)得到的预浸渍后的反应体系中，并继续在45r/min的转速条件下进行糖化反应，得到含有葡萄糖的糖液。在此过程中，每12小时取样检测发酵液中还原糖的含量。
实施例5：
与实施例4不同的是，用于木质纤维素的全菌糖化方法，不经驯化及预浸渍步骤，包括以下步骤：
(1)种子液培养：将热纤梭菌在以20g/L微晶纤维素为碳源的培养基(葡萄糖培养基除葡萄糖以外的成分)中培养24h。在步骤(1)结束后，采用实施例1的方法检测种子液的纤维小体比活力、产量和细胞生物量。
(2)全菌糖化：按照8％体积分数的接种量，将种子液直接接种到含有1kg预处理后的灌木枝条和8L的培养基溶液的反应体系中进行全菌糖化，预处理原料及全菌糖化的条件与实施例4相同。在此过程中，每12小时取样检测发酵液中还原糖的含量，计算底物中纤维素到可溶性糖的转化率。
实施例6：
与实施例1不同的是，用于木质纤维素的全菌糖化方法，包括以下步骤：
(1)种子液优化：
在厌氧条件下，将热纤梭菌以5g/L的葡萄糖为唯一碳源的培养基，即葡萄糖培养基中，按照0.1％体积分数的接种比例，进行5次传代培养；然后接种到添加0.5g/L干重的预处理木片的葡萄糖培养基中，在60℃的温度条件下诱导驯化24h，从而获得高生长速度、高纤维小体产量、高纤维小体比活力的全菌糖化种子液。所述的预处理方法为文献(Biotechnology for Biofuels，2014，7:116)中的碱法与水热相结合的预处理技术。
(2)原料预浸渍：按照1:8的固液重量体积比，将100kg预处理后的木质纤维素原料和 800L的培养基溶液在1.5r/min的转速条件下混合均匀，并持续24h；所述预处理后的木质纤维素原料与步骤(1)中相同。所述混合均匀为通过容器的整体滚动翻转实现混合均匀。
(3)全菌糖化：将步骤(1)优化的全菌糖化种子液以8％体积分数的接种量接种到步骤(2)得到的预浸渍后的反应体系中，并继续在1.5r/min的转速条件下进行糖化反应，得到含有葡萄糖的糖液。在此过程中，每12小时取样检测发酵液中还原糖的含量，计算底物中纤维素到可溶性糖的转化率。
实施例7：
与实施例1不同的是，用于木质纤维素的全菌糖化方法，包括以下步骤：
(1)种子液优化：
在厌氧条件下，将热纤梭菌以5g/L的葡萄糖为唯一碳源的培养基，即葡萄糖培养基中，按照2％体积分数的接种比例，进行2次传代培养；然后接种到添加1g/L干重的预处理稻草的葡萄糖培养基中，在60℃的温度条件下诱导驯化48h，从而获得高生长速度、高纤维小体产量、高纤维小体比活力的全菌糖化种子液。所述的预处理方法为文献(纤维素科学与技术，2002，3，47-52)中的汽爆预处理技术。
(2)原料预浸渍：按照1:11的固液重量体积比，将100kg预处理后的木质纤维素原料和1100L的培养基溶液在0.5r/min的转速条件下混合均匀，并持续48h；所述预处理后的木质纤维素原料与步骤(1)中相同。所述混合均匀为通过容器的整体滚动翻转实现混合均匀。
(3)全菌糖化：将步骤(1)优化的全菌糖化种子液以2％体积分数的接种量接种到步骤(2)得到的预浸渍后的反应体系中，并在1.6r/min的转速条件下进行糖化反应，得到含有葡萄糖的糖液。在此过程中，每12小时取样检测发酵液中还原糖的含量，计算底物中纤维素到可溶性糖的转化率。
实施例8：
与实施例1不同的是，用于木质纤维素的全菌糖化方法，包括以下步骤：
(1)种子液优化：
在厌氧条件下，将热纤梭菌以5g/L的葡萄糖为唯一碳源的培养基，即葡萄糖培养基中，按照0.5％体积分数的接种比例，进行4次传代培养；然后接种到添加2g/L干重的预处理废纸的葡萄糖培养基中，在60℃的温度条件下诱导驯化12h，从而获得高生长速度、高纤维小体产量、高纤维小体比活力的全菌糖化种子液。所述的预处理方法为文献(Bioresource Technology，2004，91，93-100)中的水热预处理技术。在步骤(1)结束后，采用实施例1 的方法检测种子液的纤维小体比活力、产量和细胞生物量。
(2)原料预浸渍：按照1:10的固液重量体积比，将0.2kg预处理后的木质纤维素原料和2L的培养基溶液在5.0r/min的转速条件下混合均匀，并持续40h；所述预处理后的木质纤维素原料与步骤(1)中相同。所述混合均匀为通过容器的整体滚动翻转实现混合均匀。
(3)全菌糖化：将步骤(1)优化的全菌糖化种子液以4％体积分数的接种量接种到步骤(2)得到的预浸渍后的反应体系中，并继续在5.0r/min的转速条件下进行糖化反应，得到含有葡萄糖的糖液。在此过程中，每12小时取样检测发酵液中还原糖的含量，计算底物中纤维素到可溶性糖的转化率。
实施例9：
与实施例8不同的是，用于木质纤维素的全菌糖化方法，不经驯化及预浸渍步骤，包括以下步骤：
(1)种子液培养：将热纤梭菌在以5g/L纤维二糖为碳源的培养基(葡萄糖培养基除葡萄糖以外的成分)中培养12h。在步骤(1)结束后，采用实施例1的方法检测种子液的纤维小体比活力、产量和细胞生物量。
(2)全菌糖化：按照4％体积分数的接种量，将种子液直接接种到含有0.2kg预处理后的废纸和2L的培养基溶液的反应体系中进行全菌糖化，预处理原料及全菌糖化的条件与实施例8相同。在此过程中，每12小时取样检测发酵液中还原糖的含量，计算底物中纤维素到可溶性糖的转化率。
实施例10：
与实施例1不同的是，用于木质纤维素的全菌糖化方法，包括以下步骤：
(1)种子液优化：
在厌氧条件下，将黄色溶纤梭菌以10g/L的葡萄糖为唯一碳源的培养基，即葡萄糖培养基中，按照1％体积分数的接种比例，进行3次传代培养；然后接种到添加0.5g/L干重的预处理麦秸的葡萄糖培养基中，在65℃的温度条件下诱导驯化24h，从而获得高生长速度、高纤维小体产量、高纤维小体比活力的全菌糖化种子液。所述的预处理方法同实施例1中的磺化预处理技术。在步骤(1)结束后，采用实施例1的方法检测种子液的纤维小体比活力、产量和细胞生物量。
(2)原料预浸渍：按照1:15的固液重量体积比，将0.2kg预处理后的木质纤维素原料和3L的培养基溶液在20r/min的转速条件下混合均匀，并持续12h；所述预处理后的木质纤维 素原料与步骤(1)中相同。所述混合均匀为通过容器中搅拌装置的搅拌实现混合均匀。
(3)全菌糖化：将步骤(1)优化的全菌糖化种子液以5％体积分数的接种量接种到步骤(2)得到的预浸渍后的反应体系中，并继续在20r/min的转速条件下进行糖化反应，得到含有葡萄糖的糖液。在此过程中，每12小时取样检测发酵液中还原糖的含量，计算底物中纤维素到可溶性糖的转化率。
实施例11：
与实施例10不同的是，用于木质纤维素的全菌糖化方法，不经驯化及预浸渍步骤，包括以下步骤：
(1)种子液培养：将黄色溶纤梭菌在以10g/L微晶纤维素为碳源的培养基(葡萄糖培养基除葡萄糖以外的成分)中培养24h。在步骤(1)结束后，采用实施例1的方法检测种子液的纤维小体比活力、产量和细胞生物量。
(2)全菌糖化：按照5％体积分数的接种量，将种子液直接接种到含有0.2kg预处理后的麦秸和3L的培养基溶液的反应体系中进行全菌糖化，预处理原料及全菌糖化的条件与实施例10相同。在此过程中，每12小时取样检测发酵液中还原糖的含量，计算底物中纤维素到可溶性糖的转化率。
实施例12：
与实施例1不同的是，用于木质纤维素的全菌糖化方法，包括以下步骤：
(1)种子液优化：
在厌氧条件下，将嗜纤维梭菌以10g/L的葡萄糖为唯一碳源的培养基，即葡萄糖培养基中，按照1％体积分数的接种比例，进行3次传代培养；然后接种到添加0.5g/L干重的预处理麦秸的葡萄糖培养基中，在37℃的温度条件下诱导驯化36h，从而获得高生长速度、高纤维小体产量、高纤维小体比活力的全菌糖化种子液。所述的预处理方法同实施例1中的磺化预处理技术。在步骤(1)结束后，采用实施例1的方法检测种子液的纤维小体比活力、产量和细胞生物量。
(2)原料预浸渍：按照1:20的固液重量体积比，将0.2kg预处理后的木质纤维素原料和4L的培养基溶液在20r/min的转速条件下混合均匀，并持续10h；所述预处理后的木质纤维素原料与步骤(1)中相同。所述混合均匀为通过容器中搅拌装置的搅拌实现混合均匀。
(3)全菌糖化：将步骤(1)优化的全菌糖化种子液以1％体积分数的接种量接种到步骤(2)得到的预浸渍后的反应体系中，并继续在20r/min的转速条件下进行糖化反应，得到含 有葡萄糖的糖液。在此过程中，每12小时取样检测发酵液中还原糖的含量，计算底物中纤维素到可溶性糖的转化率。
实施例13：
与实施例12不同的是，用于木质纤维素的全菌糖化方法，不经驯化及预浸渍步骤，包括以下步骤：
(1)种子液培养：将嗜纤维梭菌在以10g/L微晶纤维素为碳源的培养基(葡萄糖培养基除葡萄糖以外的成分)中培养36h。在步骤(1)结束后，采用实施例1的方法检测种子液的纤维小体比活力、产量和细胞生物量。
(2)全菌糖化：按照1％体积分数的接种量，将种子液直接接种到含有0.2kg预处理后的麦秸和4L的培养基溶液的反应体系中进行全菌糖化，预处理原料及全菌糖化的条件与实施例12相同。在此过程中，每12小时取样检测发酵液中还原糖的含量，计算底物中纤维素到可溶性糖的转化率。
实施例14：
与实施例1不同的是，用于木质纤维素的全菌糖化方法，包括以下步骤：
(1)种子液优化：
在厌氧条件下，将解纤维梭菌以10g/L的葡萄糖为唯一碳源的培养基，即葡萄糖培养基中，按照1％体积分数的接种比例，进行3次传代培养；然后接种到添加0.5g/L干重的预处理麦秸的葡萄糖培养基中，在34℃的温度条件下诱导驯化36h，从而获得高生长速度、高纤维小体产量、高纤维小体比活力的全菌糖化种子液。所述的预处理方法同实施例2中的预处理技术。在步骤(1)结束后，采用实施例1的方法检测种子液的纤维小体比活力、产量和细胞生物量。
(2)原料预浸渍：按照1:40的固液重量体积比，将0.1kg预处理后的木质纤维素原料和4L的培养基溶液在75r/min的转速条件下混合均匀，并持续5h；所述预处理后的木质纤维素原料与步骤(1)中相同。所述混合均匀为通过容器中搅拌装置的搅拌实现混合均匀。
(3)全菌糖化：将步骤(1)优化的全菌糖化种子液以0.5％体积分数的接种量接种到步骤(2)得到的预浸渍后的反应体系中，并继续在75r/min的转速条件下进行糖化反应，得到含有葡萄糖的糖液。在此过程中，每12小时取样检测发酵液中还原糖的含量，计算底物中纤维素到可溶性糖的转化率。
实施例15：
与实施例12不同的是，用于木质纤维素的全菌糖化方法，不经驯化及预浸渍步骤，包括以下步骤：
(1)种子液培养：将解纤维梭菌在以10g/L纤维二糖为碳源的培养基(葡萄糖培养基除葡萄糖以外的成分)中培养36h。在步骤(1)结束后，采用实施例1的方法检测种子液的纤维小体比活力、产量和细胞生物量。
(2)全菌糖化：按照0.5％体积分数的接种量，将种子液直接接种到含有0.1kg预处理后的麦秸和4L的培养基溶液的反应体系中进行全菌糖化，预处理原料及全菌糖化的条件与实施例14相同。在此过程中，每12小时取样检测发酵液中还原糖的含量，计算底物中纤维素到可溶性糖的转化率。
实施例16：
与实施例1不同的是，用于木质纤维素的全菌糖化方法，包括以下步骤：
(1)种子液优化：
在厌氧条件下，将解纤维醋弧菌以10g/L的葡萄糖为唯一碳源的培养基，即葡萄糖培养基中，按照1％体积分数的接种比例，进行3次传代培养；然后接种到添加0.5g/L干重的预处理玉米芯的葡萄糖培养基中，在37℃的温度条件下诱导驯化36h，从而获得高生长速度、高纤维小体产量、高纤维小体比活力的全菌糖化种子液。所述的预处理方法同实施例5中的汽爆法预处理技术。在步骤(1)结束后，采用实施例1的方法检测种子液的纤维小体比活力、产量和细胞生物量。
(2)原料预浸渍：按照1:50的固液重量体积比，将0.1kg预处理后的木质纤维素原料和5L的培养基溶液在100r/min的转速条件下混合均匀，并持续1h；所述预处理后的木质纤维素原料与步骤(1)中相同。所述混合均匀为通过容器中搅拌装置的搅拌实现混合均匀。
(3)全菌糖化：将步骤(1)优化的全菌糖化种子液以0.1％体积分数的接种量接种到步骤(2)得到的预浸渍后的反应体系中，并继续在100r/min的转速条件下进行糖化反应，得到含有葡萄糖的糖液。在此过程中，每12小时取样检测发酵液中还原糖的含量，计算底物中纤维素到可溶性糖的转化率。
实施例17：
与实施例16不同的是，用于木质纤维素的全菌糖化方法，不经驯化及预浸渍步骤，包括以下步骤：
(1)种子液培养：将解纤维醋弧菌在以10g/L纤维二糖为碳源的培养基(葡萄糖培养基除葡萄糖以外的成分)中培养36h。在步骤(1)结束后，采用实施例1的方法检测种子液的纤维小体比活力、产量和细胞生物量。
(2)全菌糖化：按照0.1％体积分数的接种量，将种子液直接接种到含有0.1kg预处理后的玉米芯和5L的培养基溶液的反应体系中进行全菌糖化，预处理原料及全菌糖化的条件与实施例16相同。在此过程中，每12小时取样检测发酵液中还原糖的含量，计算底物中纤维素到可溶性糖的转化率。
实施例18：
与实施例1不同的是，用于木质纤维素的全菌糖化方法，包括以下步骤：
(1)种子液优化：
在厌氧条件下，将溶纤维假拟杆菌以5g/L的葡萄糖为唯一碳源的培养基，即葡萄糖培养基中，按照2％体积分数的接种比例，进行2次传代培养；然后接种到添加1g/L干重的预处理废纸的葡萄糖培养基中，在42℃的温度条件下诱导驯化40h，从而获得高生长速度、高纤维小体产量、高纤维小体比活力的全菌糖化种子液。所述的预处理方法同实施例5中的汽爆法预处理技术。在步骤(1)结束后，采用实施例1的方法检测种子液的纤维小体比活力、产量和细胞生物量。
(2)原料预浸渍：按照1:25的固液重量体积比，将0.2kg预处理后的木质纤维素原料和5L的培养基溶液在1.6r/min的转速条件下混合均匀，并持续12h；所述预处理后的木质纤维素原料与步骤(1)中相同。所述混合均匀为通过容器的整体滚动翻转实现混合均匀。
(3)全菌糖化：将步骤(1)优化的全菌糖化种子液以5％体积分数的接种量接种到步骤(2)得到的预浸渍后的反应体系中，并继续在1.6r/min的转速条件下进行糖化反应，得到含有葡萄糖的糖液。在此过程中，每12小时取样检测发酵液中还原糖的含量，计算底物中纤维素到可溶性糖的转化率。
实施例19：
与实施例18不同的是，用于木质纤维素的全菌糖化方法，不经驯化及预浸渍步骤，包括以下步骤：
(1)种子液培养：将溶纤维假拟杆菌在以10g/L预处理废纸为碳源的培养基(葡萄糖培养基除葡萄糖以外的成分)中培养40h。在步骤(1)结束后，采用实施例1的方法检测种子液的纤维小体比活力、产量和细胞生物量。
(2)全菌糖化：按照5％体积分数的接种量，将种子液直接接种到含有0.2kg预处理后的木质纤维素原料和5L的培养基溶液的反应体系中进行全菌糖化，预处理原料及全菌糖化的条件与实施例18相同。在此过程中，每12小时取样检测发酵液中还原糖的含量，计算底物中纤维素到可溶性糖的转化率。
实施例20：
与实施例1不同的是，用于木质纤维素的全菌糖化方法，包括以下步骤：
(1)种子液优化：
在厌氧条件下，将白色瘤胃球菌以20g/L的葡萄糖为唯一碳源的培养基，即葡萄糖培养基中，按照1％体积分数的接种比例，进行2次传代培养；然后接种到添加2g/L干重的预处理玉米芯的葡萄糖培养基中，在37℃的温度条件下诱导驯化40h，从而获得高生长速度、高纤维小体产量、高纤维小体比活力的全菌糖化种子液。所述的预处理方法同实施例5中的汽爆法预处理技术。在步骤(1)结束后，采用实施例1的方法检测种子液的纤维小体比活力、产量和细胞生物量。
(2)原料预浸渍：按照1:3的固液重量体积比，将0.2kg预处理后的木质纤维素原料和0.6L的培养基溶液在2r/min的转速条件下混合均匀，并持续48h；所述预处理后的木质纤维素原料与步骤(1)中相同。所述混合均匀为通过容器的整体滚动翻转实现混合均匀。
(3)全菌糖化：将步骤(1)优化的全菌糖化种子液以10％体积分数的接种量接种到步骤(2)得到的预浸渍后的反应体系中，并继续在2r/min的转速条件下进行糖化反应，得到含有葡萄糖的糖液。在此过程中，每12小时取样检测发酵液中还原糖的含量，计算底物中纤维素到可溶性糖的转化率。
实施例21：
与实施例20不同的是，用于木质纤维素的全菌糖化方法，不经驯化及预浸渍步骤，包括以下步骤：
(1)种子液培养：将白色瘤胃球菌在以20g/L预处理玉米芯为碳源的培养基(葡萄糖培养基除葡萄糖以外的成分)中培养40h。在步骤(1)结束后，采用实施例1的方法检测种子液的纤维小体比活力、产量和细胞生物量。
(2)全菌糖化：按照10％体积分数的接种量，将种子液直接接种到含有0.2kg预处理后的木质纤维素原料和0.6L的培养基溶液的反应体系中进行全菌糖化，预处理原料及全菌糖化 的条件与实施例20相同。在此过程中，每12小时取样检测发酵液中还原糖的含量，计算底物中纤维素到可溶性糖的转化率。
实施例22：
与实施例1不同的是，用于木质纤维素的全菌糖化方法，包括以下步骤：
(1)种子液优化：
在厌氧条件下，将黄化瘤胃球菌以20g/L的葡萄糖为唯一碳源的培养基，即葡萄糖培养基中，按照1％体积分数的接种比例，进行2次传代培养；然后接种到添加2g/L干重的预处理玉米芯的葡萄糖培养基中，在40℃的温度条件下诱导驯化48h，从而获得高生长速度、高纤维小体产量、高纤维小体比活力的全菌糖化种子液。所述的预处理方法为文献(生物加工过程，2010，3，66-72)中的稀酸水解预处理技术。在步骤(1)结束后，采用实施例1的方法检测种子液的纤维小体比活力、产量和细胞生物量。
(2)原料预浸渍：按照1:5的固液重量体积比，将0.4kg预处理后的木质纤维素原料和2L的培养基溶液在10r/min的转速条件下混合均匀，并持续36h；所述预处理后的木质纤维素原料与步骤(1)中相同。所述混合均匀为通过容器的整体滚动翻转实现混合均匀。
(3)全菌糖化：将步骤(1)优化的全菌糖化种子液以10％体积分数的接种量接种到步骤(2)得到的预浸渍后的反应体系中，并继续在10r/min的转速条件下进行糖化反应，得到含有葡萄糖的糖液。在此过程中，每12小时取样检测发酵液中还原糖的含量，计算底物中纤维素到可溶性糖的转化率。
实施例23：
与实施例22不同的是，用于木质纤维素的全菌糖化方法，不经驯化及预浸渍步骤，包括以下步骤：
(1)种子液培养：将黄化瘤胃球菌在以20g/L预处理玉米芯为碳源的培养基(葡萄糖培养基除葡萄糖以外的成分)中培养48h。在步骤(1)结束后，采用实施例1的方法检测种子液的纤维小体比活力、产量和细胞生物量。
(2)全菌糖化：按照10％体积分数的接种量，将种子液直接接种到含有0.4kg预处理后的木质纤维素原料和2L的培养基溶液的反应体系中进行全菌糖化，预处理原料及全菌糖化的条件与实施例22相同。在此过程中，每12小时取样检测发酵液中还原糖的含量，计算底物中纤维素到可溶性糖的转化率。
表1.实施例1-22全菌糖化过程中的指标参数
Figure PCTCN2019097813-appb-000001
注：
1.生长速度是指生长到对数中期所需的时间。
2.延滞期是指全菌糖化过程中底物中纤维素到可溶性糖的转化率低于10％(质量比)的时间。
3.水解周期是指全菌糖化过程中底物中纤维素到可溶性糖的转化率达到80％(质量比)的时间。
由表1可知，
(1)根据四组实施例8-9、14-15、和16-17的对比可知，采用本申请所述的用于木质纤维素的全菌糖化方法，所得到的种子液的纤维小体的比活力为4.6-17.8U/mg；纤维小体的产量为4.6-6.3mg/mL；种子生长速度为10-36小时。糖液中葡萄糖的产量为10.6-54.8g/L；延滞期为0.5-1.0天；糖化周期为6.5-14天。
采用纤维二糖为碳源的情况下，纤维小体的比活力为1.3-4.1U/mg；纤维小体的产量为1.4-2.1mg/mL；种子生长速度为12-36小时。糖液中葡萄糖的产量为10.7-50.3g/L；延滞期为2-3天；糖化周期为10-17.5天。
说明，与采用纤维二糖为碳源的情况相比，采用本申请所述的用于木质纤维素的全菌糖化方法所获得的种子液的纤维小体的比活力和纤维小体的产量均大大提高；延滞期和糖化周期大大缩短；种子生长速度以及糖液中葡萄糖的产量几乎持平。
(2)根据四组实施例4-5、10-11、和12-13的对比可知，用本申请所述的用于木质纤维素的全菌糖化方法，所得到的种子液的纤维小体的比活力为4.1-17.7U/mg；纤维小体的产量为13.9-25.5mg/mL；种子生长速度为16-32小时。糖液中葡萄糖的产量为26.6-62.5g/L；延滞期为0.5-1.5天；糖化周期为7.5-13.5天。
采用微晶纤维素为碳源的情况下，纤维小体的比活力为3.3-17.1U/mg；纤维小体的产量为15.0-21.3mg/mL；种子生长速度为24-40小时。糖液中葡萄糖的产量为23.9-63.1g/L；延滞期为2.5-4.5天；糖化周期为12-18天。
说明，与采用微晶纤维素为碳源的情况相比，采用本申请所述的用于木质纤维素的全菌糖化方法所获得的种子液的纤维小体的比活力、纤维小体的产量和糖液中葡萄糖的产量几乎持平；而种子生长速度大大提高，延滞期和糖化周期大大缩短。
(3)根据四组实施例1-2、18-19、20-21和22-23的对比可知，采用本申请所述的用于木质纤维素的全菌糖化方法，所得到的种子液的纤维小体的比活力为4.1-14.8U/mg；纤维小体的产量为7.9-15.2mg/mL；种子生长速度为40-48小时。糖液中葡萄糖的产量为27.1-112.5g/L；延滞期为0.5-1.5天；糖化周期为9.5-18.5天。
采用木质纤维素为碳源的情况下，纤维小体的比活力为5.1-15.3U/mg；纤维小体的产量为8.1-14.8mg/mL；种子生长速度为66-72小时。糖液中葡萄糖的产量为24.7-103.1g/L；延滞期为2.5-5.0天；糖化周期为15-26.5天。
说明，与采用木质纤维素为碳源的情况相比，采用本申请所述的用于木质纤维素的全菌糖化方法所获得的种子液的纤维小体的比活力、纤维小体的产量和糖液中葡萄糖的产量几乎持平；而种子生长速度大大提高，延滞期和糖化周期大大缩短。
综上可知，(1)本发明所获得的种子液的纤维小体的比活力与微晶纤维素为碳源生产的纤维小体一致、高于以纤维二糖为碳源生产的纤维小体；(2)本发明所获得的种子液的纤维小体产量与纤维素为碳源生产的纤维小体一致；高于以纤维二糖为碳源生产的纤维小体；(3)本发明所获得的种子液的种子生长速度与以纤维二糖为碳源生长的种子一致，高于纤维素为碳源生长的种子，远高于预处理木质纤维素为碳源生长的种子；(4)本发明中的延滞期和糖化周期与以纤维二糖/微晶纤维素/预处理木质纤维素为碳源相比，均大大缩短。
这说明，本发明所述的用于木质纤维素的全菌糖化方法，以相对廉价的葡萄糖添加低浓 度的预处理木质纤维素生物质为碳源，用于培养热纤梭菌等产纤维小体菌株，作为全菌糖化的种子液；与现有技术相比，所述全菌糖化的种子液实现了成本降低和周期缩短的效果，克服了现有技术中二者不可兼得的难题。此外，本发明所述的木质纤维素的全菌糖化方法，采用了原料预浸渍的技术，显著缩短了全菌糖化的适应缓冲期(延滞期)，特别是当高固含量的条件下，提高全菌糖化的效率，从而降低整个糖化过程的周期和成本。

Claims (10)

  1. 用于木质纤维素的全菌糖化方法，其特征在于：包括以下步骤：
    (1)种子液优化：在厌氧条件下，将产纤维小体菌株在葡萄糖培养基中进行传代驯化；然后接种到添加了预处理后的木质纤维素原料的葡萄糖培养基中进行诱导驯化，从而获得优化后的全菌糖化种子液；所述葡萄糖培养基是以葡萄糖为唯一碳源的培养基；
    (2)原料预浸渍：按照一定的固液重量体积比，将预处理后的木质纤维素原料和培养基溶液在容器中混合均匀，并持续一定时间；所述预处理后的木质纤维素原料与步骤(1)中相同；
    (3)全菌糖化：将步骤(1)优化的全菌糖化种子液接种到步骤(2)得到的预浸渍后的反应体系中，并在0.5-100r/min的转速条件下进行糖化反应，得到含有葡萄糖的糖液。
  2. 根据权利要求1所述的用于木质纤维素的全菌糖化方法，其特征在于：步骤(1)所述葡萄糖培养基中葡萄糖的浓度为1-20g/L；所述传代驯化进行2-5次；所述木质纤维素原料的添加量为0.1-2.0g/L。
  3. 根据权利要求1所述的用于木质纤维素的全菌糖化方法，其特征在于：所述诱导驯化的温度条件为34-65℃；所述诱导驯化的时间为12-48小时。
  4. 根据权利要求1所述的用于木质纤维素的全菌糖化方法，其特征在于：步骤(2)所述的木质纤维素原料与培养基溶液的固液重量体积比为1:3-1:50；所述的木质纤维素原料和培养基溶液在0.5-100r/min的转速条件下混合均匀并持续1-48h。
  5. 根据权利要求4所述的用于木质纤维素的全菌糖化方法，其特征在于：所述容器为带有搅拌桨的厌氧发酵设备或者厌氧旋转混匀器。
  6. 根据权利要求1所述的用于木质纤维素的全菌糖化方法，其特征在于：步骤(3)所述的全菌糖化种子液的接种量为0.1-10％的体积分数。
  7. 根据权利要求1-6中任意一项所述的用于木质纤维素的全菌糖化方法，其特征在于：其中，所述的产纤维小体菌株为热纤梭菌、黄色溶纤梭菌、嗜纤维梭菌、解纤维梭菌、解纤维醋弧菌、溶纤维假拟杆菌、白色瘤胃球菌或黄化瘤胃球菌。
  8. 根据权利要求1-6中任意一项所述的用于木质纤维素的全菌糖化方法，其特征在于：所述的预处理为碱法、稀酸法、水热法、汽爆法和磺化法预处理技术中的一种或多种的组合。
  9. 根据权利要求1-6中任意一项所述的用于木质纤维素的全菌糖化方法，其特征在于：所述木质纤维素原料为玉米秸秆、麦秸、灌木条枝、木片、、玉米芯、稻草和废纸中的一种或多种的组合。
  10. 根据权利要求1-6中任意一项所述的用于木质纤维素的全菌糖化方法，其特征在于：步骤(1)所述的葡萄糖培养基为每升水中添加磷酸氢二钾2.9g/L、磷酸二氢钾1.5g/L、尿素 0.8g/L、氯化钙0.1g/L、氯化镁1.8g/L、硫酸亚铁0.0005g/L、硫化钠2g/L、玉米浆4g/L、柠檬酸三钠2g/L、葡萄糖1-20g/L、pH 6.5-7.5。
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CN109055459A (zh) * 2018-08-17 2018-12-21 中国科学院青岛生物能源与过程研究所 用于木质纤维素的全菌糖化方法
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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009143481A1 (en) * 2008-05-23 2009-11-26 University Of Georgia Research Foundation, Inc. Paenibacillus ssp. and methods for fermentation of lignocellulosic materials
CN101824395A (zh) * 2009-03-06 2010-09-08 华东理工大学 一种以固体秸秆为碳源培养发酵种子液的方法
CN101851650A (zh) * 2010-04-19 2010-10-06 中国科学院青岛生物能源与过程研究所 一种纤维素原料的糖化方法
WO2014078716A1 (en) * 2012-11-16 2014-05-22 The Regents Of The University Of California Recombinant bacillus subtilis that can grow on plant biomass
CN109055459A (zh) * 2018-08-17 2018-12-21 中国科学院青岛生物能源与过程研究所 用于木质纤维素的全菌糖化方法

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101851656B (zh) * 2010-04-19 2012-08-29 中国科学院青岛生物能源与过程研究所 一种生产纤维素乙醇的方法
WO2012109578A2 (en) * 2011-02-11 2012-08-16 The Trustees Of Dartmouth College Clostridium thermocellum strains for enhanced ethanol production and method of their use
EP3008196B1 (en) * 2013-06-14 2021-11-17 The Trustees Of Dartmouth College Methods to enhance microbial conversion of cellulosic biomass with mechanical augmentation
CN104073526B (zh) * 2014-07-01 2016-08-24 浙江大学 一种利用木质纤维原料生产丙酮/丁醇的方法
CN108118020B (zh) * 2018-01-16 2020-06-12 中国科学院青岛生物能源与过程研究所 纤维素降解微生物的培养基、制备及其应用

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009143481A1 (en) * 2008-05-23 2009-11-26 University Of Georgia Research Foundation, Inc. Paenibacillus ssp. and methods for fermentation of lignocellulosic materials
CN101824395A (zh) * 2009-03-06 2010-09-08 华东理工大学 一种以固体秸秆为碳源培养发酵种子液的方法
CN101851650A (zh) * 2010-04-19 2010-10-06 中国科学院青岛生物能源与过程研究所 一种纤维素原料的糖化方法
WO2014078716A1 (en) * 2012-11-16 2014-05-22 The Regents Of The University Of California Recombinant bacillus subtilis that can grow on plant biomass
CN109055459A (zh) * 2018-08-17 2018-12-21 中国科学院青岛生物能源与过程研究所 用于木质纤维素的全菌糖化方法

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
HAROUN, B.M. ET AL.: "Significance of Acclimatization for Biohydrogen Production from Synthetic Lignocellulose Hydrolysate in Continuous-flow Systems", INTERNATIONAL JOURNAL OF HYDROGEN ENERGY, vol. 41, no. 32, 23 June 2016 (2016-06-23), pages 14003 - 14014, XP055364183, DOI: 10.1016/j.ijhydene.2016.05.289 *
YOAV, S. ET AL.: "How does Cellulosome Composition Influence Deconstruction of Lignocellulosic Substrates in Clostridium (Ruminiclostridium) Thermocellum DSM 1313?", BIOTECHNOLOGY FOR BIOFUELS, vol. 10, no. 222, 31 December 2017 (2017-12-31), pages 1 - 16, XP055686456 *

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