CN109097416B - Lignocellulose one-pot biotransformation method - Google Patents
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Abstract
The invention provides a lignocellulose one-pot biotransformation method, which comprises the following steps: (1) mechanical treatment: pulverizing lignocellulose raw material, cleaning, putting into a closed reactor, adding water, and soaking. (2) Chemical treatment: adding ammonium sulfite into the reactor, and after the sulfonation treatment is finished, allowing the black liquor to flow out; (3) cleaning materials: adding water into the reactor, and uniformly mixing; introducing high-pressure nitrogen to make the black liquor flow out; (4) saccharification: adding a saccharification culture medium into the container, and sterilizing at high temperature and high pressure; inoculating seed liquid of the strain producing the cellulose body, and saccharifying to obtain sugar liquid containing glucose. (5) Fermentation: inoculating activated aerobic heterotrophic microorganism seed liquid into the obtained fermentation medium, and fermenting. The method simplifies the process steps and the requirements on equipment, reduces the cost of carbon sources and the cost of enzyme, solves the problem of comprehensive utilization of agricultural and forestry wastes, and has great environmental benefit and social benefit.
Description
Technical Field
The invention belongs to the technical field of biology, relates to a bioconversion method of lignocellulose, and particularly relates to a whole-bacterium saccharification method for lignocellulose.
Background
In recent years, the harvesting of renewable energy, materials or chemicals from biomass has received widespread attention from countries throughout the world. The production of second generation bio-based fuels or chemicals from lignocellulosic biomass has become a key to the large-scale substitution of petroleum-based products. Lignocellulose biomass is the most abundant renewable biomass resource in the earth, and has great application and development prospects when being converted into energy, chemicals or materials. Particularly, China is a big agricultural country, and the annual crop straw yield exceeds 9 hundred million tons. On the other hand, the problems of environmental pollution, traffic safety accidents and the like caused by straw burning also promote the nation to put forward a series of plans, schemes and measures for comprehensive utilization of straws and promote the industrialized development of comprehensive utilization of agricultural waste biomass.
The conversion of lignocellulose into fuel or chemicals usually needs three step units of pretreatment, saccharification and fermentation, the common technical conditions of the different step units are greatly different, and the whole operation process is complex, high in energy consumption and simultaneously generates a large amount of waste water. Therefore, related technologies for integrating saccharification and fermentation links, including three processes, namely, fractional hydrolysis fermentation (SHF), Simultaneous Saccharification Fermentation (SSF), and integrated bioprocessing (CBP), have been developed internationally. Wherein, SHF and SSF both need to firstly produce free cellulase system of fungal origin in independent reactors, and then carry out enzymatic hydrolysis and fermentation of cellulose on the pretreated raw material. CBP integrates the production of cellulase and each relatively independent technical link of enzymolysis and fermentation into the same step, and is carried out in the same reactor (Curr Opin Biotechnol 2005,16: 577-.
In addition, researchers have also conducted research into pretreatment techniques and downstream process integration techniques. Kataeva et al found that thermophilic bacteria Caldcellulosriptor bescii could hydrolyze substrate without pretreatment (Energy & Environmental science.2013.6: 2186;. invention patent application No. 201010149132.2 discloses "a method for saccharifying cellulose raw material". The method is based on a cellulosome secreted by thermophilic anaerobes, which can effectively degrade crystalline cellulose with a complex structure. the application enables cellulose material without pretreatment by physical or chemical methods to be directly fermented or enzymatically treated and efficiently hydrolyzed into reducing sugars, however, both of them have problems of low efficiency and complex final product composition, and have no value for application.A invention patent application No. 201610202657.5 discloses a "method for preparing fermentable sugar or bioethanol by biomass' one pot method". The method comprises the following steps of 1: (1) adding mechanically treated and sieved biomass into a sodium hydroxide/urea solution system under low temperature and stirring to room temperature; (2) adding acid to adjust the temperature Adjusting pH, adding enzyme for hydrolysis; determining the sugar content in the enzymolysis liquid, and calculating the enzymolysis conversion rate of sugar in the biomass raw material; (3) and after 24-48 h of enzymatic hydrolysis, inoculating a strain into the enzymatic hydrolysate, fermenting to prepare bioethanol, and measuring the yield of the target product. The application completes pretreatment, enzymatic hydrolysis and fermentation in one pot, is convenient to operate and short in time consumption, and reduces loss of lignocellulose substrates and generation of a large amount of waste water in the process. However, this method requires the addition of acid for the neutralization of the lye, the amount of chemicals used is large and the yield of the end product is low. In addition, free cellulase preparations are adopted in the enzymolysis process, and the existing enzyme preparation technology is monopolized by developed national companies, so that the production process adopting the cellulase preparations is generally high in cost and has no economical efficiency.
The sulfonation method is a method for removing lignin industrially and mostly, namely, the lignin is sulfonated by sulfite or bisulfite, and the sulfonated lignin is easier to remove due to the increase of hydrophilicity. Lvjiaqing and the like adopt ammonium sulfite to pretreat wheat straw material preparation waste residues (China paper making, 2015,34,1-6), use 16 percent of ammonium sulfite to pretreat the wheat straw material waste residues at 165 ℃, obtain a substrate by PFI pulp grinding, and the total sugar conversion rate of enzyme hydrolysis can reach 67 percent. Yu et al found (Bioresource Technology,2016,199, 188-. However, the lignocellulose raw material pretreated by the sulfonation method is rich in free lignin components, namely black liquor is formed, the pH value is high, the lignin is obviously inhibited to enzyme preparations, and the next step of enzymolysis and saccharification can be carried out only by repeatedly cleaning the raw material by special pulp washing equipment after blanking; and the lignocellulose raw material is subjected to sulfonation pretreatment alone, mixed sugar is obtained after enzyme hydrolysis, and the utilization efficiency of hemicellulose is low. The invention patent application 201610133959.1 discloses a "method for pre-treating and separating lignocellulosic biomass" comprising the steps of: 1) firstly, mechanically crushing lignocellulose biomass to a proper particle size, and then pre-impregnating; 2) mixing the solid obtained in the step 1) with water or steam to carry out hydrothermal pretreatment; 3) mixing the solid material which is obtained in the step 2) and is subjected to hydrothermal pretreatment and cleaning with a sulfonation pretreatment liquid medicine for sulfonation pretreatment. The application combines hydrothermal pretreatment and sulfonation pretreatment, so that hemicellulose and lignin can be effectively removed, the cellulose content of a substrate is obviously improved, and the enzymatic hydrolysis efficiency of the substrate is greatly improved.
Compared with the process depending on enzyme preparations, the biological saccharification strategy based on the whole-bacteria catalysis of the cellulose has the advantages of simplifying the flow, reducing the equipment requirement and the like, and is a process route most suitable for the biotransformation and utilization of cellulose biomass. Cellulosome is an extracellular multienzyme complex with a complex structure and composition, and is one of the most efficient cellulose degradation systems known in nature. Currently, the catalytic saccharification of lignocellulose mainly utilizes thermophilic anaerobes based on clostridium thermocellum, a cellulosome, as a whole bacteria catalyst. To date, no one-pot method for catalyzing and saccharifying lignocellulose by using whole bacteria has been reported.
Disclosure of Invention
Aiming at the problems of complicated steps, poor technical matching, high cost, serious pollution and the like in the utilization of lignocellulose in the prior art, the invention provides the method for the one-pot biotransformation of lignocellulose, which has the advantages of low cost, simple process and the like, reduces the discharge of waste water and has good industrial application prospect.
A lignocellulose one-pot biotransformation method comprises the following steps:
(1) mechanical treatment: mechanically crushing the lignocellulose raw material, cleaning with water, then putting the lignocellulose raw material into a closed reactor from a feeding port above the closed reactor, adding water according to the solid-liquid weight volume ratio of 1:2-1:6, uniformly mixing, and soaking.
Wherein the particle size of the fragments of the mechanically crushed lignocellulose raw material is not more than 2cm, and the soaking time is 2-4 h. The lignocellulose raw material is one or a combination of a plurality of corn stalks, wheat straws, shrub branches, wood chips, corncobs, rice straws and waste paper.
(2) Chemical treatment: adding a proper amount of ammonium sulfite into the closed reactor, and carrying out sulfonation treatment for 1-4h at the temperature of 160-190 ℃. After the reaction is finished, opening a discharge opening at the bottom of the closed reactor, and allowing the black liquor to flow out of a storage tank through a filtering device arranged at the discharge opening; the storage tank is connected with the closed reactor through a pipeline. When the pressure in the closed reactor is reduced to the atmospheric pressure, a vacuum pump at the top of the storage tank is used for pumping vacuum, so that negative pressure is formed in the storage tank, and black liquor further flows out; and closing the discharge opening until the water content of the materials in the closed reactor reaches 30-50%.
Wherein the final concentration of the ammonium sulfite in the closed reactor is 9-15% (w/v).
(3) Cleaning materials: adding water into the closed reactor, and uniformly mixing; introducing high-pressure nitrogen into the closed reactor to make the pressure in the reactor reach 10-12 standard atmospheric pressures, then opening a discharge opening at the bottom of the closed reactor to make the black liquor flow out into a storage tank through a filtering device equipped with the discharge opening. When the pressure in the closed reactor is reduced to the atmospheric pressure, a vacuum pump at the top of the storage tank is used for pumping vacuum, so that negative pressure is formed in the storage tank, and black liquor further flows out; and closing the discharge opening until the water content of the materials in the closed reactor reaches 30-50%. The amount of the added water is 1-2 times of the volume of the material after the step (2) treatment.
Wherein the material washing step can be repeated 1-4 times until the pH of the material is not higher than 7.5. Black liquor is removed by suction filtration washing, so that the addition of acid is avoided, and the use of chemicals is reduced; and black liquor can be used for preparing organic fertilizer to be returned to the field through modification, so that the process economy is improved, and the wastewater discharge is reduced. In addition, after the material subjected to sulfonation pretreatment is subjected to suction filtration and washing, the requirement of a saccharification step is completely met, and the key step for realizing the one-pot method is realized.
(4) Saccharification: adding a saccharification culture medium into a closed container according to the solid-liquid weight-volume ratio of 1:2-1:25, and sterilizing at high temperature and high pressure; inoculating seed liquid of the strain producing the cellulosome, and performing saccharification reaction at 34-65 ℃ and pH of 5.8-6.5 by adopting an intermittent stirring mode to obtain sugar liquid containing glucose. And the mixing mode of intermittent stirring is adopted, so that the energy consumption and the equipment loss in the saccharification stage can be greatly reduced on the premise of ensuring the mass transfer efficiency, and the saccharification efficiency is improved.
Wherein the batch stirring is: stirring for 5-10 minutes every 1-6 hours, wherein the rotation speed of the batch stirring is 0.5-20 rpm. During saccharification, the pH is controlled to 5.8-6.5 by feeding sodium hydroxide.
The strain capable of producing the cellulosome is clostridium thermocellum, clostridium flavum, clostridium cellulophilus, clostridium cellulolyticum, vibrio cellulolyticum, bacteroides cellulolyticus, ruminococcus albus or ruminococcus xanthans; the inoculation amount is 2-10% (v/v).
The saccharification culture medium is prepared by adding 2.9g/L dipotassium phosphate, 1.5g/L potassium dihydrogen phosphate, 0.8g/L urea, 0.1g/L calcium chloride, 1.8g/L magnesium chloride, 0.0005g/L ferrous sulfate, 2g/L sodium sulfide, 4g/L corn steep liquor and 2g/L, pH 6.5.5-7.5 of trisodium citrate into each liter of water.
(5) Fermentation: when the concentration of glucose in the closed reactor reaches 25-180g/L, introducing sterile air, and adding culture medium components to obtain a fermentation culture medium; then inoculating activated aerobic heterotrophic microorganism seed solution, stirring well, fermenting at 15-38 deg.C under nitrogen supplement and pH of 6.5-9.0 for 1.5-7 days to obtain aerobic heterotrophic microorganism fermentation liquor, and measuring biomass.
Wherein the oil-producing microorganism is Schizochytrium, Nannochloropsis, Mortierella alpina, Chlorella, Phaffia rhodozyma or Candida, and the inoculation amount is 5-10% (v/v). The stirring speed is 100-200rpm, and the ventilation volume is 0.2-2 vvm.
The culture medium comprises the following components: 0.2-30g/L of yeast extract, 0.2-20g/L of peptone, 6-30g/L of corn steep liquor, 0.5-8g/L of monopotassium phosphate, 0.5-5g/L of magnesium sulfate, 0-30g/L of sea crystal, 0-200 mg/L of vitamin B13, vitamin B63-200mg/L, vitamin B121-50 mg/L and 1-50mg/L of biotin.
The lignocellulose one-pot biotransformation device comprises a closed reactor 5 and a storage tank 8 communicated with a vacuum device 7; the upper part of the closed reactor 5 is provided with an air inlet 2 and a feed inlet 3, a heating device, a temperature control device and a stirring paddle 4 connected with a motor 1 are arranged inside the closed reactor 5, and the bottom of the closed reactor 5 is provided with a discharge opening 6; the discharge opening 6 is communicated with the inlet of a storage tank 8; and a filtering device is arranged on the discharge opening 6, and an outlet is arranged at the bottom of the storage tank 8. By adopting the device, the one-pot method biotransformation of lignocellulose can be realized, and the problem that a device matched with the one-pot method is lacked in the prior art is solved.
The invention has the beneficial effects that:
(1) the method for the one-pot biotransformation of lignocellulose connects the pretreatment, saccharification and fermentation processes in series and is carried out in the same reactor, thereby greatly simplifying the process steps and the requirements on equipment.
(2) The invention adopts the cellulase preparation based on the cellulosome-producing bacteria to realize the saccharification of lignocellulose, avoids the addition of free enzyme preparation and obviously reduces the enzyme cost in the saccharification stage.
(3) The invention combines lignocellulose biomass as raw material with oil fermentation production, on one hand, the cost of carbon source is reduced, on the other hand, the problem of comprehensive utilization of agricultural and forestry wastes is solved, and the invention has great environmental benefit and social benefit.
Drawings
FIG. 1 is a schematic structural diagram of a lignocellulose one-pot bioconversion device of the present invention.
Detailed Description
The present invention will be further described with reference to the following examples.
Example 1: lignocellulose one-pot biotransformation method
A lignocellulose one-pot biotransformation method comprises the following steps:
(1) mechanical treatment: mechanically crushing the corn straws until the particle size of the fragments is not more than 2 cm; cleaning with water, then putting the mixture into a closed reactor from a feeding port above the closed reactor, adding water according to the solid-liquid weight-volume ratio of 1:2, uniformly mixing, and soaking for 4 hours.
(2) Chemical treatment: ammonium sulfite was added to the closed reactor to a final concentration of 11% (w/v), and sulfonation was carried out at 175 ℃ for 3.5 hours. After the reaction is finished, opening a discharge opening at the bottom of the closed reactor, and allowing the black liquor to flow out of a storage tank through a filtering device arranged at the discharge opening; the storage tank is connected with the closed reactor through a pipeline. When the pressure in the closed reactor is reduced to the atmospheric pressure, a vacuum pump at the top of the storage tank is used for pumping vacuum, so that negative pressure is formed in the storage tank, and black liquor further flows out; and closing the discharge opening until the water content of the materials in the closed reactor reaches 30-50%.
(3) Cleaning materials: adding water with the volume 1 time that of the material into the closed reactor; introducing high-pressure nitrogen into the closed reactor to make the pressure in the reactor reach 12 standard atmospheric pressures, then opening a discharge opening at the bottom of the closed reactor to make the black liquor flow out into a storage tank through a filtering device equipped with the discharge opening. When the pressure in the closed reactor is reduced to the atmospheric pressure, a vacuum pump at the top of the storage tank is used for pumping vacuum, so that negative pressure is formed in the storage tank, and black liquor further flows out; and closing the discharge opening until the water content of the materials in the closed reactor reaches 30-50%. The material washing step was repeated 2 times until the pH of the material was not higher than 7.5.
(4) Saccharification: adding a saccharification culture medium into a closed container according to the solid-liquid weight-volume ratio of 1:6, and sterilizing at high temperature and high pressure; inoculating the clostridium thermocellum seed liquid, wherein the inoculation amount is 5% (v/v); carrying out saccharification reaction at 60 deg.C and pH of 5.8-6.5 by intermittent stirring to obtain glucose-containing sugar solution.
The intermittent stirring is as follows: stirring was carried out for 5 minutes every 1 hour, and the rotation speed of the batch stirring was 5 rpm. During saccharification, the pH is controlled to 5.8-6.5 by feeding sodium hydroxide.
The saccharification culture medium is prepared by adding 2.9g/L dipotassium phosphate, 1.5g/L potassium dihydrogen phosphate, 0.8g/L urea, 0.1g/L calcium chloride, 1.8g/L magnesium chloride, 0.0005g/L ferrous sulfate, 2g/L sodium sulfide, 4g/L corn steep liquor and 2g/L, pH 6.5.5-7.5 of trisodium citrate into each liter of water.
(5) Fermentation: when the concentration of glucose in the closed reactor reaches 70-80g/L, introducing sterile air, and adding culture medium components to obtain the fermentation culture medium. Then inoculating activated schizochytrium limacinum seed liquid with the inoculation amount of 5% (v/v), and uniformly stirring; fermenting at 25 deg.C under rotation speed of 100rpm and ventilation of 0.2vvm for 3 days under nitrogen supplementation and pH of 6.5 to obtain aerobic heterotrophic microorganism fermentation broth, and measuring biomass.
The culture medium comprises the following components: 0.2-30g/L of yeast extract, 0.2-20g/L of peptone, 6-30g/L of corn steep liquor, 0.5-8g/L of monopotassium phosphate, 0.5-5g/L of magnesium sulfate, 0-30g/L of sea crystal, 0-200 mg/L of vitamin B13, vitamin B63-200mg/L, vitamin B121-50 mg/L and 1-50mg/L of biotin.
Example 2: lignocellulose one-pot biotransformation method
In contrast to example 1, a method for the one-pot bioconversion of lignocellulose comprises the following steps:
(1) mechanical treatment: mechanically crushing the wheat straws until the particle size of the fragments is not more than 2 cm; cleaning with water, then putting into a closed reactor from a feeding port above the closed reactor, adding water according to the solid-liquid weight-volume ratio of 1:3, uniformly mixing, and soaking for 2-4 h.
(2) Chemical treatment: ammonium sulfite was added to the closed reactor to a final concentration of 12% (w/v), and sulfonation was carried out at 180 ℃ for 4 hours. After the reaction is finished, the black liquor flows out to a storage tank until the water content of the materials in the closed reactor reaches 30-50%, and a discharge opening is closed.
(3) Cleaning materials: adding water with the volume 1 time that of the material into the closed reactor; introducing high-pressure nitrogen into the closed reactor to ensure that the pressure in the closed reactor reaches 12 standard atmospheric pressures, then opening a discharge opening to ensure that black liquor flows out into a storage tank until the water content of materials in the closed reactor reaches 30-50%, and closing the discharge opening.
The above material washing steps were repeated 4 times until the pH of the material was not higher than 7.5.
(4) Saccharification: adding a saccharification culture medium into a closed container according to the solid-liquid weight-volume ratio of 1:3.5, and sterilizing at high temperature and high pressure; inoculating the clostridium thermocellum seed liquid, wherein the inoculation amount is 6% (v/v); carrying out saccharification reaction at 60 deg.C and pH of 5.8-6.5 by intermittent stirring to obtain glucose-containing sugar solution.
The intermittent stirring is as follows: stirring was carried out for 5 minutes every 2 hours, and the rotation speed of the batch stirring was 2 rpm. During saccharification, the pH is controlled to 5.8-6.5 by feeding sodium hydroxide.
(5) Fermentation: when the glucose concentration in the closed reactor reaches 140-150g/L, introducing sterile air, and adding the culture medium components to obtain the fermentation culture medium. Then inoculating activated nannochloropsis oculata seed liquid with the inoculation amount of 8% (v/v), and stirring uniformly; fermenting at 30 deg.C under the conditions of rotation speed of 150rpm, ventilation of 1.0vvm, nitrogen supplement and pH8.0 for 6 days to obtain aerobic heterotrophic microorganism fermentation broth, and measuring biomass.
The culture medium comprises the following components: 0.2-30g/L of yeast extract, 0.2-20g/L of peptone, 6-30g/L of corn steep liquor, 0.5-8g/L of monopotassium phosphate, 0.5-5g/L of magnesium sulfate, 0-30g/L of sea crystal, 0-200 mg/L of vitamin B13, vitamin B63-200mg/L, vitamin B121-50 mg/L and 1-50mg/L of biotin.
Example 3: lignocellulose one-pot biotransformation method
In contrast to example 1, a method for the one-pot bioconversion of lignocellulose comprises the following steps:
(1) mechanical treatment: mechanically crushing shrub branches until the particle size of the fragments is not more than 2 cm; cleaning with water, then putting into a closed reactor from a feeding port above the closed reactor, adding water according to the solid-liquid weight-volume ratio of 1:4, uniformly mixing, and soaking for 2-4 h.
(2) Chemical treatment: ammonium sulfite was added to the closed reactor to a final concentration of 13% (w/v), and sulfonation was carried out at 185 ℃ for 1 hour. After the reaction is finished, the black liquor flows out to a storage tank until the water content of the materials in the closed reactor reaches 30-50%, and a discharge opening is closed.
(3) Cleaning materials: adding water with the volume 1.5 times that of the material into the closed reactor; introducing high-pressure nitrogen into the closed reactor to ensure that the pressure in the closed reactor reaches 12 standard atmospheric pressures, then opening a discharge opening to ensure that black liquor flows out into a storage tank until the water content of materials in the closed reactor reaches 30-50%, and closing the discharge opening.
The above material washing steps were repeated 4 times until the pH of the material was not higher than 7.5.
(4) Saccharification: adding a saccharification culture medium into a closed container according to the solid-liquid weight-volume ratio of 1:5, and sterilizing at high temperature and high pressure; inoculating the clostridium thermocellum seed liquid, wherein the inoculation amount is 8% (v/v); carrying out saccharification reaction at 60 deg.C and pH of 5.8-6.5 by intermittent stirring to obtain glucose-containing sugar solution.
The intermittent stirring is as follows: stirring was carried out for 10 minutes every 3 hours, and the rotation speed of the batch stirring was 0.5 rpm. During saccharification, the pH is controlled to 5.8-6.5 by feeding sodium hydroxide.
(5) Fermentation: when the concentration of glucose in the closed reactor reaches 90-110g/L, introducing sterile air, and adding culture medium components to obtain a fermentation culture medium. Then inoculating activated mortierella alpina seed liquid with the inoculation amount of 10% (v/v), and uniformly stirring; fermenting at 28 deg.C under rotation speed of 200rpm and ventilation of 2.0vvm for 6.5 days under nitrogen supplementation and pH of 7.5 to obtain aerobic heterotrophic microorganism fermentation broth, and measuring biomass.
The culture medium comprises the following components: 0.2-30g/L of yeast extract, 0.2-20g/L of peptone, 6-30g/L of corn steep liquor, 0.5-8g/L of monopotassium phosphate, 0.5-5g/L of magnesium sulfate, 0-30g/L of sea crystal, 0-200 mg/L of vitamin B13, vitamin B63-200mg/L, vitamin B121-50 mg/L and 1-50mg/L of biotin.
Example 4: lignocellulose one-pot biotransformation method
In contrast to example 1, a method for the one-pot bioconversion of lignocellulose comprises the following steps:
(1) mechanical treatment: mechanically crushing the wood chips until the particle size of the chips is not more than 2 cm; cleaning with water, then putting into a closed reactor from a feeding port above the closed reactor, adding water according to the solid-liquid weight volume ratio of 1:5, uniformly mixing, and soaking for 2-4 h.
(2) Chemical treatment: ammonium sulfite was added to the closed reactor to a final concentration of 14% (w/v), and sulfonation was carried out at 190 ℃ for 1.5 h. After the reaction is finished, the black liquor flows out to a storage tank until the water content of the materials in the closed reactor reaches 30-50%, and a discharge opening is closed.
(3) Cleaning materials: adding water with the volume 1.5 times that of the material into the closed reactor; introducing high-pressure nitrogen into the closed reactor to ensure that the pressure in the reactor reaches 10 standard atmospheric pressures, then opening a discharge opening to ensure that black liquor flows out into a storage tank until the water content of materials in the closed reactor reaches 30-50%, and closing the discharge opening.
The above material washing steps were repeated 4 times until the pH of the material was not higher than 7.5.
(4) Saccharification: adding a saccharification culture medium into a closed container according to the solid-liquid weight-volume ratio of 1:8, and sterilizing at high temperature and high pressure; inoculating the clostridium thermocellum seed liquid with the inoculation amount of 10% (v/v); carrying out saccharification reaction at 60 deg.C and pH of 5.8-6.5 by intermittent stirring to obtain glucose-containing sugar solution.
The intermittent stirring is as follows: stirring was carried out for 10 minutes every 6 hours, and the rotation speed of the batch stirring was 20 rpm. During saccharification, the pH is controlled to 5.8-6.5 by feeding sodium hydroxide.
(5) Fermentation: when the concentration of glucose in the closed reactor reaches 60-70g/L, introducing sterile air, and adding culture medium components to obtain the fermentation culture medium. Then inoculating activated chlorella seed liquid with the inoculation amount of 6% (v/v), and uniformly stirring; fermenting at rotation speed of 150rpm, ventilation amount of 0.5vvm, temperature of 15 deg.C, nitrogen supplement, pH of 7.0 for 2.5 days to obtain aerobic heterotrophic microorganism fermentation broth, and measuring biomass.
The culture medium comprises the following components: 0.2-30g/L of yeast extract, 0.2-20g/L of peptone, 6-30g/L of corn steep liquor, 0.5-8g/L of monopotassium phosphate, 0.5-5g/L of magnesium sulfate, 0-30g/L of sea crystal, 0-200 mg/L of vitamin B13, vitamin B63-200mg/L, vitamin B121-50 mg/L and 1-50mg/L of biotin.
Example 5: lignocellulose one-pot biotransformation method
In contrast to example 1, a method for the one-pot bioconversion of lignocellulose comprises the following steps:
(1) mechanical treatment: mechanically crushing the straws until the particle size of the fragments is not more than 2 cm; cleaning with water, then putting into a closed reactor from a feeding port above the closed reactor, adding water according to the solid-liquid weight-volume ratio of 1:6, uniformly mixing, and soaking for 2-4 h.
(2) Chemical treatment: ammonium sulfite was added to the closed reactor to a final concentration of 15% (w/v), and sulfonation was carried out at 160 ℃ for 2 hours. After the reaction is finished, the black liquor flows out to a storage tank until the water content of the materials in the closed reactor reaches 30-50%, and a discharge opening is closed.
(3) Cleaning materials: adding 2 times of water by volume of the material into the closed reactor; introducing high-pressure nitrogen into the closed reactor to ensure that the pressure in the reactor reaches 10 standard atmospheric pressures, then opening a discharge opening to ensure that black liquor flows out into a storage tank until the water content of materials in the closed reactor reaches 30-50%, and closing the discharge opening.
The above material washing steps were repeated 4 times until the pH of the material was not higher than 7.5.
(4) Saccharification: adding a saccharification culture medium into a closed container according to the solid-liquid weight-volume ratio of 1:10, and sterilizing at high temperature and high pressure; inoculating the clostridium thermocellum seed liquid with the inoculation amount of 2% (v/v); carrying out saccharification reaction at 60 deg.C and pH of 5.8-6.5 by intermittent stirring to obtain glucose-containing sugar solution.
The intermittent stirring is as follows: stirring was carried out for 5 minutes every 4 hours, and the rotation speed of the batch stirring was 10 rpm. During saccharification, the pH is controlled to 5.8-6.5 by feeding sodium hydroxide.
(5) Fermentation: when the concentration of glucose in the closed reactor reaches 50-60g/L, introducing sterile air, and adding culture medium components to obtain the fermentation culture medium. Then inoculating activated candida seed liquid, wherein the inoculation amount is 8% (v/v), and uniformly stirring; fermenting at rotation speed of 150rpm, ventilation amount of 1.2vvm, temperature of 38 deg.C, nitrogen supplement, pH of 9.0 for 5 days to obtain aerobic heterotrophic microorganism fermentation broth, and measuring biomass.
The culture medium comprises the following components: 0.2-30g/L of yeast extract, 0.2-20g/L of peptone, 6-30g/L of corn steep liquor, 0.5-8g/L of monopotassium phosphate, 0.5-5g/L of magnesium sulfate, 0-30g/L of sea crystal, 0-200 mg/L of vitamin B13, vitamin B63-200mg/L, vitamin B121-50 mg/L and 1-50mg/L of biotin.
Example 6: lignocellulose one-pot biotransformation method
In contrast to example 1, a method for the one-pot bioconversion of lignocellulose comprises the following steps:
(1) mechanical treatment: mechanically crushing the waste paper until the particle size of the fragments is not more than 2 cm; cleaning with water, then putting into a closed reactor from a feeding port above the closed reactor, adding water according to the solid-liquid weight-volume ratio of 1:2, uniformly mixing, and soaking for 2-4 h.
(2) Chemical treatment: ammonium sulfite was added to the closed reactor to a final concentration of 9% (w/v), and sulfonation was carried out at 165 ℃ for 2.5 hours. After the reaction is finished, the black liquor flows out to a storage tank until the water content of the materials in the closed reactor reaches 30-50%, and a discharge opening is closed.
(3) Cleaning materials: adding 2 times of water by volume of the material into the closed reactor; introducing high-pressure nitrogen into the closed reactor to ensure that the pressure in the reactor reaches 11 standard atmospheric pressures, then opening a discharge opening to ensure that black liquor flows out into a storage tank until the water content of materials in the closed reactor reaches 30-50%, and closing the discharge opening.
The above material washing steps were repeated 4 times until the pH of the material was not higher than 7.5.
(4) Saccharification: adding a saccharification culture medium into a closed container according to the solid-liquid weight-volume ratio of 1:2, and sterilizing at high temperature and high pressure; inoculating yellow clostridium cellulolyticum seed liquid, wherein the inoculation amount is 6% (v/v); carrying out saccharification reaction at 65 deg.C and pH of 5.8-6.5 by intermittent stirring to obtain glucose-containing sugar solution.
The intermittent stirring is as follows: stirring was carried out for 10 minutes every 5 hours, and the rotation speed of the batch stirring was 15 rpm. During saccharification, the pH is controlled to 5.8-6.5 by feeding sodium hydroxide.
(5) Fermentation: when the glucose concentration in the closed reactor reaches 160-. Then inoculating activated phaffia rhodozyma seed liquid with the inoculation amount of 10% (v/v), and uniformly stirring; fermenting at 18 deg.C under rotation speed of 150rpm and ventilation of 2.0vvm for 7 days under nitrogen supplementation and pH of 7.5 to obtain aerobic heterotrophic microorganism fermentation broth, and measuring biomass.
The culture medium comprises the following components: 0.2-30g/L of yeast extract, 0.2-20g/L of peptone, 6-30g/L of corn steep liquor, 0.5-8g/L of monopotassium phosphate, 0.5-5g/L of magnesium sulfate, 0-30g/L of sea crystal, 0-200 mg/L of vitamin B13, vitamin B63-200mg/L, vitamin B121-50 mg/L and 1-50mg/L of biotin.
Example 7: lignocellulose one-pot biotransformation method
In contrast to example 1, a method for the one-pot bioconversion of lignocellulose comprises the following steps:
(1) mechanical treatment: mechanically crushing the wheat straws until the particle size of the fragments is not more than 2 cm; cleaning with water, then putting into a closed reactor from a feeding port above the closed reactor, adding water according to the solid-liquid weight-volume ratio of 1:3, uniformly mixing, and soaking for 2-4 h.
(2) Chemical treatment: ammonium sulfite was added to the closed reactor to a final concentration of 10% (w/v), and sulfonation was carried out at 170 ℃ for 3 hours. After the reaction is finished, the black liquor flows out to a storage tank until the water content of the materials in the closed reactor reaches 30-50%, and a discharge opening is closed.
(3) Cleaning materials: adding water with the volume 1.2 times that of the material into the closed reactor; introducing high-pressure nitrogen into the closed reactor to ensure that the pressure in the reactor reaches 11 standard atmospheric pressures, then opening a discharge opening to ensure that black liquor flows out into a storage tank until the water content of materials in the closed reactor reaches 30-50%, and closing the discharge opening.
The above material washing steps were repeated 4 times until the pH of the material was not higher than 7.5.
(4) Saccharification: adding a saccharification culture medium into a closed container according to the solid-liquid weight-volume ratio of 1:15, and sterilizing at high temperature and high pressure; inoculating clostridium cellulophilus seed liquid, wherein the inoculation amount is 6% (v/v); carrying out saccharification reaction at 37 deg.C and pH of 5.8-6.5 by intermittent stirring to obtain glucose-containing sugar solution.
The intermittent stirring is as follows: stirring was carried out for 8 minutes every 3 hours, and the rotation speed of the batch stirring was 12 rpm. During saccharification, the pH is controlled to 5.8-6.5 by feeding sodium hydroxide.
(5) Fermentation: when the concentration of glucose in the closed reactor reaches 30-50g/L, introducing sterile air, and adding culture medium components to obtain the fermentation culture medium. Then inoculating activated schizochytrium limacinum seed liquid with the inoculation amount of 5% (v/v), and uniformly stirring; fermenting at 28 deg.C under conditions of rotation speed of 100rpm, ventilation of 0.8vvm, nitrogen supplement and pH7.0 for 3 days to obtain aerobic heterotrophic microorganism fermentation broth, and measuring biomass.
The culture medium comprises the following components: 0.2-30g/L of yeast extract, 0.2-20g/L of peptone, 6-30g/L of corn steep liquor, 0.5-8g/L of monopotassium phosphate, 0.5-5g/L of magnesium sulfate, 0-30g/L of sea crystal, 0-200 mg/L of vitamin B13, vitamin B63-200mg/L, vitamin B121-50 mg/L and 1-50mg/L of biotin.
Example 8: lignocellulose one-pot biotransformation method
In contrast to example 1, a method for the one-pot bioconversion of lignocellulose comprises the following steps:
(1) mechanical treatment: mechanically crushing the wheat straws until the particle size of the fragments is not more than 2 cm; cleaning with water, then putting into a closed reactor from a feeding port above the closed reactor, adding water according to the solid-liquid weight-volume ratio of 1:4, uniformly mixing, and soaking for 2-4 h.
(2) Chemical treatment: ammonium sulfite was added to the closed reactor to a final concentration of 11% (w/v), and sulfonation was carried out at 175 ℃ for 3.5 hours. After the reaction is finished, the black liquor flows out to a storage tank until the water content of the materials in the closed reactor reaches 30-50%, and a discharge opening is closed.
(3) Cleaning materials: adding water with the volume 1.2 times that of the material into the closed reactor; introducing high-pressure nitrogen into the closed reactor to ensure that the pressure in the reactor reaches 11 standard atmospheric pressures, then opening a discharge opening to ensure that black liquor flows out into a storage tank until the water content of materials in the closed reactor reaches 30-50%, and closing the discharge opening.
The above material washing steps were repeated 4 times until the pH of the material was not higher than 7.5.
(4) Saccharification: adding a saccharification culture medium into a closed container according to the solid-liquid weight-volume ratio of 1:10, and sterilizing at high temperature and high pressure; inoculating seed liquid of clostridium cellulolyticum, wherein the inoculation amount is 6% (v/v); carrying out saccharification reaction at 34 deg.C and pH of 5.8-6.5 by intermittent stirring to obtain glucose-containing sugar solution.
The intermittent stirring is as follows: stirring was carried out for 10 minutes every 3 hours, and the rotation speed of the batch stirring was 8 rpm. During saccharification, the pH is controlled to 5.8-6.5 by feeding sodium hydroxide.
(5) Fermentation: when the concentration of glucose in the closed reactor reaches 40-60g/L, introducing sterile air, and adding culture medium components to obtain the fermentation culture medium. Then inoculating activated schizochytrium limacinum seed liquid with the inoculation amount of 8% (v/v), and uniformly stirring; fermenting at rotation speed of 100rpm and aeration rate of 1.5vvm at 35 deg.C under nitrogen supplement and pH of 7.0 for 5.5 days to obtain aerobic heterotrophic microorganism fermentation broth, and measuring biomass.
The culture medium comprises the following components: 0.2-30g/L of yeast extract, 0.2-20g/L of peptone, 6-30g/L of corn steep liquor, 0.5-8g/L of monopotassium phosphate, 0.5-5g/L of magnesium sulfate, 0-30g/L of sea crystal, 0-200 mg/L of vitamin B13, vitamin B63-200mg/L, vitamin B121-50 mg/L and 1-50mg/L of biotin.
Example 9: lignocellulose one-pot biotransformation method
In contrast to example 1, a method for the one-pot bioconversion of lignocellulose comprises the following steps:
(1) mechanical treatment: mechanically crushing the wheat straws until the particle size of the fragments is not more than 2 cm; cleaning with water, then putting into a closed reactor from a feeding port above the closed reactor, adding water according to the solid-liquid weight volume ratio of 1:5, uniformly mixing, and soaking for 2-4 h.
(2) Chemical treatment: ammonium sulfite was added to the closed reactor to a final concentration of 12% (w/v), and sulfonation was carried out at 180 ℃ for 4 hours. After the reaction is finished, the black liquor flows out to a storage tank until the water content of the materials in the closed reactor reaches 30-50%, and a discharge opening is closed.
(3) Cleaning materials: adding water with the volume 1.8 times that of the material into the closed reactor; introducing high-pressure nitrogen into the closed reactor to ensure that the pressure in the reactor reaches 10.5 standard atmospheric pressures, then opening a discharge opening to ensure that black liquor flows out into a storage tank until the water content of materials in the closed reactor reaches 30-50%, and closing the discharge opening.
The above material washing steps were repeated 4 times until the pH of the material was not higher than 7.5.
(4) Saccharification: adding a saccharification culture medium into a closed container according to the solid-liquid weight-volume ratio of 1:4.5, and sterilizing at high temperature and high pressure; inoculating fiber-decomposing vibrio aceticus seed liquid, wherein the inoculation amount is 8% (v/v); carrying out saccharification reaction at 37 deg.C and pH of 5.8-6.5 by intermittent stirring to obtain glucose-containing sugar solution.
The intermittent stirring is as follows: stirring was carried out for 8 minutes every 4 hours, and the rotation speed of the batch stirring was 6 rpm. During saccharification, the pH is controlled to 5.8-6.5 by feeding sodium hydroxide.
(5) Fermentation: when the glucose concentration in the closed reactor reaches 110-130g/L, introducing sterile air, and adding the culture medium components to obtain the fermentation culture medium. Then inoculating activated schizochytrium limacinum seed liquid with the inoculation amount of 8% (v/v), and uniformly stirring; fermenting at 25 deg.C under rotation speed of 100rpm and ventilation of 1.8vvm for 3.5 days under nitrogen supplement and pH of 7.0 to obtain aerobic heterotrophic microorganism fermentation broth, and measuring biomass.
The culture medium comprises the following components: 0.2-30g/L of yeast extract, 0.2-20g/L of peptone, 6-30g/L of corn steep liquor, 0.5-8g/L of monopotassium phosphate, 0.5-5g/L of magnesium sulfate, 0-30g/L of sea crystal, 0-200 mg/L of vitamin B13, vitamin B63-200mg/L, vitamin B121-50 mg/L and 1-50mg/L of biotin.
Example 10: lignocellulose one-pot biotransformation method
In contrast to example 1, a method for the one-pot bioconversion of lignocellulose comprises the following steps:
(1) mechanical treatment: mechanically crushing the corncobs until the particle size of the fragments is not more than 2 cm; cleaning with water, then putting into a closed reactor from a feeding port above the closed reactor, adding water according to the solid-liquid weight-volume ratio of 1:6, uniformly mixing, and soaking for 2-4 h.
(2) Chemical treatment: ammonium sulfite was added to the closed reactor to a final concentration of 13% (w/v), and sulfonation was carried out at 185 ℃ for 1 hour. After the reaction is finished, the black liquor flows out to a storage tank until the water content of the materials in the closed reactor reaches 30-50%, and a discharge opening is closed.
(3) Cleaning materials: adding water with the volume 1.8 times that of the material into the closed reactor; introducing high-pressure nitrogen into the closed reactor to ensure that the pressure in the reactor reaches 10.5 standard atmospheric pressures, then opening a discharge opening to ensure that black liquor flows out into a storage tank until the water content of materials in the closed reactor reaches 30-50%, and closing the discharge opening.
The above material washing steps were repeated 4 times until the pH of the material was not higher than 7.5.
(4) Saccharification: adding a saccharification culture medium into a closed container according to the solid-liquid weight-volume ratio of 1:5, and sterilizing at high temperature and high pressure; inoculating a fiber dissolving pseudomonas seed solution, wherein the inoculation amount is 8% (v/v); carrying out saccharification reaction at 42 deg.C and pH of 5.8-6.5 by intermittent stirring to obtain glucose-containing sugar solution.
The intermittent stirring is as follows: stirring was carried out for 10 minutes every 6 hours, and the rotation speed of the batch stirring was 15 rpm. During saccharification, the pH is controlled to 5.8-6.5 by feeding sodium hydroxide.
(5) Fermentation: when the concentration of glucose in the closed reactor reaches 60-80g/L, introducing sterile air, and adding culture medium components to obtain the fermentation culture medium. Then inoculating activated mortierella alpina seed liquid with the inoculation amount of 10% (v/v), and uniformly stirring; fermenting at 26 deg.C under rotation speed of 200rpm and ventilation of 1.5vvm for 3.5 days under nitrogen supplementation and pH of 7.0 to obtain aerobic heterotrophic microorganism fermentation broth, and measuring biomass.
The culture medium comprises the following components: 0.2-30g/L of yeast extract, 0.2-20g/L of peptone, 6-30g/L of corn steep liquor, 0.5-8g/L of monopotassium phosphate, 0.5-5g/L of magnesium sulfate, 0-30g/L of sea crystal, 0-200 mg/L of vitamin B13, vitamin B63-200mg/L, vitamin B121-50 mg/L and 1-50mg/L of biotin.
Example 11: lignocellulose one-pot biotransformation method
In contrast to example 1, a method for the one-pot bioconversion of lignocellulose comprises the following steps:
(1) mechanical treatment: mechanically crushing the waste paper until the particle size of the fragments is not more than 2 cm; cleaning with water, then putting into a closed reactor from a feeding port above the closed reactor, adding water according to the solid-liquid weight-volume ratio of 1:3, uniformly mixing, and soaking for 2-4 h.
(2) Chemical treatment: ammonium sulfite was added to the closed reactor to a final concentration of 14% (w/v), and sulfonation was carried out at 190 ℃ for 1.5 h. After the reaction is finished, the black liquor flows out to a storage tank until the water content of the materials in the closed reactor reaches 30-50%, and a discharge opening is closed.
(3) Cleaning materials: adding 2 times of water by volume of the material into the closed reactor; introducing high-pressure nitrogen into the closed reactor to ensure that the pressure in the reactor reaches 11.5 standard atmospheric pressures, then opening a discharge opening to ensure that black liquor flows out into a storage tank until the water content of materials in the closed reactor reaches 30-50%, and closing the discharge opening.
The above material washing steps were repeated 4 times until the pH of the material was not higher than 7.5.
(4) Saccharification: adding a saccharification culture medium into a closed container according to the solid-liquid weight-volume ratio of 1:25, and sterilizing at high temperature and high pressure; inoculating a white rumen coccus seed solution, wherein the inoculation amount is 8% (v/v); carrying out saccharification reaction at 37 deg.C and pH of 5.8-6.5 by intermittent stirring to obtain glucose-containing sugar solution.
The intermittent stirring is as follows: stirring was carried out for 6 minutes every 1 hour, and the rotation speed of the batch stirring was 5 rpm. During saccharification, the pH is controlled to 5.8-6.5 by feeding sodium hydroxide.
(5) Fermentation: when the concentration of glucose in the closed reactor reaches 25-35g/L, introducing sterile air, and adding culture medium components to obtain the fermentation culture medium. Then inoculating activated mortierella alpina seed liquid with the inoculation amount of 6% (v/v), and uniformly stirring; fermenting at 38 deg.C under rotation speed of 200rpm and ventilation of 1.5vvm for 1.5 days under nitrogen supplementation and pH of 6.5 to obtain aerobic heterotrophic microorganism fermentation broth, and measuring biomass.
The culture medium comprises the following components: 0.2-30g/L of yeast extract, 0.2-20g/L of peptone, 6-30g/L of corn steep liquor, 0.5-8g/L of monopotassium phosphate, 0.5-5g/L of magnesium sulfate, 0-30g/L of sea crystal, 0-200 mg/L of vitamin B13, vitamin B63-200mg/L, vitamin B121-50 mg/L and 1-50mg/L of biotin.
Example 12: lignocellulose one-pot biotransformation method
In contrast to example 1, a method for the one-pot bioconversion of lignocellulose comprises the following steps:
(1) mechanical treatment: mechanically crushing the corncobs until the particle size of the fragments is not more than 2 cm; cleaning with water, then putting into a closed reactor from a feeding port above the closed reactor, adding water according to the solid-liquid weight-volume ratio of 1:4, uniformly mixing, and soaking for 2-4 h.
(2) Chemical treatment: ammonium sulfite was added to the closed reactor to a final concentration of 15% (w/v), and sulfonation was carried out at 160 ℃ for 2 hours. After the reaction is finished, the black liquor flows out to a storage tank until the water content of the materials in the closed reactor reaches 30-50%, and a discharge opening is closed.
(3) Cleaning materials: adding 2 times of water by volume of the material into the closed reactor; introducing high-pressure nitrogen into the closed reactor to ensure that the pressure in the reactor reaches 11.5 standard atmospheric pressures, then opening a discharge opening to ensure that black liquor flows out into a storage tank until the water content of materials in the closed reactor reaches 30-50%, and closing the discharge opening.
The above material washing steps were repeated 4 times until the pH of the material was not higher than 7.5.
(4) Saccharification: adding a saccharification culture medium into a closed container according to the solid-liquid weight-volume ratio of 1:3, and sterilizing at high temperature and high pressure; inoculating a xanthate rumen coccus seed solution with the inoculation amount of 8% (v/v); carrying out saccharification reaction at 37 deg.C and pH of 5.8-6.5 by intermittent stirring to obtain glucose-containing sugar solution.
The intermittent stirring is as follows: stirring was carried out for 6 minutes every 1 hour, and the rotation speed of the batch stirring was 2 rpm. During saccharification, the pH is controlled to 5.8-6.5 by feeding sodium hydroxide.
(5) Fermentation: when the glucose concentration in the closed reactor reaches 100-120g/L, introducing sterile air, and adding culture medium components to obtain a fermentation culture medium. Then inoculating activated mortierella alpina seed liquid with the inoculation amount of 8% (v/v), and uniformly stirring; fermenting at rotation speed of 200rpm and ventilation volume of 2.0vvm at 15 deg.C under nitrogen supplement and pH of 7.5 for 7 days to obtain aerobic heterotrophic microorganism fermentation broth, and measuring biomass.
The culture medium comprises the following components: 0.2-30g/L of yeast extract, 0.2-20g/L of peptone, 6-30g/L of corn steep liquor, 0.5-8g/L of monopotassium phosphate, 0.5-5g/L of magnesium sulfate, 0-30g/L of sea crystal, 0-200 mg/L of vitamin B13, vitamin B63-200mg/L, vitamin B121-50 mg/L and 1-50mg/L of biotin.
Example 13: lignocellulose one-pot biotransformation device
The lignocellulose one-pot biotransformation device comprises a closed reactor 5 and a storage tank 8 communicated with a vacuum device 7; the upper part of the closed reactor 5 is provided with an air inlet 2 and a feed inlet 3, a heating device, a temperature control device and a stirring paddle 4 connected with a motor 1 are arranged inside the closed reactor 5, and the bottom of the closed reactor 5 is provided with a discharge opening 6; the discharge opening 6 is communicated with the inlet of a storage tank 8; and a filtering device is arranged on the discharge opening 6, and an outlet is arranged at the bottom of the storage tank 8. By adopting the device, the one-pot method biotransformation of lignocellulose can be realized, and the problem that a device matched with the one-pot method is lacked in the prior art is solved.
TABLE 1 examples 1-12 lignocellulose bioconversion results by one-pot method
As can be seen from Table 1, the cellulose saccharification rate by the lignocellulose one-pot biotransformation method of the present invention is 80.9-90.8%, and the biomass obtained by culturing Schizochytrium, Nannochloropsis, Mortierella alpina, Chlorella, Phaffia rhodozyma and Candida with the sugar solution can reach or exceed the level of the existing stepwise technology. The conversion rate of the glucose, namely the conversion rate of the glucose in the sugar solution to the biomass is 31.2-51.2 percent, which reaches the higher level of the prior fermentation technology.
The lignocellulose one-pot biotransformation method provided by the invention has the advantages that the pretreatment, saccharification and fermentation processes are carried out in series in the same reactor, on the premise that the biomass and glucose conversion rate reach higher levels in the prior art, the process steps and the requirements on equipment are simplified, the production cost is reduced, and the lignocellulose one-pot biotransformation method has important significance for industrialization. Moreover, the invention avoids the addition of free enzyme preparation, and obviously reduces the cost of enzyme used in the saccharification stage.
Claims (7)
1. The lignocellulose one-pot biotransformation method is characterized in that: the method comprises the following steps:
(1) mechanical treatment: mechanically crushing a lignocellulose raw material, cleaning, putting into a closed reactor, adding water according to the solid-liquid weight volume ratio of 1:2-1:6, uniformly mixing, and soaking;
(2) chemical treatment: adding a proper amount of ammonium sulfite into the closed reactor, and carrying out sulfonation treatment for 1-4h at the temperature of 160-190 ℃; the final concentration of the ammonium sulfite in the closed reactor is 9-15% by mass volume ratio; after the reaction is finished, opening a discharge opening at the bottom of the closed reactor to enable the black liquor to flow into a storage tank; when the pressure in the closed reactor is reduced to the atmospheric pressure, a vacuum pump at the top of the storage tank is used for pumping vacuum, so that negative pressure is formed in the storage tank, and black liquor further flows out; closing the discharge opening until the water content of the materials in the closed reactor reaches 30-50%;
(3) cleaning materials: adding water into the closed reactor, and uniformly mixing; introducing high-pressure nitrogen into the closed reactor to ensure that the pressure in the reactor reaches 10-12 standard atmospheric pressures, and then opening a discharge opening at the bottom of the closed reactor to ensure that black liquor flows into a storage tank; when the pressure in the closed reactor is reduced to the atmospheric pressure, a vacuum pump at the top of the storage tank is used for pumping vacuum, so that negative pressure is formed in the storage tank, and black liquor further flows out; closing the discharge port until the water content of the materials in the closed reactor reaches 30-50%; repeating for 1-4 times until the pH of the material is not higher than 7.5;
(4) saccharification: adding a saccharification culture medium into the closed reactor according to the solid-liquid weight-volume ratio of 1:2-1:25, and sterilizing at high temperature and high pressure; inoculating seed liquid of the strain producing the cellulosome, and performing saccharification reaction at the temperature of 34-65 ℃ and the pH of 5.8-6.5 by adopting an intermittent stirring mode to obtain sugar liquid containing glucose; the strain capable of producing the cellulosome is clostridium thermocellum, clostridium flavum, clostridium cellulophilus, clostridium cellulolyticum, vibrio cellulolyticus, pseudomonas cellulolyticus, ruminococcus albus or ruminococcus xanthans; the intermittent stirring is specifically as follows: stirring for 5-10 minutes every 1-6 hours, wherein the rotation speed of the batch stirring is 0.5-20 rpm;
(5) fermentation: when the concentration of glucose in the closed reactor reaches 25-180g/L, introducing sterile air, and adding culture medium components to obtain a fermentation culture medium; then inoculating activated aerobic heterotrophic microorganism seed solution, stirring well, fermenting at 15-38 deg.C under nitrogen supplement and pH of 6.5-9.0 for 1.5-7 days to obtain aerobic heterotrophic microorganism fermentation liquor, and measuring biomass.
2. The lignocellulose one-pot bioconversion process of claim 1, wherein: the amount of the added water in the step (3) is 1-2 times of the volume of the material treated in the step (2).
3. The lignocellulose one-pot bioconversion process of claim 2, wherein: the particle size of fragments of the mechanically crushed lignocellulose raw material in the step (1) is not more than 2cm, and the soaking time is 2-4 h; the lignocellulose raw material is one or a combination of a plurality of corn stalks, wheat straws, shrub branches, wood chips, corncobs, rice straws and waste paper.
4. The lignocellulose one-pot bioconversion process of claim 2, wherein: the inoculation amount of the seed liquid of the cellulosome-producing strain in the step (4) is 2-10% by volume.
5. The lignocellulose one-pot bioconversion process as recited in any one of claims 1-4, wherein: the saccharification culture medium specifically comprises: 2.9g/L dipotassium hydrogen phosphate, 1.5g/L potassium dihydrogen phosphate, 0.8g/L urea, 0.1g/L calcium chloride, 1.8g/L magnesium chloride, 0.0005g/L ferrous sulfate, 2g/L sodium sulfide, 4g/L corn steep liquor and 2g/L, pH 6.5.5-7.5 of trisodium citrate are added into each liter of water.
6. The lignocellulose one-pot bioconversion process as recited in any one of claims 1-4, wherein: the aerobic heterotrophic microorganism in the step (5) is schizochytrium limacinum, nannochloropsis, mortierella alpina, chlorella, phaffia rhodozyma or candida, and the inoculation amount is 5-10% by volume; the stirring speed is 100-200rpm, and the ventilation volume is 0.2-2 vvm.
7. The lignocellulose one-pot bioconversion process as recited in any one of claims 1-4, wherein: the culture medium in the step (5) comprises the following components: 0.2-30g/L of yeast extract, 0.2-20g/L of peptone, 6-30g/L of corn steep liquor, 0.5-8g/L of monopotassium phosphate, 0.5-5g/L of magnesium sulfate, 0-30g/L of sea crystal, 0-200 mg/L of vitamin B13, vitamin B63-200mg/L, vitamin B121-50 mg/L and 1-50mg/L of biotin.
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Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102864188A (en) * | 2011-07-07 | 2013-01-09 | 中国科学院青岛生物能源与过程研究所 | Method for producing biodiesel from lignocellulose |
| CN102888348A (en) * | 2012-07-12 | 2013-01-23 | 中国科学院青岛生物能源与过程研究所 | Schizochytrium limacinum and method or fermenting and producing DHA (Docosahexaenoic Acid) grease utilizing high density of schizochytrium limacinum |
| CN103468562A (en) * | 2013-09-13 | 2013-12-25 | 天津市滨海新区聚荣科技有限公司 | Material mixing and fermenting device |
| CN105087687A (en) * | 2014-05-04 | 2015-11-25 | 中国科学院大连化学物理研究所 | Method for preparing microbial oil from paper pulp produced by using ammonium sulfite process |
| CN205127900U (en) * | 2015-08-07 | 2016-04-06 | 中国石油天然气股份有限公司 | Polymer slurry discharging and separating device |
| CN105779512A (en) * | 2016-05-16 | 2016-07-20 | 中国科学院广州能源研究所 | Method for preparing fuel ethanol by carrying out mixed hydrolysis on wood fiber wastes |
| CN205676451U (en) * | 2016-06-08 | 2016-11-09 | 安聪聪 | A kind of tablet vinegar fermentor device |
| CN205821288U (en) * | 2016-06-14 | 2016-12-21 | 许吉钙 | A kind of compound saccharifying device for beer |
| CN107080138A (en) * | 2017-06-12 | 2017-08-22 | 六安市智奇工业设计有限公司 | A kind of red date drink preparation installation for fermenting |
| CN207412862U (en) * | 2017-09-21 | 2018-05-29 | 天津坤禾生物科技股份有限公司 | A kind of filter device for being exclusively used in microbial fermentation solution |
| CN108118020A (en) * | 2018-01-16 | 2018-06-05 | 中国科学院青岛生物能源与过程研究所 | Culture medium, preparation and its application of cellulose degradation microorganism |
-
2018
- 2018-08-17 CN CN201810939277.9A patent/CN109097416B/en active Active
Patent Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102864188A (en) * | 2011-07-07 | 2013-01-09 | 中国科学院青岛生物能源与过程研究所 | Method for producing biodiesel from lignocellulose |
| CN102888348A (en) * | 2012-07-12 | 2013-01-23 | 中国科学院青岛生物能源与过程研究所 | Schizochytrium limacinum and method or fermenting and producing DHA (Docosahexaenoic Acid) grease utilizing high density of schizochytrium limacinum |
| CN103468562A (en) * | 2013-09-13 | 2013-12-25 | 天津市滨海新区聚荣科技有限公司 | Material mixing and fermenting device |
| CN105087687A (en) * | 2014-05-04 | 2015-11-25 | 中国科学院大连化学物理研究所 | Method for preparing microbial oil from paper pulp produced by using ammonium sulfite process |
| CN205127900U (en) * | 2015-08-07 | 2016-04-06 | 中国石油天然气股份有限公司 | Polymer slurry discharging and separating device |
| CN105779512A (en) * | 2016-05-16 | 2016-07-20 | 中国科学院广州能源研究所 | Method for preparing fuel ethanol by carrying out mixed hydrolysis on wood fiber wastes |
| CN205676451U (en) * | 2016-06-08 | 2016-11-09 | 安聪聪 | A kind of tablet vinegar fermentor device |
| CN205821288U (en) * | 2016-06-14 | 2016-12-21 | 许吉钙 | A kind of compound saccharifying device for beer |
| CN107080138A (en) * | 2017-06-12 | 2017-08-22 | 六安市智奇工业设计有限公司 | A kind of red date drink preparation installation for fermenting |
| CN207412862U (en) * | 2017-09-21 | 2018-05-29 | 天津坤禾生物科技股份有限公司 | A kind of filter device for being exclusively used in microbial fermentation solution |
| CN108118020A (en) * | 2018-01-16 | 2018-06-05 | 中国科学院青岛生物能源与过程研究所 | Culture medium, preparation and its application of cellulose degradation microorganism |
Non-Patent Citations (2)
| Title |
|---|
| 几种主要的生物反应器介绍;许建和等;《生物催化工程》;华东理工大学出版社;20081031;第201-202页 * |
| 化学法;陆强等;《液体生物燃料技术与工程》;上海科学技术出版社;20130131;第140页第148页第153-155页 * |
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