CN113652465A - Method for preparing biochemical fulvic acid by fermenting rice straws with cellulose degradation composite bacterial system - Google Patents
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Abstract
The invention discloses a method for preparing biochemical fulvic acid by fermenting rice straws with a cellulose degradation composite bacterial system. The invention improves the accessibility of the cellulose based on the extrusion and expansion treatment of the structural characteristics of the rice straws; the fermentation process conditions are optimized through continuous liquid fermentation of a compound strain; on the basis, the non-heating ultrasonic wave auxiliary technology strengthens fermentation, realizes the high-efficiency conversion of fermentation substrates, obtains fermentation products rich in fulvic acid, strengthens the synergistic effect among strains in the continuous fermentation process, prevents the strain degeneration, and greatly shortens the fermentation period compared with solid state fermentation. The invention innovates the rice straw resource utilization mode, improves the efficiency of producing the biochemical fulvic acid by fermenting the rice straw, reduces the production cost, protects the environment, has strong practicability and is easy for large-scale production.
Description
Technical Field
The invention relates to the technical field of agricultural biology, in particular to a method for preparing biochemical fulvic acid by fermenting rice straws with cellulose degradation composite strains.
Background
Rice is an important grain crop in China and plays an extremely important role in grain production. A large amount of straws are generated in the rice planting process, and the rice straws are wastes left after rice harvesting, are also important agricultural resources, and can be used for producing feed, paper towels, biomass energy sources, organic fertilizer sources and the like. The rice planting area in China is large, 3000 hectares are achieved, the rice straw production amount is large, and the annual yield is about 2 hundred million tons. At present, mechanical returning is a main utilization mode of rice straws, but the problems of high energy consumption, poor treatment effect and the like exist, and the resource utilization of the rice straws is severely restricted. The stubble height after harvesting rice is about 40-50 cm generally, and the rice stubble is easy to wind the operation parts of the tillage machine when next-stubble crops are planted due to high rice stubble and high humidity in the field, so that the working performance and the operation efficiency are influenced; meanwhile, the rice straw stubble burying and returning effect is poor, the rotting period is long, and the utilization efficiency of organic matters is difficult to exert. In the practical agricultural production, in order to save farming season and cost and facilitate the planting of next-stubble crops, people mostly adopt a method of burning on the spot to treat the rice straws. Therefore, resources are wasted, serious environmental pollution is caused, and the method is not beneficial to environmental protection and sustainable development of agriculture. Therefore, researches and innovations on the rice straw resource utilization technology are urgent and necessary.
The cellulose content of the rice straw is about 30 percent, the hemicellulose content is about 25 percent, the lignin content is about 10 percent, and the rice straw also contains elements such as nitrogen, phosphorus, potassium, calcium, magnesium, zinc, iron, copper and the like. Cellulose, hemicellulose, lignin, nitrogen, phosphorus, potassium, calcium, magnesium, zinc, iron, copper and the like are important resources and are widely applied to the fields of agriculture, buildings, chemical industry and the like. By vigorously developing a rice straw resource utilization technology and recycling cellulose, hemicellulose, lignin, nitrogen, phosphorus, potassium, calcium, magnesium, zinc, iron, copper and the like in rice straws, the rice straws are changed into valuables, so that the rice straws really become an important available resource and the environmental pollution of the rice straws is certainly and greatly reduced. The development of the rice straw resource utilization technology is the national demand of China for actively coping with the serious challenge of agricultural non-point source pollution. The development of the rice straw recycling technology can realize the high-efficiency recycling of the waste rice straw, recycle resources, change waste into valuable and protect the environment. Therefore, the development of the rice straw resource utilization technology is significant.
Disclosure of Invention
Aiming at the problems of serious environmental pollution and resource loss in rice straw burning. The invention aims to provide a rice straw resource utilization method, which is characterized in that a rice straw pretreatment method, a method for preparing biochemical fulvic acid by fermenting rice straws by a high-efficiency cellulose degradation composite bacterial system are established, and a non-heating ultrasonic wave auxiliary technology is adopted to strengthen rice straw fermentation, so that rice straw biotransformation is realized, and biochemical fulvic acid products are obtained.
The purpose of the invention is realized by the following technical scheme.
The method for preparing the biochemical fulvic acid by fermenting the rice straws with the cellulose degradation composite bacterial system comprises the following steps of:
(1) after rice straws are cleaned and subjected to impurity removal, crushing by a rice straw crusher, wherein the amount of the treated rice straws is 4-10 tons per day, the size of the crushed rice straws is controlled to be 2-8 mm, and the water content of the crushed rice straws is adjusted to be 20-40%; then carrying out extrusion and puffing treatment on the rice straw powder, wherein the extrusion and puffing conditions are as follows: the temperature is 100-140 ℃, the clearance of a die hole is 3-6 mm, the rotating speed of a screw is 50-100 r/min, and the distance between the end part of the screw and a template is 10-20 mm;
(2) continuously fermenting the puffed rice straw composite bacterial system, and forming a seed preservation culture medium (per liter): potato 200 g, glucose 20 g and agar 15 g. Activation medium composition (/ liter): 200 g of potato and 20 g of glucose. The culture medium for fermentation seed production comprises the following components (liter): 10-40 g of extruded and puffed rice straw, 0.2-2 g of ammonium sulfate, 0.5-3 g of sodium nitrate, 0.1-0.6 g of dipotassium hydrogen phosphate, 0.05-0.5 g of sodium dihydrogen phosphate, 0.05-0.3 g of calcium chloride monohydrate, 0.01-0.2 g of ferrous sulfate heptahydrate, and 0.1-0.5 g of magnesium sulfate heptahydrate. Fermentation medium composition (/ liter): 100-200 g of extruded and puffed rice straw, 0.2-2 g of ammonium sulfate, 0.5-3 g of sodium nitrate, 0.1-0.6 g of dipotassium hydrogen phosphate, 0.05-0.5 g of sodium dihydrogen phosphate, 0.05-0.3 g of calcium chloride monohydrate, 0.01-0.2 g of ferrous sulfate heptahydrate, and 0.1-0.5 g of magnesium sulfate heptahydrate. The volume of the fermentation tank is 10 liters, the working volume is 5 liters, the inoculation amount is 5% -15%, and the fermentation strains are inoculated according to the proportion: the high-efficiency cellulose degradation composite bacteria system starts continuous fermentation when the content of fulvic acid in a fermentation tank reaches 8% -16%, the volume of fermentation liquor is maintained at 5 liters, the fermentation temperature is 32-40 ℃, the pH value is 5.8-6.8, and the rotation speed is 80-160 r/min. During continuous fermentation, a non-heating ultrasonic auxiliary technology is adopted to promote the fermentation of the rice straws.
The efficient cellulose degradation composite bacterial system in the step (2) of the invention is bacillus subtilis, streptomycete, trichoderma reesei and aspergillus niger.
The inoculation in the step (2) of the invention is carried out according to the proportion, namely the ratio of the bacillus subtilis to the streptomyces to the trichoderma reesei to the aspergillus niger is 5-1: 2-1: 4-1: 10-2.
The non-heating ultrasonic auxiliary technology in the step (2) of the invention is characterized in that fermentation liquor is ultrasonically treated every 5-8 days after the fermentation is started, and the ultrasonic conditions are as follows: the ultrasonic power is 240-450W, the ultrasonic time is 20-36 min, and the working time/intermittent time of one ultrasonic cycle is 10-25 s/5 s.
Compared with the prior art, the invention has the beneficial effects that:
(1) the invention establishes a method for preparing biochemical fulvic acid by fermenting rice straws with a cellulose degradation composite bacterial system, develops a rice straw recycling method, realizes the recycling of the rice straws through continuous fermentation biotransformation, greatly improves the recycling efficiency of the rice straws, achieves the purposes of resource recovery and environmental protection, is environment-friendly in the whole recycling process, and is a novel method which has strong practicability and meets the industrial requirements.
(2) The invention combines the non-heating ultrasonic auxiliary technology for assistance in the continuous fermentation process, realizes the high-efficiency conversion of the fermentation substrate to obtain the fermentation product rich in fulvic acid, strengthens the synergistic effect among strains in the continuous fermentation process, prevents the strain degeneration and greatly shortens the fermentation period compared with the solid fermentation.
(3) The invention extrudes and puffs the rice straws, and improves the accessibility of the strains to the cellulose.
Detailed Description
The invention will be further illustrated by the following examples. The examples merely represent preferred embodiments of the invention, which are described in greater detail and detail, but are not to be construed as limiting the scope of the invention. It should be noted that, for those skilled in the art, various changes, modifications and substitutions can be made without departing from the spirit of the present invention, and these are all within the scope of the present invention.
In the following examples, reagents such as glucose and agar and potatoes are commercially available, and Bacillus subtilis, Streptomyces, Trichoderma reesei, and Aspergillus niger were isolated and screened autonomously in the laboratory and stored.
The rice straw used in the following examples was collected from suburb rice fields in Nanchang.
Example 1.
After rice straws are cleaned and decontaminated, the rice straw pulverizer pulverizes the rice straws, the amount of the rice straws processed per day is 4 tons, the size of the pulverized rice straws is controlled to be 2 millimeters, and the water content of the pulverized rice straws is adjusted to be 20 percent. Then carrying out extrusion and puffing treatment on the rice straw powder, wherein the extrusion and puffing conditions are as follows: the temperature is 100 ℃, the clearance of a die hole is 3 mm, the rotating speed of a screw is 50 r/min, and the distance between the end part of the screw and a template is 10 mm, so that the puffed material is obtained. Continuously fermenting the puffed rice straw composite bacterial system, and forming a seed preservation culture medium (per liter): potato 200 g, glucose 20 g and agar 15 g. Activation medium composition (/ liter): 200 g of potato and 20 g of glucose. The culture medium for fermentation seed production comprises the following components (liter): 10 g of extruded and puffed rice straw, 0.2 g of ammonium sulfate, 0.5 g of sodium nitrate, 0.1 g of dipotassium phosphate, 0.05 g of sodium dihydrogen phosphate, 0.05 g of calcium chloride monohydrate, 0.01 g of ferrous sulfate heptahydrate and 0.1 g of magnesium sulfate heptahydrate. Fermentation medium composition (/ liter): 100 g of extruded and puffed rice straw, 0.2 g of ammonium sulfate, 0.5 g of sodium nitrate, 0.1 g of dipotassium phosphate, 0.05 g of sodium dihydrogen phosphate, 0.05 g of calcium chloride monohydrate, 0.01 g of ferrous sulfate heptahydrate and 0.1 g of magnesium sulfate heptahydrate. The volume of the fermentation tank is 10 liters, the working volume is 5 liters, the inoculation amount is 5 percent, the inoculation is carried out according to the proportion of 5:2:4:10, and the fermentation strain: the continuous fermentation is started when the content of fulvic acid in a fermentation tank reaches 8 percent, the volume of fermentation liquor is maintained at 5 liters, the fermentation temperature is 32 ℃, the pH value is 5.8, and the rotating speed is 80 r/min. The non-heating ultrasonic wave auxiliary technology strengthens fermentation, namely fermentation liquid is ultrasonically treated every 5 days at the beginning of fermentation, and the ultrasonic conditions are as follows: ultrasonic power 240 watts, ultrasonic time 20 minutes, working time/rest time of one ultrasonic cycle 10 seconds/5 seconds. The fermentation was continued for 3 months and the yield of fulvic acid was determined and the results are shown in Table 1.
Example 2.
After rice straws are cleaned and decontaminated, the rice straw crusher crushes the rice straws, the amount of the rice straws processed per day is 10 tons, the size of the crushed rice straws is controlled to be 8 millimeters, and the water content of the crushed rice straws is adjusted to 40 percent. Then carrying out extrusion and puffing treatment on the rice straw powder, wherein the extrusion and puffing conditions are as follows: the temperature is 140 ℃, the clearance of a die hole is 6 mm, the rotating speed of the screw is 100 r/min, and the distance between the end part of the screw and the template is 20 mm, so that the puffed material is obtained. Continuously fermenting the puffed rice straw composite bacterial system, and forming a seed preservation culture medium (per liter): potato 200 g, glucose 20 g and agar 15 g. Activation medium composition (/ liter): 200 g of potato and 20 g of glucose. The culture medium for fermentation seed production comprises the following components (liter): 40 g of extruded and puffed rice straw, 2 g of ammonium sulfate, 3 g of sodium nitrate, 0.6 g of dipotassium phosphate, 0.5 g of sodium dihydrogen phosphate, 0.3 g of calcium chloride monohydrate, 0.2 g of ferrous sulfate heptahydrate and 0.5 g of magnesium sulfate heptahydrate. Fermentation medium composition (/ liter): 200 g of extruded and puffed rice straw, 2 g of ammonium sulfate, 3 g of sodium nitrate, 0.6 g of dipotassium phosphate, 0.5 g of sodium dihydrogen phosphate, 0.3 g of calcium chloride monohydrate, 0.2 g of ferrous sulfate heptahydrate and 0.5 g of magnesium sulfate heptahydrate. The volume of the fermentation tank is 10 liters, the working volume is 5 liters, the inoculation amount is 15 percent, the inoculation is carried out according to the proportion of 1:1:1:2, and the fermentation strain: the continuous fermentation is started when the content of fulvic acid in a fermentation tank reaches 16 percent, the volume of fermentation liquor is maintained at 5 liters, the fermentation temperature is 40 ℃, the pH value is 6.8, and the rotating speed is 160 r/min. The non-heating ultrasonic wave auxiliary technology strengthens fermentation, namely fermentation liquid is ultrasonically treated every 8 days at the beginning of fermentation, and the ultrasonic conditions are as follows: ultrasonic power 450 watts, ultrasonic time 36 minutes, working time/rest time of one ultrasonic cycle 25 seconds/5 seconds. The fermentation was continued for 3 months and the yield of fulvic acid was determined and the results are shown in Table 1.
Example 3.
After rice straws are cleaned and decontaminated, the rice straw crusher crushes the rice straws, the amount of the rice straws processed per day is 7 tons, the size of the crushed rice straws is controlled to be 5 millimeters, and the water content of the crushed rice straws is adjusted to 30 percent. Then carrying out extrusion and puffing treatment on the rice straw powder, wherein the extrusion and puffing conditions are as follows: the temperature is 120 ℃, the clearance of a die hole is 6 mm, the rotating speed of a screw is 75 r/min, and the distance between the end part of the screw and a template is 15 mm, so that the puffed material is obtained. Continuously fermenting the puffed rice straw composite bacterial system, and forming a seed preservation culture medium (per liter): potato 200 g, glucose 20 g and agar 15 g. Activation medium composition (/ liter): 200 g of potato and 20 g of glucose. The culture medium for fermentation seed production comprises the following components (liter): 30 g of extruded and puffed rice straw, 1 g of ammonium sulfate, 1.5 g of sodium nitrate, 0.3 g of dipotassium phosphate, 0.3 g of sodium dihydrogen phosphate, 0.15 g of calcium chloride monohydrate, 0.1 g of ferrous sulfate heptahydrate and 0.3 g of magnesium sulfate heptahydrate. Fermentation medium composition (/ liter): 150 g of extruded and puffed rice straw, 0.1 g of ammonium sulfate, 0.2 g of sodium nitrate, 0.3 g of dipotassium phosphate, 0.25 g of sodium dihydrogen phosphate, 0.25 g of calcium chloride monohydrate, 0.1 g of ferrous sulfate heptahydrate and 0.3 g of magnesium sulfate heptahydrate. The volume of the fermentation tank is 10 liters, the working volume is 5 liters, the inoculation amount is 10 percent, the inoculation is carried out according to the proportion of 3:1.5:2.5:6, and the fermentation strain: the continuous fermentation is started when the content of fulvic acid in a fermentation tank reaches 12 percent, the volume of fermentation liquor is maintained at 5 liters, the fermentation temperature is 36 ℃, the pH value is 6.3, and the rotating speed is 120 r/min. The non-heating ultrasonic wave auxiliary technology strengthens fermentation, namely fermentation liquid is ultrasonically treated every 8 days at the beginning of fermentation, and the ultrasonic conditions are as follows: ultrasonic power 360 watts, ultrasonic time 28 minutes, working time/rest time of one ultrasonic cycle 20 seconds/5 seconds. The fermentation was continued for 3 months and the yield of fulvic acid was determined and the results are shown in Table 1.
Comparative example 1.
Seed retention medium composition (/ liter): potato 200 g, glucose 20 g and agar 15 g. Activation medium composition (/ liter): 200 g of potato and 20 g of glucose. The culture medium for fermentation seed production comprises the following components (liter): 30 g of rice straw, 1 g of ammonium sulfate, 1.5 g of sodium nitrate, 0.3 g of dipotassium phosphate, 0.3 g of sodium dihydrogen phosphate, 0.15 g of calcium chloride monohydrate, 0.1 g of ferrous sulfate heptahydrate and 0.3 g of magnesium sulfate heptahydrate. Fermentation medium composition (/ liter): 150 g of rice straw, 0.1 g of ammonium sulfate, 0.2 g of sodium nitrate, 0.3 g of dipotassium phosphate, 0.25 g of sodium dihydrogen phosphate, 0.25 g of calcium chloride monohydrate, 0.1 g of ferrous sulfate heptahydrate and 0.3 g of magnesium sulfate heptahydrate. Fermentation tank volume 10 liter, working volume 5 liter, inoculum size 10%, fermentation bacterial: the bacillus subtilis is continuously fermented, the volume of the fermentation liquid is maintained at 5 liters, the fermentation temperature is 36 ℃, the pH value is 6.3, and the rotating speed is 120 r/min. The fermentation was continued for 3 months and the yield of fulvic acid was determined and the results are shown in Table 1.
Comparative example 2.
750 g of rice straw, 10 liters of fermentation tank, 5 liters of working volume and 10 percent of inoculation amount, inoculating according to the proportion of 3:1.5:2.5:6, and fermenting the strains: continuously fermenting the bacillus subtilis, the streptomyces, the trichoderma reesei and the aspergillus niger, wherein the volume of fermentation liquid is maintained at 5 liters, the fermentation temperature is 36 ℃, the pH value is 6.3, and the rotating speed is 120 r/min. The fermentation was continued for 3 months and the yield of fulvic acid was determined and the results are shown in Table 1.
Table 1 examples and comparative results
| Examples | Yield of fulvic acid (kilogram) |
| Example 1 | 12 |
| Example 2 | 9 |
| Example 3 | 18 |
| Comparative example 1 | 0.5 |
| Comparative example 2 | 0.2 |
Claims (6)
1. The method for preparing the biochemical fulvic acid by fermenting the rice straws with the cellulose degradation compound bacteria system is characterized by comprising the following steps:
(1) pretreatment of rice straws: cleaning, impurity removing, crushing and drying the rice straws in sequence to form rice straw powder with the particle size of 2-8 mm and the water content of 20% -40%, and then carrying out extrusion and puffing treatment on the rice straw powder;
(2) fermenting the puffed rice straw powder by using a composite bacterial system: the volume of a fermentation tank is 10 liters, the working volume is 5 liters, strains are inoculated according to the proportion of 5% -15% of the inoculation amount, the strains are cellulose degradation composite strains, continuous fermentation is started when the content of fulvic acid in the fermentation tank reaches 8% -16%, the volume of fermentation liquor is maintained at 5 liters, the fermentation temperature is 32-40 ℃, the pH value is 5.8-6.8, and the rotation speed is 80-160 r/min; during continuous fermentation, a non-heating ultrasonic auxiliary technology is adopted to promote the fermentation of the rice straws.
2. The method for preparing the biochemical fulvic acid by fermenting the rice straws with the cellulose degradation compound bacteria system according to claim 1, wherein the extrusion puffing conditions in the step (1) are as follows: the temperature is 100-140 ℃, the clearance of a die hole is 3-6 mm, the rotating speed of a screw is 50-100 r/min, and the distance between the end part of the screw and a template is 10-20 mm.
3. The method for preparing fulvic acid by fermenting rice straw with cellulose degradation composite bacteria according to claim 1, wherein the culture medium of each stage of culture and fermentation of the strain comprises:
seed retention medium composition (/ liter): 200 g of potato, 20 g of glucose and 15 g of agar;
activation medium composition (/ liter): 200 g of potato and 20 g of glucose;
the culture medium for fermentation seed production comprises the following components (liter): 10-40 g of extruded and puffed rice straw powder, 0.2-2 g of ammonium sulfate, 0.5-3 g of sodium nitrate, 0.1-0.6 g of dipotassium phosphate, 0.05-0.5 g of sodium dihydrogen phosphate, 0.05-0.3 g of calcium chloride monohydrate, 0.01-0.2 g of ferrous sulfate heptahydrate, and 0.1-0.5 g of magnesium sulfate heptahydrate;
the composition (/ liter) of the multi-strain mixed fermentation medium is as follows: 100-200 g of extruded and puffed rice straw powder, 0.2-2 g of ammonium sulfate, 0.5-3 g of sodium nitrate, 0.1-0.6 g of dipotassium phosphate, 0.05-0.5 g of sodium dihydrogen phosphate, 0.05-0.3 g of calcium chloride monohydrate, 0.01-0.2 g of ferrous sulfate heptahydrate, and 0.1-0.5 g of magnesium sulfate heptahydrate.
4. The method for preparing fulvic acid by fermenting rice straws with the cellulose-degrading composite bacterial system according to claim 1, wherein the cellulose-degrading composite bacterial system in the step (2) is composed of bacillus subtilis, streptomyces, trichoderma reesei and aspergillus niger.
5. The method for preparing fulvic acid by fermenting rice straws with the cellulose-degrading composite bacterial strain according to claim 4, wherein the inoculation ratio of the cellulose-degrading composite bacterial strain in the step (2) is as follows: the ratio of bacillus subtilis to streptomyces to trichoderma reesei to aspergillus niger is 5-1: 2-1: 4-1: 10-2.
6. The method for preparing the biochemical fulvic acid by fermenting the rice straws with the cellulose degradation composite bacterial system according to claim 1, wherein the non-heating ultrasonic wave auxiliary technology in the step (2) is that the fermentation broth is ultrasonically treated every 5-8 days after the fermentation is started; the conditions of the ultrasonic treatment were: the ultrasonic power is 240-450 watts, the ultrasonic time is 20-36 minutes, and the working time/intermittent time of one ultrasonic cycle is 10-25 seconds/5 seconds.
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