阅读说明：本技术 使用秸秆生产柠檬酸的废弃物制备牛蒡生物有机肥的方法 (method for preparing burdock bio-organic fertilizer from waste of citric acid production from straws ) 是由 孙科 于 2019-10-09 设计创作，主要内容包括：本发明涉及微生物发酵生产有机肥技术领域,具体涉及使用秸秆生产柠檬酸的废弃物制备牛蒡生物有机肥的方法,包括原材料处理、菌种活化、菌种驯化、高密度发酵及牛蒡生物有机肥的发酵的过程制备得到了牛蒡生物有机肥,本发明以水稻秸秆发酵生产柠檬酸产生的废渣和废液为原料进行生物有机肥发酵研究,通过对固氮菌、解磷菌、解钾菌和生防菌的活化及驯化,采用高密度发酵,控制发酵条件,最后生产出牛蒡生物有机肥,本发明制备的牛蒡生物有机肥能够改善牛蒡的种植条件,降低生产成本,增加牛蒡的产量,提高牛蒡的品质。(the invention relates to the technical field of producing organic fertilizers by microbial fermentation, in particular to a method for preparing burdock bio-organic fertilizer by using wastes generated in the production of citric acid by straws, which comprises the steps of raw material treatment, strain activation, strain domestication, high-density fermentation and fermentation of the burdock bio-organic fertilizer to obtain the burdock bio-organic fertilizer.)

1. The method for preparing the burdock bio-organic fertilizer by using the waste of citric acid produced by straws is characterized by comprising the following steps:

(1) raw material treatment: sterilizing a mixture of waste water and waste residue generated in the production of citric acid from straws;

(2) Activating strains: respectively activating the phosphate solubilizing bacteria strain, the potassium solubilizing bacteria strain, the azotobacter strain and the biocontrol bacteria strain to obtain a phosphate solubilizing bacteria activation bacteria seed liquid, a potassium solubilizing bacteria activation bacteria seed liquid, an azotobacter activation bacteria seed liquid and a biocontrol bacteria activation bacteria seed liquid;

(3) Domesticating strains: respectively acclimating the azotobacter activating bacteria seed liquid, the phosphorus-solubilizing bacteria activating bacteria seed liquid, the potassium-solubilizing bacteria activating bacteria seed liquid and the biocontrol bacteria activating bacteria seed liquid obtained in the step (2) to obtain azotobacter acclimating bacteria suspension, phosphorus-solubilizing bacteria acclimating bacteria suspension, potassium-solubilizing bacteria acclimating bacteria suspension and biocontrol bacteria acclimating bacteria suspension which can bear the acid environment of the mixture obtained in the step (1);

(4) high-density fermentation: inoculating the domesticated bacteria suspension prepared in the step (3) into a domesticated bacteria high-density fermentation culture medium together for fermentation culture, and stopping fermentation after 5-8 days to obtain a mixed strain;

(5) fermentation of the burdock bio-organic fertilizer: uniformly mixing the mixture obtained in the step (1) with a carbon source to obtain a fermentation substrate, then inoculating the mixed strain obtained by fermentation in the step (4), introducing sterile air of 0.5-1.0vvm at the temperature of 25-35 ℃, and stirring and fermenting for 24-36 hours to obtain the burdock bio-organic fertilizer;

Wherein the carbon source is one of glucose, starch and sucrose, and the mass ratio of the carbon source to the mixture is 3-5: 100.

2. the method for preparing the burdock bio-organic fertilizer by using the wastes generated in the production of the citric acid from the straws as claimed in claim 1, wherein the domesticated bacteria high-density fermentation culture medium in the step (4) is composed of the following raw materials in percentage by mass: tryptone, 12.5%; 5% of starch; 0.8 percent of yeast extract; glucose, 7%; sodium acetate, 0.2%; potassium dihydrogen phosphate, 0.1%; magnesium sulfate, 0.02%; biotin, 0.05% and the balance of water.

3. The method for preparing burdock bio-organic fertilizer by using waste of citric acid production from straws as claimed in claim 1, which is characterized in thatThe azotobacter culture medium used for activating azotobacter in the step (2) is composed of the following raw materials in parts by weight: KH (Perkin Elmer)₂PO₄0.2 part; k₂HPO₄0.8 part; MgSO (MgSO)₄·7H₂0.2 part of O; CaSO₄·2H₂0.1 part of O; na (Na)₂MoO₄·2H₂0.05 part of O; 0.5 part of yeast extract; 20 parts of mannitol; FeCl₃0.02 portion; 1000 parts of distilled water.

4. the method for preparing the burdock bio-organic fertilizer by using the wastes generated in the production of the citric acid from the straws as claimed in claim 3, wherein the acclimation process of the nitrogen-fixing bacteria in the step (3) is as follows:

Inoculating activated nitrogen-fixing bacteria seed liquid into the first nitrogen-fixing bacteria culture liquid, and culturing at 20-35 deg.C for 1-2 days; inoculating the azotobacter cultured in the first azotobacter culture solution into the second azotobacter culture solution, and culturing at 20-35 deg.C for 1-2 days; finally, inoculating the nitrogen-fixing bacteria cultured in the second nitrogen-fixing bacteria culture solution into the wastewater generated after the straws are used for producing citric acid, and culturing for 1-2 days at the temperature of 20-35 ℃ to obtain domesticated nitrogen-fixing bacteria; centrifuging and washing the obtained domesticated azotobacter under the aseptic condition, and mixing the thallus precipitate with water to obtain azotobacter domesticated bacteria suspension;

The first azotobacter culture solution is obtained by mixing wastewater and an azotobacter culture medium according to the volume ratio of 4: 6; the second azotobacter culture solution is obtained by mixing the wastewater and the azotobacter culture medium according to the volume ratio of 7: 3.

5. the method for preparing the burdock bio-organic fertilizer by using the wastes generated in the production of the citric acid from the straws as claimed in claim 1, wherein the acclimation process of the phosphate solubilizing bacteria in the step (3) is as follows:

Inoculating activated phosphate solubilizing bacteria seed liquid into the first phosphate solubilizing bacteria culture liquid, culturing for 2 days at 20-35 ℃, wherein the first day is divided into 2 periods, one period is formed every 12 hours, each period is firstly anaerobic for 4 hours, then sterile air is provided for 8 hours, and the second day is sterile air for 24 hours; inoculating the phosphate solubilizing bacteria cultured in the first phosphate solubilizing bacteria culture solution to a second phosphate solubilizing bacteria culture solution, culturing for 2 days at 20-35 ℃, wherein the first day is divided into 2 periods, one period is formed every 12 hours, each period is anaerobic for 6 hours, then sterile air is provided for 6 hours, and the second day is sterile air for 24 hours; finally, inoculating the phosphate solubilizing bacteria cultured in the second phosphate solubilizing bacteria culture solution into the wastewater generated after the straws are used for producing the citric acid, and culturing for 1-2 days in sterile air at the temperature of 20-35 ℃ to obtain domesticated phosphate solubilizing bacteria; centrifuging and washing the obtained domesticated phosphate solubilizing bacteria under the aseptic condition, and mixing the thallus precipitate with water to obtain a phosphate solubilizing bacteria domesticated bacteria suspension;

The first phosphate solubilizing bacteria culture solution is obtained by mixing wastewater and an LB culture medium according to the volume ratio of 4: 6; the second phosphate solubilizing bacteria culture solution is obtained by mixing wastewater and an LB culture medium according to the volume ratio of 7: 3.

the domestication process of the potassium bacteria in the step (3) is the same as that of the phosphorus bacteria.

6. the method for preparing the burdock bio-organic fertilizer by using the wastes generated in the production of the citric acid from the straws as claimed in claim 1, wherein the acclimation process of the biocontrol bacteria in the step (3) is as follows:

Inoculating activated biocontrol bacteria seed liquid into the first biocontrol bacteria culture solution, and introducing sterile air for aerobic culture at normal temperature for 1-3 days; inoculating the biocontrol bacteria cultured in the first biocontrol bacteria culture solution into a second biocontrol bacteria culture solution, and introducing sterile air for aerobic culture at normal temperature for 1-3 days; finally, inoculating the biocontrol bacteria cultured in the second biocontrol bacteria culture solution into the wastewater generated after the straws are used for producing the citric acid, and introducing sterile air for aerobic culture for 1-3 days at normal temperature to obtain domesticated biocontrol bacteria; centrifuging and washing the obtained domesticated biocontrol bacteria under the aseptic condition, and mixing the thallus precipitate with water to obtain biocontrol bacteria domesticated bacteria suspension;

the first biocontrol bacterium culture solution is obtained by mixing wastewater and a PD culture medium according to the volume ratio of 4: 6; the second biocontrol bacteria culture solution is obtained by mixing wastewater and a PD culture medium according to the volume ratio of 7: 3.

7. The method for preparing burdock bio-organic fertilizer by using the wastes generated in the production of citric acid from straws as claimed in claim 1, wherein the sterilization treatment in the step (1) is a high temperature sterilization treatment under the conditions of 100-120 ℃ for 5-10 min.

8. the method for preparing the burdock bio-organic fertilizer by using the wastes generated in the production of the citric acid from the straws as claimed in any one of claim 1, wherein the wastewater obtained in the step (3) is a filtrate obtained by filtering the wastewater through a filter screen with the pore size of 0.5-1 mm.

9. the method for preparing the burdock bio-organic fertilizer by using the wastes generated in the production of the citric acid by the straws as claimed in claim 1, wherein the fermentation conditions of the step (4) are as follows: culturing and fermenting in a fermentation tank with the temperature of 30-35 ℃ at 200r/min and 120-.

Technical Field

The invention relates to the technical field of organic fertilizer production by microbial fermentation, in particular to a method for preparing a burdock bio-organic fertilizer by using wastes generated in citric acid production by straws.

Background

currently, resource conservation and environmental protection become basic national policies for development in China, green ecology also become the internal requirements for development of modern agriculture, and with the continuous improvement of the requirements of China on the quality of agricultural products and sustainable development of agriculture, people pay more attention to the protection of agricultural ecological environment, and environment-friendly fertilizers are increasingly concerned and gradually become the mainstream trend of the development of fertilizers in China.

Most of soil at junctions of Peizian county and Fengxi county in Xuhuai region is neutral sandy loam, the soil layer is deep, the soil is loose, the water drainage and irrigation are convenient, the burdock is just the soil type suitable for the growth of deep-rooted vegetables, the planting of the burdock in Xuhuai region accounts for about 50% of the area of the burdock in China, the regions are concentrated, the planting and management technology is mature, but the ecological environment of the soil is destroyed by using chemical fertilizers all year round in the planting of the burdock, so that the phenomena of soil hardening and increase of plant diseases and insect pests are gradually generated, and the yield and the quality of the burdock are reduced.

In the invention patent with application number CN105886556A, a method for producing citric acid by fermenting crop straws with mixed bacteria is disclosed, which produces citric acid by fermenting rice straws with fiber instead of grain, and the strains in the mixed bacteria fermentation include: phanerochaete chrysosporium, Trichoderma reesei and Aspergillus niger, although achieving the purpose of producing citric acid by fermenting rice straw, 10-15m is produced when 1 ton of citric acid is produced after fermentation³the organic wastewater contains a large amount of sugar and reducing substances, and has the characteristics of high COD (amount of the reducing substances) concentration (more than 10000 m/L) and low pH value of 3.5-5, however, how to degrade the high-concentration organic wastewater at present becomes a bottleneck restricting the high-speed advance of the citric acid industry.

Disclosure of Invention

in order to overcome the defects of the prior art, the invention provides a method for preparing a burdock bio-organic fertilizer by using wastes generated in the process of producing citric acid by using straws, the invention takes waste residues and waste liquid generated in the process of producing citric acid by fermenting rice straws as raw materials to carry out fermentation research of the burdock bio-organic fertilizer, and the burdock bio-organic fertilizer is finally produced by domesticating and activating nitrogen-fixing bacteria, phosphate-solubilizing bacteria, potassium-solubilizing bacteria and biocontrol bacteria, adopting high-density fermentation and controlling fermentation conditions.

In order to achieve the purpose, the invention adopts the technical scheme that:

the method for preparing the burdock bio-organic fertilizer by using the waste of citric acid produced by straws comprises the following steps:

(1) raw material treatment: sterilizing a mixture of waste water and waste residue generated in the production of citric acid from straws;

(2) Activating strains: respectively activating the phosphate solubilizing bacteria strain, the potassium solubilizing bacteria strain, the azotobacter strain and the biocontrol bacteria strain to obtain a phosphate solubilizing bacteria activation bacteria seed liquid, a potassium solubilizing bacteria activation bacteria seed liquid, an azotobacter activation bacteria seed liquid and a biocontrol bacteria activation bacteria seed liquid;

(3) domesticating strains: respectively acclimating the azotobacter activating bacteria seed liquid, the phosphorus-solubilizing bacteria activating bacteria seed liquid, the potassium-solubilizing bacteria activating bacteria seed liquid and the biocontrol bacteria activating bacteria seed liquid obtained in the step (2) to obtain azotobacter acclimating bacteria suspension, phosphorus-solubilizing bacteria acclimating bacteria suspension, potassium-solubilizing bacteria acclimating bacteria suspension and biocontrol bacteria acclimating bacteria suspension which can bear the acid environment of the mixture obtained in the step (1);

(4) high-density fermentation: inoculating the domesticated bacteria suspension prepared in the step (3) into a domesticated bacteria high-density fermentation culture medium together for fermentation culture, and stopping fermentation after 5-8 days to obtain a mixed strain;

(5) Fermentation of the burdock bio-organic fertilizer: uniformly mixing the mixture obtained in the step (1) with a carbon source to obtain a fermentation substrate, then inoculating the mixed strain obtained by fermentation in the step (4), introducing sterile air of 0.5-1.0vvm at the temperature of 25-35 ℃, and stirring and fermenting for 24-36 hours to obtain the burdock bio-organic fertilizer;

Wherein the carbon source is one of glucose, starch and sucrose, and the mass ratio of the carbon source to the mixture is 3-5: 100.

preferably, the domesticated bacteria high-density fermentation medium in the step (4) is composed of the following raw materials in percentage by mass: tryptone, 12.5%; 5% of starch; 0.8 percent of yeast extract; glucose, 7%; sodium acetate, 0.2%; potassium dihydrogen phosphate, 0.1%; magnesium sulfate, 0.02%; biotin, 0.05% and the balance of water.

Preferably, the nitrogen-fixing bacteria culture medium used for activating the nitrogen-fixing bacteria in the step (2) is composed of the following raw materials in parts by weight: KH (Perkin Elmer)₂PO₄0.2 part; k₂HPO₄0.8 part; MgSO (MgSO)₄·7H₂0.2 part of O; CaSO₄·2H₂0.1 part of O; na (Na)₂MoO₄·2H₂0.05 part of O; 0.5 part of yeast extract; 20 parts of mannitol; FeCl₃0.02 portion; 1000 parts of distilled water.

preferably, the acclimatization process of the azotobacter in the step (3) is as follows:

Inoculating the activated azotobacter seed liquid into the first azotobacter culture liquid, and culturing at 20-35 deg.C for 1-2 days; inoculating the azotobacter cultured in the first azotobacter culture solution into the second azotobacter culture solution, and culturing at 20-35 deg.C for 1-2 days; finally, inoculating the nitrogen-fixing bacteria cultured in the second nitrogen-fixing bacteria culture solution into the wastewater generated after the straws are used for producing citric acid, and culturing for 1-2 days at the temperature of 20-35 ℃ to obtain domesticated nitrogen-fixing bacteria; centrifuging and washing the obtained domesticated azotobacter under the aseptic condition, and mixing the thallus precipitate with water to obtain azotobacter domesticated bacteria suspension;

the first azotobacter culture solution is obtained by mixing wastewater and an azotobacter culture medium according to the volume ratio of 4: 6; the second azotobacter culture solution is obtained by mixing the wastewater and the azotobacter culture medium according to the volume ratio of 7: 3.

Preferably, the acclimatization process of the phosphate solubilizing bacteria in the step (3) is as follows:

Inoculating activated phosphate solubilizing bacteria seed liquid into the first phosphate solubilizing bacteria culture liquid, culturing for 2 days at 20-35 ℃, wherein the first day is divided into 2 periods, one period is formed every 12 hours, each period is firstly anaerobic for 4 hours, then sterile air is provided for 8 hours, and the second day is sterile air for 24 hours; inoculating the phosphate solubilizing bacteria cultured in the first phosphate solubilizing bacteria culture solution to a second phosphate solubilizing bacteria culture solution, culturing for 2 days at 20-35 ℃, wherein the first day is divided into 2 periods, one period is formed every 12 hours, each period is anaerobic for 6 hours, then sterile air is provided for 6 hours, and the second day is sterile air for 24 hours; finally, inoculating the phosphate solubilizing bacteria cultured in the second phosphate solubilizing bacteria culture solution into the wastewater generated after the straws are used for producing the citric acid, and culturing for 1-2 days in sterile air at the temperature of 20-35 ℃ to obtain domesticated phosphate solubilizing bacteria; centrifuging and washing the obtained domesticated phosphate solubilizing bacteria under the aseptic condition, and mixing the thallus precipitate with water to obtain a phosphate solubilizing bacteria domesticated bacteria suspension;

The first phosphate solubilizing bacteria culture solution is obtained by mixing wastewater and an LB culture medium according to the volume ratio of 4: 6; the second phosphate solubilizing bacteria culture solution is obtained by mixing wastewater and an LB culture medium according to the volume ratio of 7: 3.

The domestication process of the potassium bacteria in the step (3) is the same as that of the phosphorus bacteria.

preferably, the acclimatization process of the biocontrol bacteria in the step (3) is as follows:

inoculating the activated biocontrol bacteria seed liquid into the first biocontrol bacteria culture liquid, and introducing sterile air for aerobic culture at normal temperature for 1-3 days; inoculating the biocontrol bacteria cultured in the first biocontrol bacteria culture solution into a second biocontrol bacteria culture solution, and introducing sterile air for aerobic culture at normal temperature for 1-3 days; finally, inoculating the biocontrol bacteria cultured in the second biocontrol bacteria culture solution into the wastewater generated after the straws are used for producing the citric acid, and introducing sterile air for aerobic culture for 1-3 days at normal temperature to obtain domesticated biocontrol bacteria; centrifuging and washing the obtained domesticated biocontrol bacteria under the aseptic condition, and mixing the thallus precipitate with water to obtain biocontrol bacteria domesticated bacteria suspension;

the first biocontrol bacterium culture solution is obtained by mixing wastewater and a PD culture medium according to the volume ratio of 4: 6; the second biocontrol bacteria culture solution is obtained by mixing wastewater and a PD culture medium according to the volume ratio of 7: 3.

Preferably, the sterilization treatment in the step (1) adopts a high-temperature sterilization treatment method, and the conditions are 100 ℃ and 120 ℃ for sterilization for 5-10 min.

Preferably, the wastewater in the step (3) is a filtrate which is filtered by a filter screen with the aperture of 0.5-1 mm.

Preferably, the fermentation conditions of step (4) are: culturing and fermenting in a fermentation tank with the temperature of 30-35 ℃ at 200r/min and 120-.

compared with the prior art, the invention has the beneficial effects that:

1. the method comprises the steps of raw material treatment, strain activation, strain acclimation, high-density fermentation and fermentation of the burdock bio-organic fertilizer, wherein in the preparation process of the organic fertilizer, high-temperature instantaneous sterilization is firstly carried out in the raw material treatment process, mixed bacteria in waste water and waste residue after citric acid is produced by straws are killed, the influence of the mixed bacteria on nitrogen-fixing bacteria, phosphate-solubilizing bacteria, potassium-solubilizing bacteria and biocontrol bacteria to be inoculated is avoided, then the strains in the preservation state are put into a proper culture medium to be cultured in the strain activation process, pure and strong cultures are obtained through step-by-step expansion culture, namely cultures with vigorous activity and enough inoculation quantity are obtained, and then acclimation under different conditions is respectively carried out on the phosphate-solubilizing bacteria activation strains, the potassium-solubilizing bacteria activation strains, the nitrogen-fixing bacteria activation strains and the biocontrol bacteria activation strains to ensure that the nitrogen-fixing bacteria and the phosphate-solubilizing bacteria are acclimated under different conditions, The potassium bacteria and the biocontrol bacteria can grow and play a role in the acidic environment of the wastewater to obtain azotobacter domesticated bacteria suspension, phosphorus bacteria domesticated bacteria suspension, potassium bacteria domesticated bacteria suspension and biocontrol bacteria domesticated bacteria suspension with excellent acid resistance, then the domesticated bacteria suspension is subjected to high-density fermentation to obtain strains which are enough for fermentation, the high-density fermentation not only reduces the volume of a culture container and the consumption of a culture medium and improves the separation and extraction efficiency in downstream engineering, but also shortens the production period, reduces the equipment investment and reduces the production cost, so that the specific production rate of products is improved, and finally the strains subjected to high-density fermentation and the wastewater and waste residues obtained after the straws produce citric acid are fermented together to finally prepare the burdock biological organic fertilizer.

2. the burdock bio-organic fertilizer prepared by the invention is an acidic and aqueous fertilizer, not only can neutralize alkaline soil generated by long-term application of chemical fertilizer, but also relieves soil hardening due to moisture in the organic fertilizer during fertilization, and organic substances contained in the organic fertilizer promote the growth and development of plants, so that the burdock bio-organic fertilizer provided by the invention can improve the planting conditions of burdock, increase the yield of burdock and improve the quality of burdock.

3. the method adopts the waste water and waste residues generated after the citric acid is produced from the straws as the main substances of the fertilizer, not only solves the problem of waste water treatment after the citric acid is produced from the straws, but also changes the organic wastes into valuables, realizes resource utilization, greatly reduces the production cost on production, simultaneously realizes the recycling of the organic waste water, has higher economic, ecological and social benefits, and is a powerful guarantee for realizing sustainable development of agriculture.

Detailed Description

the biocontrol bacterium, the azotobacter, the phosphate solubilizing bacterium and the potassium solubilizing bacterium are respectively selected from the following groups:

The azotobacter is azotobacter chroococcum, the phosphate solubilizing bacteria are saccharomyces cerevisiae or bacillus megaterium, and the potassium solubilizing bacteria are bacillus mucilaginosus; the test tube strains of azotobacter chroococcum, saccharomyces cerevisiae, bacillus megaterium and bacillus mucilaginosus are independently preserved in a strain preservation cabinet at the temperature of 3-6 ℃, and the azotobacter, the potassium bacteria and the azotobacter used in the method are the same as the azotobacter, the phosphorus bacteria and the potassium bacteria in the method for preparing the biological organic fertilizer by co-fermentation of the azotobacter, the phosphorus bacteria and the potassium bacteria, wherein the application number of the azotobacter, the potassium bacteria and the azotobacter is 201610372100.6, the application date of the method is 2016.05.31, and the name of the method is' the method for preparing the biological organic fertilizer by co-fermentation.

The waste water and waste residue of the invention are both from the invention patent with the application number of CN105886556A and the application date of 2016.08.24, which is named as a method for producing citric acid by mixed bacteria fermented crop straws.

the biocontrol strain in the invention is Trichoderma viride (Trichoderma viride), Trichoderma harzianum (Trichoderma harziamum), Trichoderma aureoviride (Trichoderma auxeoviride) and Trichoderma koningii (Trichoderma koningii); the biocontrol bacterium used in the invention is the same as the biocontrol bacterium in the application No. 201010612049.4, application date 2010.12.20 and the name of trichoderma biocontrol agent produced by sweet potato starch wastewater and mushroom residues.