CN115611670A - Active bacillus subtilis biological bacterial fertilizer - Google Patents
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- CN115611670A CN115611670A CN202211169864.7A CN202211169864A CN115611670A CN 115611670 A CN115611670 A CN 115611670A CN 202211169864 A CN202211169864 A CN 202211169864A CN 115611670 A CN115611670 A CN 115611670A
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- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
- C05F17/00—Preparation of fertilisers characterised by biological or biochemical treatment steps, e.g. composting or fermentation
- C05F17/20—Preparation of fertilisers characterised by biological or biochemical treatment steps, e.g. composting or fermentation using specific microorganisms or substances, e.g. enzymes, for activating or stimulating the treatment
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- C05—FERTILISERS; MANUFACTURE THEREOF
- C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
- C05F7/00—Fertilisers from waste water, sewage sludge, sea slime, ooze or similar masses
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
- C12R2001/125—Bacillus subtilis ; Hay bacillus; Grass bacillus
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- Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
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Abstract
The invention discloses an active bacillus subtilis biological bacterial fertilizer, and belongs to the technical field of solid organic waste treatment and biological bacterial fertilizer production. Aiming at the problems of short storage time and quick reduction of microbial activity of microbial fertilizer, the method takes the biogas residues obtained by the synergistic anaerobic digestion of kitchen wastes and sludge with low price and wide sources as raw materials for producing the biological bacterial fertilizer, selects bacillus subtilis as a fermentation strain to prepare the high-activity bacillus subtilis biological bacterial fertilizer, does not produce secondary pollution in the fermentation process, and has low energy consumption and cost, and the viable count of the bacterial fertilizer is 0.43 multiplied by 10 8 cfu/g, number of spores 0.22X 10 8 cfu/g. And some extracellular electronic enzymes released by the bacillus subtilis can better degrade biogas residues, and simultaneously release spores resisting external adverse environment, and have high temperature resistance and acid resistanceThe fertilizer efficiency of the prepared microbial fertilizer can be more stable.
Description
Technical Field
The invention belongs to the technical field of solid organic waste treatment and biological bacterial fertilizer production, and particularly relates to a high-activity long-shelf-life bacillus subtilis biological bacterial fertilizer prepared from biogas residues obtained after kitchen waste and excess sludge are subjected to synergistic anaerobic digestion.
Background
At present, the amount of kitchen waste and excess sludge in China is increasing year by year. The anaerobic fermentation technology has good resource utilization effect, and becomes a main means for treating the kitchen waste and the excess sludge in a resource, reduction and harmless manner. Because the two are cooperated with each other to generate a large amount of biogas residues in the anaerobic fermentation process, the nutritional ingredients of the biogas residues are unstable and the heavy metal content is high, and secondary pollution can be caused if the biogas residues are not properly treated. At present, the comprehensive utilization approach of the biogas residues at home and abroad is mainly used as organic fertilizer to be applied to soil, but the application of economic and environmental problems is limited, so that the search for a more appropriate biogas residue treatment technology is particularly necessary.
The biogas residues are prepared into the biological bacterial fertilizer, so that the waste can be recycled, and the method is an economical, feasible and environment-friendly utilization mode of recycling, reducing and harmlessly utilizing the biogas residues. Compared with the traditional fertilizer, the biological bacterial fertilizer is a novel green pollution-free fertilizer, and the raw material is rich in more organic matters capable of being absorbed by plants through a series of life activities and metabolic products of added strains, so that the problems of lack of organic matters and humus, reduction of crop yield and the like of soil caused by using a large amount of fertilizer are solved. The fertilizer has the advantages of reducing environmental pollution, high fertilizer efficiency, no toxicity, low cost, saving energy and the like, can gradually realize less or no chemical fertilizer application in farmlands, optimizes the soil quality, ensures the improvement of the crop quality and the increase of the crop yield, and can promote the sustainable development of ecological agriculture in China.
In the prior art, as the microbial fertilizer is developed later in China, the selection of strains and the control of conditions in the fermentation process are relatively backward, and the effects of the microbial fertilizer in the aspects of improving the soil fertilizer efficiency, increasing the plant yield and preventing plant diseases and insect pests are not optimistic. In addition, the microbial activity in the microbial fertilizer is greatly influenced by the environment, so the microbial fertilizer has poor stability, and the amount of the microorganisms is gradually reduced along with the delay of the storage time, thereby the using effect is poor.
Disclosure of Invention
Aiming at the problems in the prior art, the technical problem to be solved by the invention is to provide an active bacillus subtilis biological bacterial fertilizer.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows:
an active bacillus subtilis biological bacterial fertilizer is prepared by utilizing kitchen waste and sludge to cooperate with biogas residues after anaerobic digestion, and the preparation method specifically comprises the following steps:
1) Inoculating the bacillus subtilis strain into a beef extract peptone liquid culture medium, and performing shaking culture on a shaker at 30 ℃ until the growth logarithmic phase to obtain bacillus subtilis liquid;
2) Performing high-pressure steam sterilization on the biogas residue obtained after the synergistic anaerobic digestion of the kitchen waste and the sludge, cooling to room temperature, adding sterile water, adjusting the initial water content of the biogas residue to be 50-60%, adding bacillus subtilis liquid with the bacterial inoculation amount of 10-20%, and performing standing culture at the constant temperature of 30 ℃ until the pH value is 8.1-8.3 to obtain the active bacillus subtilis biological bacterial fertilizer.
Further, in the step 1), the formula of the beef extract peptone liquid medium is as follows: each 1L of water contained 3g of beef extract, 10.0g of peptone and 5g of NaCl, and the pH was adjusted to 7.2.
Further, in the step 1), the effective viable count of the bacillus subtilis liquid is 4.2 multiplied by 10 8 cfu/mL。
Further, in the step 2), the initial water content of the biogas residues is adjusted to 50-55%.
Further, in the step 2), the initial water content of the biogas residues is adjusted to 55%.
Further, in the step 2), bacillus subtilis liquid with the bacterial inoculation amount of 15% is added.
Further, in the step 2), standing and culturing for 13 days at the constant temperature of 30 ℃ to obtain the active bacillus subtilis biological bacterial fertilizer.
Further, in the step 2), the viable count of the active bacillus subtilis biological bacterial fertilizer is 0.43×10 8 cfu/g, number of spores 0.22X 10 8 cfu/g。
Compared with the prior art, the invention has the beneficial effects that:
(1) Aiming at the problems of short storage time of microbial fertilizers, quick reduction of microbial activity and the like, the method takes the kitchen waste and the biogas residues obtained after the excess sludge is subjected to synergistic anaerobic digestion, which are low in price and wide in source, as raw materials for producing the biological bacterial fertilizers, selects bacillus subtilis which can tolerate condition change as a fermentation strain according to the characteristics of the raw materials, and prepares the high-activity bacillus subtilis biological bacterial fertilizer, so that secondary pollution is not generated in the fermentation process, the energy consumption is low, and the cost is low.
(2) Some extracellular enzymes released by the bacillus subtilis can better degrade biogas residues, and simultaneously release spores resisting external adverse environments, so that the microbial fertilizer has the characteristics of high temperature resistance and acid resistance, and the fertilizer efficiency of the prepared microbial fertilizer can be more stable.
Drawings
FIG. 1 is a process flow diagram for preparing biogas residue from kitchen waste and excess sludge of a sewage treatment plant;
FIG. 2 is a graph showing the effect of different initial water contents on the growth of viable count;
FIG. 3 is a graph showing the effect of different initial moisture contents on spore number growth;
FIG. 4 is a graph showing the effect of different initial inoculum sizes on viable count growth;
FIG. 5 is a graph showing the effect of different initial inoculum sizes on spore number growth;
FIG. 6 is a graph showing pH changes during the cultivation with different initial water contents;
FIG. 7 is a graph showing the pH change during the cultivation process with different initial inoculum sizes.
Detailed Description
The invention is further described with reference to specific examples. These examples are intended to illustrate the invention and are not intended to limit the scope of the invention. Modifications or substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and scope of the invention. In the following examples, unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.
Example 1 method for preparing biological bacterial fertilizer by using Bacillus subtilis
1. Determination of fermentation conditions of Bacillus subtilis
A ring of bacillus subtilis is picked up and cultured in 100ml of beef extract peptone liquid medium for 72h at 30 ℃ and 180r/min in a shaking table, and the growth curve is measured.
2. Preparation of Bacillus subtilis liquid
Taking a ring of bacillus subtilis strain from a dish under a clean bench, inoculating the bacillus subtilis strain into a 250ml triangular flask filled with 100ml beef extract peptone liquid culture medium, and carrying out shaking culture on a shaker (180 r/min) at 30 ℃ for 22h to a logarithmic growth phase to obtain a bacillus subtilis liquid with the effective viable count of 3.2 multiplied by 10 8 (cfu/mL)。
3. Preparation of bacillus subtilis biological bacterial fertilizer by utilizing kitchen waste and excess sludge in cooperation with anaerobic digestion biogas residue
And (3) putting the biogas residues into a 250ml narrow-mouth conical flask for high-pressure steam sterilization (121 ℃,20 min), placing the conical flask on a sterile operation platform after the sterilization is finished, cooling the conical flask to room temperature, adding sterile water, adjusting the initial water content to be 50% -60%, adding bacillus subtilis seed solution, sealing the conical flask with gauze, placing the conical flask in a constant-temperature incubator for standing and culturing for 13 days, and coating the conical flask on a beef extract peptone solid medium by using a plate counting method to monitor the viable count, the spore count and the pH value in the fermentation process.
Wherein, the bacillus subtilis is purchased from Nanjing Lexus Biotechnology Limited company, and the product number is as follows: and bacillus subtilis CMCC (B) 63501, which is cultured by beef extract peptone medium and preserved for later use.
The biogas residues are from Jiangsu Hongrun biomass energy science and technology Limited company, tailings are generated by kitchen wastes and excess sludge of a sewage treatment plant through the tail ends of processes of kitchen source pretreatment, sludge pyrohydrolysis, high solid content anaerobic digestion, biogas residue deep dehydration and solar drying and the like, and the process is shown in figure 1. The physicochemical properties of the biogas residues are shown in table 1 below.
TABLE 1
| Organic matter (%) | N(％) | P(％) | K(％) | pH | |
| Biogas residue | 36 | 4.4 | 7 | 0.7 | 7.4 |
The media formulations used in this example were as follows:
the formula of the beef extract peptone liquid culture medium is as follows: providing 3g of beef extract, 10.0g of peptone and 5g of NaCl, adding into 1000ml of ultrapure water, adjusting pH to 7.2 with NaCl and NaOH, and carrying out autoclaving at 121 ℃ for 15min to obtain a beef extract peptone liquid culture medium.
The formula of the beef extract peptone solid medium is as follows: providing 3g of beef extract, 10.0g of peptone 5g of NaCl and 15g of agar, adding the mixture into 1000ml of ultrapure water, adjusting the pH value to 7.2 by using NaCl and NaOH, and carrying out autoclaving at 121 ℃ for 15min to obtain the beef extract peptone solid culture medium.
The analytical method used in this example was as follows:
viable count in the samples was determined using the dilution coating plate method: the method is carried out according to a detection method of 5.1 effective viable bacteria in the microbial fertilizer product inspection regulations (NY 2321-2013).
The method for measuring the pH in the culture process comprises the following steps: the pH value is detected according to a method for detecting the pH value in 5.7 in the microbial fertilizer product inspection regulations (NY 2321-2013).
Example 2 Effect of different initial Water content on Strain growth
Adding 35g of kitchen waste and biogas residue obtained after anaerobic digestion of excess sludge into a 250mL of fine-mouthed conical flask, sterilizing for 20min by high-pressure steam at 121 ℃, cooling to room temperature, adding 28mL of sterile water, 35mL of sterile water and 43.75mL of sterile water respectively to adjust the initial water content to be 50%, 55% and 60% respectively, inoculating the bacterial liquid in example 1 accounting for 15% of the total mass (1 mL/10g of the total mass), wherein the adding amounts of the bacterial liquid are 9.45mL, 10.5mL and 11.8mL respectively, shaking uniformly, sealing with gauze, placing in an incubator at 30 ℃ for standing culture, and turning the materials once every 48 h. And counting the number of viable bacteria by plate culture. The number of viable bacteria in 13 days of culture is 0.32 × 10 8 cfu/g、0.22×10 8 cfu/g、0.13×10 8 cfu/g. The number of spores was 0.32X 10 8 cfu/g、0.22×10 8 cfu/g、0.13×108cfu/g。
EXAMPLE 3 Effect of different initial inoculum size on growth of strains
Adding 35g of kitchen waste, biogas residue obtained after anaerobic digestion of excess sludge and 35mL of water into a 250mL of narrow-mouth conical flask, sterilizing for 20min by high-pressure steam at 121 ℃, cooling to room temperature, inoculating 7mL, 10.5mL and 14.5mL of bacillus subtilis liquid in example 1 respectively, adjusting initial inoculation amounts to be respectively the total mass (10%, 15% and 20%), shaking uniformly, sealing with gauze, placing in an incubator at 30 ℃, standing for culture, and turning materials once every 48 h. And counting the number of viable bacteria by plate culture. The number of viable bacteria in 13 days of culture is 0.35 × 10 8 cfu/g、0.43×10 8 cfu/g、0.62×10 8 cfu/g. The number of viable bacteria of Bacillus subtilis is 0.046 × 10 8 cfu/g、0.22×10 8 cfu/g、0.14×10 8 cfu/g。
Through the optimization, 35g of kitchen waste and excess sludge are subjected to anaerobic digestionAdding the biogas residue into a 250mL conical flask with a fine opening, sterilizing with high pressure steam at 121 deg.C for 20min, cooling to room temperature, adding 35mL water (initial water content is 55%) and 10.5mL (initial inoculation amount is 15%) Bacillus subtilis liquid, and culturing in 30 deg.C constant temperature incubator for 13 days to obtain bacterial fertilizer with optimal effect, and viable count of 0.43 × 10 8 cfu/g, number of spores 0.22X 10 8 cfu/g。
EXAMPLE 4 solid culture control of bacterial manure pH
The pH change was monitored during the culture in each of examples 2 and 3, and 1g of the sample was sampled, and ultrapure water was added in a mass ratio of 1: 5 (sample: water), shaken and allowed to stand for 30min, measured by a pH meter, and read after each set was stabilized. In example 2, the pH of the fermentation product was slightly alkaline at the initial stage of the culture, and gradually increased with the progress of the fermentation process, and reached about 8.3 in the ninth day of the fermentation, and then gradually increased over the following days, fluctuating between 8.1 and 8.49. In example 3, the pH of the fermentation product was slightly alkaline at the initial stage of the culture, gradually increased to about 8.28 in 1 to 9 days, and then fluctuated up and down between 8.1 and 8.3.
Comparative example
Weighing 35g of kitchen waste and biogas residue obtained after anaerobic digestion of excess sludge, sterilizing in a 250ml narrow-mouth conical flask, adding sterile water to adjust the initial water content to 55%, and adding 2 × 10 effective viable bacteria 8 cfu/mL of Bacillus subtilis and 2X 10 8 cfu/mL azotobacter chroococcum is mixed according to the volume ratio of 1: 1, and 15% (1 mL/10g total mass) of bacteria are inoculated. Shaking, sealing with gauze, placing in 30 deg.C incubator, standing for culture, and turning once every 48 hr. And counting the number of viable bacteria by plate culture. The viable count of Bacillus subtilis after 11 days of culture is 0.38 × 10 8 cfu/g, the number of spores of bacillus is 0.52 multiplied by 10 8 cfu/g, viable count of Azotobacter fuscous is 0.56 × 10 8 cfu/g。
Claims (8)
1. The active bacillus subtilis biological bacterial fertilizer is characterized by being prepared from biogas residues obtained after kitchen waste and sludge are subjected to synergistic anaerobic digestion, and the preparation method specifically comprises the following steps:
1) Inoculating the bacillus subtilis strain into a beef extract peptone liquid culture medium, and carrying out shake culture on a shaker at 30 ℃ until the logarithmic phase of growth is reached to obtain a bacillus subtilis liquid;
2) Performing high-pressure steam sterilization on the biogas residue obtained after the synergistic anaerobic digestion of the kitchen waste and the sludge, cooling to room temperature, adding sterile water, adjusting the initial water content of the biogas residue to be 50-60%, adding bacillus subtilis liquid with the bacterial inoculation amount of 10-20%, and performing standing culture at the constant temperature of 30 ℃ until the pH value is 8.1-8.3 to obtain the active bacillus subtilis biological bacterial fertilizer.
2. The active bacillus subtilis biological bacterial fertilizer as claimed in claim 1, wherein in the step 1), the formula of the beef extract peptone liquid medium is as follows: each 1L of water contained 3g of beef extract, 10.0g of peptone and 5g of NaCl, and the pH was adjusted to 7.2.
3. The active bacillus subtilis biological bacterial fertilizer of claim 1, wherein in the step 1), the effective viable count of the bacillus subtilis liquid is 4.2 x 10 8 cfu/mL。
4. The active bacillus subtilis biological bacterial fertilizer of claim 1, wherein in the step 2), the initial water content of the biogas residues is adjusted to 50-55%.
5. The active bacillus subtilis biological bacterial fertilizer of claim 4, wherein in the step 2), the initial water content of the biogas residues is adjusted to 55%.
6. The active bacillus subtilis biological bacterial fertilizer of claim 1, wherein in the step 2), the bacillus subtilis liquid with the bacterial inoculation amount of 15 percent is added.
7. The active bacillus subtilis biological bacterial fertilizer of claim 1, wherein in the step 2), the active bacillus subtilis biological bacterial fertilizer is obtained by standing and culturing for 13 days at a constant temperature of 30 ℃.
8. The active bacillus subtilis biological bacterial fertilizer of claim 7, wherein the viable count of the active bacillus subtilis biological bacterial fertilizer in the step 2) is 0.43 x 10 8 cfu/g, number of spores 0.22X 10 8 cfu/g。
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101734964A (en) * | 2008-11-26 | 2010-06-16 | 四川鹤岛农业科技有限公司 | Preparation of microorganism organic fertilizer from methane residue |
| CN103848698A (en) * | 2014-03-27 | 2014-06-11 | 青岛福瑞斯生物能源科技开发有限公司 | Biological organic fertilizer prepared by aerobic fermentation of biogas residue and preparation method thereof |
| CN113215201A (en) * | 2021-06-09 | 2021-08-06 | 重庆大学 | Coupling process for mixing pig manure with rice straw solid-state biogas fermentation and biogas residue aerobic composting |
| CN115043678A (en) * | 2022-07-15 | 2022-09-13 | 山东鼎创生物科技有限公司 | Biogas residue solid-state fermentation bio-organic fertilizer and preparation method and application thereof |
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- 2022-09-26 CN CN202211169864.7A patent/CN115611670A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101734964A (en) * | 2008-11-26 | 2010-06-16 | 四川鹤岛农业科技有限公司 | Preparation of microorganism organic fertilizer from methane residue |
| CN103848698A (en) * | 2014-03-27 | 2014-06-11 | 青岛福瑞斯生物能源科技开发有限公司 | Biological organic fertilizer prepared by aerobic fermentation of biogas residue and preparation method thereof |
| CN113215201A (en) * | 2021-06-09 | 2021-08-06 | 重庆大学 | Coupling process for mixing pig manure with rice straw solid-state biogas fermentation and biogas residue aerobic composting |
| CN115043678A (en) * | 2022-07-15 | 2022-09-13 | 山东鼎创生物科技有限公司 | Biogas residue solid-state fermentation bio-organic fertilizer and preparation method and application thereof |
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