Composite high-temperature microbial inoculum and method for promoting humification of compost by using composite high-temperature microbial inoculum

Abstract

The invention discloses a composite high-temperature bacterial agent and a method for promoting humification of compost by using the composite high-temperature bacterial agent. The composite high-temperature bacterial agent comprises at least one or two of bacillus thermotolans and Novibacillus thermophilus. The complex microbial inoculant disclosed by the invention is a thermophilic complex microbial inoculant, can directly utilize nutrient substances in organic solid wastes such as municipal sludge, livestock and poultry manure, agricultural residues, kitchen wastes and the like to grow and breed, and meanwhile, humus in compost is reduced; hydroxyl free radicals are continuously generated along with the change of anaerobic / aerobic alternating conditions of the compost, and the humification process of the compost is promoted through the free radicals.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology and environmental technology, and particularly relates to a compound high-temperature bacterial agent and a method for promoting humification of compost. BACKGROUND

[0002] Urban sludge, household garbage, livestock and poultry manure, and agricultural and forestry residues are the main sources of current large-scale organic solid waste, with an annual production of several billion tons, and have the characteristics of large quantity and wide range. Organic waste is not only a major source of environmental pollution, but also a huge biomass resource library. Due to the rich organic matter and nitrogen, phosphorus, potassium and other nutrient components, the resource utilization potential is huge. Composting is an effective means to realize the resource utilization of organic solid waste. However, the fermentation temperature of traditional composting is low, which leads to a slow humification process, seriously affecting the efficiency of composting and the quality of composting products.

[0003] Humification refers to the process of converting fresh organic matter (mainly plant and animal residues) into humus through biochemical or chemical reactions under the action of microorganisms. Composting is a process of artificial humification. The process of affecting composting includes temperature, pH, C / N, moisture content, etc. Composting can promote the humification of organic matter, which is an important means of resource utilization of organic solid waste, and also an important "barrier" to prevent pollutants in organic solid waste from entering the soil and other environments. The traditional composting theory believes that the essence of composting is the process of microbial-driven organic humification and pollutant degradation, and the generation of heat energy.

[0004] Free radicals, also known as "radicals" in chemistry, are atoms or groups with unpaired electrons formed by homolysis of covalent bonds of molecules under external conditions such as light and heat, and have strong chemical reactivity. According to the length of its existence time, it can be divided into transient free radicals and persistent free radicals (Environmental persistent free radicals, EPFRs). Reactive oxygen species (Reactive oxygen species, ROS) is a representative free radical in transient free radicals, which widely exists in the environment, has strong reactivity, and participates in the transformation of many elements in the environment. Mainly including hydroxyl radical (·OH), singlet oxygen (O2), superoxide radical (·O2 1 - ​·OH is a common transient free radical in the environment and the most powerful active oxygen free radical in oxidation (E^φ=2.8 V), which was first discovered by Haber and Weiss in 1934 in what is now known as the Fenton reaction. ·OH is composed of one hydrogen atom and one oxygen atom, which makes them highly active and easily steal hydrogen atoms from other molecules to form water molecules. Therefore, it is one of the strongest oxidizing agents in nature, which can immediately react with surrounding chemicals (including organic pollutants and inhibitors) without selectivity. ·OH is ubiquitous in various types of environments, including natural water bodies, atmosphere, biological systems, and interstellar space, etc. The gas phase, liquid phase, and heterogeneous reactions of ·OH with organic compounds are usually complex chemical processes through many partially oxidized free radical intermediates.

[0005] ·OH plays an important role in the redox transformation of humic acid. In the aerobic-anaerobic alternating environment, the iron redox cycle driven by iron-reducing bacteria such as Shewanella sp. and Geobacter sp. can promote the continuous production of ·OH in the environment, achieving effective removal of environmental pollutants. High-temperature composting of organic solid waste not only provides nutrients for microbial growth, but also forms an aerobic-anaerobic reaction interface required for microbial-driven iron redox cycle reactions in compost particles due to uneven mass transfer, which makes the oxygen in the core of the compost particles extremely low. The aerobic-anaerobic reaction interface changes dynamically with changes in compost temperature, moisture content, aeration amount, turning, etc. Studies have shown that thermophilic bacteria in the composting process can accelerate the redox process of soluble iron, which directly leads to the continuous production of ·OH in the compost, thereby accelerating the degradation of organic matter in the compost and improving the maturity and humification of the compost. Composting is one of the main methods of garbage disposal, and composting can convert organic waste into stable humus to achieve the resource and harmlessness of organic waste. The composting environment contains the basic substances for free radical production and meets the environmental conditions for free radical production, which is a "hot zone" for free radical production.

[0006] The study adjusts the composting process and raw materials, the aerobic / anaerobic cycle program of the compost itself, and uses the humus inside the compost to produce a large amount of hydroxyl radicals, which work together with the microorganisms in the compost to achieve the rapid advancement of the humification process.

[0007] Bacillus thermophilus (B. thermophilus) Bacillus thermotolerans ) is a group of facultative anaerobic, gram-positive bacteria that can grow at 20-60°C, with an optimal growth temperature of 50°C. Under anaerobic conditions, they can grow with organic matter as an electron donor and AQDS as an electron acceptor, reducing the quinone of AQDS.

[0008] Neocallimastix patricium (N. patricium) Novibacillus thermophilusIt grows at 30–65°C (optimal 50°C), pH 6.5–10.5 (optimal pH 7.5–8.0), and 0–10% NaCl (optimal 5.0–7.0% NaCl); anaerobic growth is possible with a variety of electron donors and acceptors. AQDS, fumarate, or nitrate are used as electron acceptors, while lactic acid, acetate, sucrose, and D-glucose are used as electron donors. The aforementioned thermophilic microorganisms are widely distributed in high-temperature environments such as composting, volcanoes, and hot springs. They can all grow facultatively and, under anaerobic conditions, can utilize various organic substances as electron donors to reduce humic substances, while simultaneously promoting the biodegradation of organic matter. They have been applied to ultra-high temperature composting of urban sludge and the harmless disposal of animal carcasses, effectively improving the degradation efficiency of organic solid waste. However, to date, there are no research reports on the role of thermophilic humic acid-reducing bacteria and thermophilic microorganisms in promoting the composting humification process. Summary of the Invention

[0009] The purpose of this invention is to provide a compound microbial agent that can enhance the composting and humification process.

[0010] The technical solution adopted in this invention is: A compound high-temperature microbial agent and its method for enhancing the humification process in composting, comprising the following steps: preparing the compound high-temperature microbial agent, and mixing the compound high-temperature microbial agent with organic solid waste in a certain proportion for composting.

[0011] The preparation of the compound high-temperature bacterial agent described in step 1 includes the following steps: activating thermophilic humic reducing bacteria to prepare a seed liquid, inoculating it into a fermentation medium for anaerobic fermentation to prepare the compound high-temperature bacterial agent.

[0012] (1) Preferred, thermophilic humic reducing bacteria are facultative aerobic bacteria.

[0013] (2) Preferably, the thermophilic humic reducing bacteria are Bacillus thermotolerans ,and Novibacillus thermophilus At least one or two of them.

[0014] (3) Preferred thermophilic humic reducing bacteria Bacillus thermotolerans and Novibacillus thermophilus The method for preparing seed solution is as follows: Use a 10% concentration seed solution at a 2% inoculum rate. 8 cfu / mL Bacillus thermotolerans and Novibacillus thermophilus The shake-flask bacterial culture was inoculated into a seed tank containing CCTCC No. 0033 medium, and incubated at 50°C with air introduced at a flow rate of 50-80 m³ / h. 3 Fermentation time is 24-48 hours under the conditions of 160-250 rpm and stirring speed of 1h.

[0015] The composition of the CCTCC No.0033 culture medium is as follows: 10.0 g / L tryptone, 5.0 g / L yeast extract, 10.0 g / L NaCl, pH 7.0, 0.15 MPa, sterilized at 121℃ for 30 min.

[0016] (4) Preferably, the preparation method of the compound thermophilic bacteria agent is as follows: first, Bacillus thermotolerans and Novibacillus thermophilus The seed culture was inoculated into the anaerobic fermenter at a rate of 1.5% and anaerobic fermented at 50°C for 48-96 hours. After fermentation, 4% soybean meal powder was added as a conditioning agent, stirred evenly, and then filtered through a plate and frame filter press to achieve a moisture content of 45%-55%.

[0017] Preferably, the anaerobic fermentation medium is prepared as follows: 2 g / L (NH4)SO4, 0.2 g / L MgSO4·7H2O, 0.01 g / L CaCl2·2H2O, 0.001 g / L FeSO4·7H2O, 1.5 g / L Na3PO4·12H2O, 1.5 g / L KH2PO4, 1 g / L glucose, 1-5% humic acid, adjust the pH to 7.25±0.1, stir evenly, introduce nitrogen / carbon dioxide mixture into the medium, sterilize at 100℃ for 45 minutes, and cool to 50℃ for later use.

[0018] Method for enhancing the humification process in composting with compound thermophilic bacteria agent: Mix the compound thermophilic bacteria agent and organic solid waste materials at a weight ratio of 1%~5%: 95%~99%.

[0019] Preferably, the organic solid waste material can be one or a mixture of several of the following: urban sludge, livestock and poultry manure, and domestic waste.

[0020] The beneficial effects of this invention are: The compound microbial agent of this invention is a facultative anaerobic compound microbial agent with reducing humic substances. It can directly utilize the nutrients in organic solid wastes such as urban sludge, livestock and poultry manure, agricultural residues, and kitchen waste for growth and reproduction. As the anaerobic / aerobic alternation conditions of composting change, hydroxyl radicals are continuously generated, which promote the humification process of composting through free radicals. Attached Figure Description

[0021] Figure 1 These are the temperature change curves of different composting treatments in Example 4; Figure 2 These are the three-dimensional fluorescence spectra of the different composting treatments in Example 4; Figure 3 This is the curve showing the change in hydroxyl radical content in sludge composting in Example 4; Figure 4It is the humification index of the different composting treatments in Example 4. Detailed Implementation

[0022] At 2% Bacillus thermotolerans SgZ-8 T The bacterial culture was inoculated into CCTCC No. 0033 medium and incubated at 50°C with an aeration rate of 60-100 m³ / h. 3 / h, stirring speed 200 rpm, fermentation time 12 hours, to obtain Bacillus thermotolerans SgZ-8 T Seed liquid; The anaerobic fermentation medium is prepared as follows: 2 g / L (NH4)SO4, 0.2 g / L MgSO4·7H2O, 0.01 g / L CaCl2·2H2O, 0.001 g / L FeSO4·7H2O, 1.5 g / L Na3PO4·12H2O, 1.5 g / L KH2PO4, 1 g / L glucose, and 1-5% humic acid. The pH is adjusted to 7.25±0.1. The mixture is stirred evenly, nitrogen gas is introduced into the medium, and the medium is sterilized at 100℃ for 45 minutes. The medium is then cooled to 50℃ for later use.

[0023] Preparation of compound thermophilic bacteria agent: Bacillus thermotolerans SgZ-8 T The bacterial culture was inoculated into the anaerobic fermenter at an inoculum rate of 1.5% and anaerobic fermented at 50℃ for 72 hours. After fermentation, 5% soybean meal powder was added as a conditioning agent, stirred evenly, and then filtered through a plate and frame filter press to achieve a moisture content of 45%~55%.

[0024] final Bacillus thermotolerans SgZ-8 T The bacterial count is approximately 1×10⁻⁶. 8 cfu / g.

[0025] At 2% Novibacillus thermophilus SG-1 T The bacterial culture was inoculated into No. 0033 medium and incubated at 50°C with an aeration rate of 60-100 m³ / h. 3 / h, stirring speed 200 rpm, fermentation time 20 hours, to obtain Novibacillus thermophilus SG-1 T Seed liquid; The anaerobic fermentation medium is prepared as follows: 2 g / L (NH4)SO4, 0.2 g / L MgSO4·7H2O, 0.01 g / L CaCl2·2H2O, 0.001 g / L FeSO4·7H2O, 1.5 g / L Na3PO4·12H2O, 1.5 g / L KH2PO4, 1 g / L glucose, and 1-5% humic acid. The pH is adjusted to 7.25±0.1. The mixture is stirred evenly, nitrogen gas is introduced into the medium, and the medium is sterilized at 100℃ for 45 minutes. The medium is then cooled to 50℃ for later use.

[0026] Preparation of compound thermophilic bacteria agent: Novibacillus thermophilus SG-1 T The bacterial culture was inoculated into the anaerobic fermenter at an inoculum rate of 1.5% and anaerobic fermented at 50℃ for 72 hours. After fermentation, 5% soybean meal powder was added as a conditioning agent, stirred evenly, and then filtered through a plate and frame filter press to achieve a moisture content of 45%~55%.

[0027] final Novibacillus thermophilus SG-1 T The bacterial count is approximately 0.8 × 10⁻⁶. 8 cfu / g.

[0028] At a concentration of 2%, Bacillus thermotolerans SgZ-8 T The bacterial culture was inoculated into CCTCC No. 0033 medium and incubated at 50°C with an aeration rate of 60-100 m³ / h. 3 / h, stirring speed 200 rpm, fermentation time 12 hours, to obtain Bacillus thermotolerans SgZ-8 T Seed liquid; At a concentration of 10, use 2% 9 cfu / mL Novibacillus thermophilus SG-1 T The bacterial culture was inoculated into No. 0033 medium and incubated at 50°C with an aeration rate of 60-100 m³ / h. 3 / h, stirring speed 200 rpm, fermentation time 20 hours, to obtain Novibacillus thermophilus SG-1 T Seed liquid; The anaerobic fermentation medium is prepared as follows: 2 g / L (NH4)SO4, 0.2 g / L MgSO4·7H2O, 0.01 g / L CaCl2·2H2O, 0.001 g / L FeSO4·7H2O, 1.5 g / L Na3PO4·12H2O, 1.5 g / L KH2PO4, 1 g / L glucose, and 1-5% humic acid. The pH is adjusted to 7.25±0.1. The mixture is stirred evenly, nitrogen gas is introduced into the medium, and the medium is sterilized at 100℃ for 45 minutes. The medium is then cooled to 50℃ for later use.

[0029] Preparation of compound thermophilic bacteria agent: Bacillus thermotolerans SgZ-8 T bacterial solution and Novibacillus thermophilus SG-1 T The bacterial culture was inoculated into the anaerobic fermenter at an inoculum rate of 1.5% and anaerobic fermented at 50℃ for 72 hours. After fermentation, 5% soybean meal powder was added as a conditioning agent, stirred evenly, and then filtered through a plate and frame filter press to achieve a moisture content of 45%~55%.

[0030] final Bacillus thermotolerans SgZ-8 T and Novibacillus thermophilus SG-1 T The bacterial count is approximately 8.3 × 10⁻⁶. 7 cfu / g and 7×10 7 cfu / g.

[0031] Example 4: The effect of compound thermophilic bacteria agent on promoting composting and humification of sludge in high-temperature composting.

[0032] Prepare according to the method in Example 3 respectively Bacillus thermotolerans SgZ-8 T and Novibacillus thermophilus SG-1 T Seed liquid; The compound thermophilic bacteria agent was prepared according to the method in Example 3.

[0033] After plate counting and test tube analysis, the total bacterial count in the obtained compound thermophilic bacteria agent was approximately 5.2 × 10⁻⁶. 8 cfu / g, Bacillus thermotolerans SgZ-8 T and Novibacillus thermophilus SG-1 T The bacterial count was approximately 3.3 × 10⁻⁶. 8 cfu / g, 1.9×10 8 cfu / g.

[0034] Concentrated sludge from a wastewater treatment plant in Fujian Province (basic properties of the sludge are shown in Table 3) was collected and placed in a fermenter. The pH was adjusted to 7.0 ± 0.5 by adding 0.2% foaming agent, 2 g / L yeast extract, 0.5 g / L ammonium chloride, 0.5 g / L glucose, 0.5 g / L trypsinone, and 0.3 g / L potassium dihydrogen phosphate, and the sludge was adjusted to a solids content of 4%. The mixture was sterilized at 100℃ for 45 minutes and then cooled to 50℃ for later use.

[0035] Dewatered sludge and rice husks were initially mixed evenly at a weight ratio of 5:1 (the basic properties of the sludge high-temperature compost material are shown in Table 2). The sludge compost material and the prepared compound thermophilic bacteria agent were then piled and fermented at a weight ratio of 95%:5%. A conventional treatment was also conducted without inoculation of the compound thermophilic bacteria agent. Both the conventional and inoculated treatments involved turning the compost pile every 5 days or when the temperature exceeded 60℃. The compost was analyzed and tested after the composting process was completed.

[0036] Table 2. Basic Properties of Sludge Compost Materials pH Moisture content (%) Total carbon (%) Total nitrogen (%) C / N 7.37 66.52 54.5 3.6 15:1 Temperature change curves for different composting treatments are as follows: Figure 1 As shown. Compared with the control treatment, the temperature increased significantly and the duration of the high-temperature period was longer after adding the compound thermophilic agent of the present invention. Three-dimensional fluorescence spectroscopy showed a significant increase in the degree of humification. Figure 2 The ·OH content in the treatment group was highest during the high-temperature period. Figure 3 ), the humification index increased ( Figure 4 This indicates that the addition of microbial agents promoted the humification process in composting.

Claims

1. A compound microbial agent containing thermophilic bacterial strains. Bacillus thermotolerans ,and Novibacillus thermophilus At least one or two of them.

2. The compound microbial agent according to claim 1, characterized in that: Bacillus thermotolerans for Bacillus thermotolerans SgZ-8 T Collection number: CCTCC No.AB 2012108T.

3. The compound microbial agent according to claim 1, characterized in that: Novibacillus thermophilus for Novibacillus thermophilus SG-1 T Collection number: CGMCC No.15303.

4. The compound microbial agent according to claim 1, characterized in that: thermophilic bacteria strains Bacillus thermotolerans SgZ-8 T and Novibacillus thermophilus SG-1 T The bacterial content was (10) 8 ~10 9 cfu / g); (10 8 ~10 9 cfu / g).

5. A compound high-temperature microbial agent and a method for promoting composting and humification of the same, comprising adding the compound microbial agent of claims 1 to 4 to organic solid waste for composting.

6. The method according to claim 5, characterized in that: The maximum temperature for composting should not exceed 100℃.

7. The method according to claim 5, characterized in that: During the composting process, the high-temperature period of no less than 45°C shall last for no less than 8 days.

8. The method according to claim 5, characterized in that: Organic solid waste materials are selected from at least one of urban sludge, livestock and poultry manure, agricultural residues, and kitchen waste.

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