Microbial Composite Bacteria for Sludge Reduction in Biochemical System, Its Preparation Method and Application of Microbial Agent for Sludge Reduction in Biochemical System

Abstract

The invention discloses a biochemical system sludge reduction microbial composite bacteria, a preparation method thereof, and an application of a biochemical system sludge reduction microbial agent, belonging to the field of sewage treatment and microorganisms, and solving the problem of how to reduce excess sludge in a sewage treatment process at low cost without generating secondary pollution or solid waste. The invention discloses Bacillus smithii (preservation number: CGMCC No.31841); Heyndrickxia coagulans (preservation number: CGMCC No.31842); Parageobacillus caldoxylosilyticus (preservation number: CGMCC No.31844); Aeribacillus pallidus (preservation number: CGMCC No.31845); Pallidibacillus thermolactis (preservation number: CGMCC No.31846); The microbial composite bacteria and bacterial agents composed of No. 31847 are applied to the biochemical system to reduce the amount of residual sludge in the sewage treatment process from the source. It has the advantages of high activity, low investment, low energy consumption, easy operation and no secondary pollution.

Description

Technical Field

[0001] The present invention relates to the field of sewage treatment and microbial technology, in particular to a biochemical system sludge reduction microbial composite bacteria and a preparation method thereof, and application of a biochemical system sludge reduction microbial agent. Background Art

[0002] Sludge, a typical wastewater treatment product, is an extremely complex, heterogeneous mass composed of organic debris, bacterial cells, inorganic particles, and colloids. Its key characteristics are a high moisture content (over 99%), a high organic content, a tendency to decompose and stink, and fine particles, a low specific gravity, and a colloidal liquid state.

[0003] Sludge treated in environmental protection projects generally refers to the sediment and froth produced during sewage treatment at sewage treatment plants. Its composition is complex, containing numerous microorganisms, pathogens, heavy metals, and organic pollutants. Because it contains numerous harmful substances, it can become secondary pollution to sewage treatment plants if not properly handled.

[0004] The current status of sludge treatment in my country is as follows:

[0005] 1. Sanitary landfill: The most commonly used traditional sludge disposal method is landfill. This method is simple, easy to implement, and low-cost. However, it fails to achieve the ultimate goals of sludge disposal, such as harmlessness, stabilization, volume reduction, and resource utilization, and it can also easily cause secondary pollution. Furthermore, with current land shortages and the gradual replacement of landfills with incineration plants, this treatment method is also facing elimination.

[0006] 2. Land Utilization: Sludge can be used for agricultural purposes, for forestry and horticulture, and for the improvement of abandoned mine sites. However, since toxic and harmful microorganisms and heavy metals are present in sludge, it must be stabilized and rendered harmless before land use, otherwise it will cause secondary contamination of the soil.

[0007] Sludge incineration: This method involves burning sludge mixed with auxiliary fuels in a specific ratio. Sludge incineration can achieve waste reduction goals, completely oxidizing organic matter and leaving virtually no heavy metals in the ash. However, the exhaust gas produced by sludge incineration can cause secondary environmental pollution. Furthermore, due to the high moisture content and low calorific value of sludge, the addition of large amounts of auxiliary fuel is required, making it uneconomical and unsuitable.

[0008] 4. Sludge Composting: Composting is the process of fermenting sludge using microorganisms. By adding a certain proportion of leavening agents and conditioners, such as straw, rice straw, sawdust, or household garbage, to the sludge, the microbial community, under a moist environment, oxidizes and decomposes various organic substances, converting them into humus-like substances. Humus-like substances are a high-quality organic compound fertilizer or can be used to produce organic bacterial fertilizer.

[0009] 5. Sludge Thermal Drying: Thermal drying uses heat to dry sludge. Its high-temperature sterilization kills pathogens and parasite eggs, allowing the sludge to dry quickly and preventing odor from affecting the surrounding environment. Thermal drying significantly reduces the volume of sludge by 75-80%, resulting in highly efficient granular fertilizer that is easy to store and transport.

[0010] Of course, there are also new technologies for sludge treatment in China. For example, the applicant's prior patent application number is 202210432505.X, the publication number is CN114835265A, and the invention name is: A method for treating high-ammonia nitrogen wastewater. This method only uses a fully biological treatment method for ultra-high ammonia nitrogen wastewater with a concentration of 1000-6000 mg / L and a pH value greater than 9.5, providing a biological treatment pathway for the treatment of high-ammonia nitrogen wastewater, but its scope of use is limited.

[0011] Another example is the use of solar-powered low-temperature composite membrane sludge treatment technology. Sludge with a moisture content of 80% to 85% is evenly distributed within a sealed solar-powered composite hydrophobic membrane box. Using solar energy, it undergoes low-temperature evaporation and deep drying, producing a sludge residue with a moisture content of less than 8%. This residue can be used to make activated carbon, and the evaporated water can be reused or discharged in compliance with emission standards. Biomass-coupled power generation, which utilizes biomass resources such as sludge with existing coal-fired power plants for power generation, can promote sludge reduction, harmlessness, resource utilization, and large-scale disposal. These methods all focus on sludge treatment or physical and chemical methods to reduce sludge, but fail to achieve sludge reduction at the source. Summary of the Invention

[0012] In the field of sewage treatment technology, how to solve the technical problem of how to reduce the residual sludge in the sewage treatment process from the source by preparing microbial composite bacteria or microbial agents for sludge reduction in biochemical systems, achieving low cost and without generating secondary pollution or solid waste. The present invention provides a biochemical system sludge reduction microbial composite bacteria and a preparation method thereof and the application of a biochemical system sludge reduction microbial agent. The present invention applies microbial technology to the biochemical system, and achieves the reduction of the residual sludge in the sewage treatment process from the source; the technology improves the efficiency of sludge reduction, reduces operating costs, solves the problems of large investment and environmental pollution, and truly realizes a low-cost, harmless and environmentally friendly treatment technology revolution of sludge, and has very broad application prospects in sewage treatment and sludge treatment.

[0013] The purpose of the present invention is specifically achieved through the following technical solutions:

[0014] The present invention discloses Bacillus smithii, which is characterized in that its preservation number is: CGMCC No.31841, the preservation date is: September 3, 2024, the name of the preservation unit is: General Microbiology Center of China Culture Collection Administration (CGMCC), and the address is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0015] The present invention discloses Heyndrickxia coagulans, which is characterized in that its preservation number is: CGMCC No. 31842, the preservation date is: September 3, 2024, the name of the preservation unit is: General Microbiology Center of China Culture Collection Administration (CGMCC), and the address is: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0016] The present invention discloses Parageobacillus caldoxylosilyticus, characterized in that its preservation number is: CGMCC No.31844, the preservation date is: September 3, 2024, the name of the preservation unit is: General Microbiology Center of China Culture Collection Administration (CGMCC), and the address is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0017] The present invention discloses Aeribacillus pallidus, which is characterized in that its preservation number is CGMCC No.31845, the preservation date is September 3, 2024, the name of the preservation unit is China General Microbiology Center (CGMCC), and the address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0018] The present invention discloses a thermophilic lactic acid bacillus (Pallidibacillus thermolactis), which is characterized in that its preservation number is: CGMCC No.31847, the preservation date is: September 3, 2024, the name of the preservation unit is: General Microbiology Center of China Culture Collection Administration (CGMCC), and the address is: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0019] The invention discloses a biochemical system sludge reduction microbial composite bacteria, comprising: Bacillus smithii, Heyndrickxia coagulans, Parageobacillus caldoxylosilyticus, Aeribacillus pallidus and Pallidibacillus thermolactis.

[0020] Among them, the effective viable cell count ratio of Bacillus smithii: Heyndrickxiacoagulans: Parageobacillus caldoxylosilyticus: Aeribacillus pallidus: Pallidibacillus thermolactis is: (10-25%): (25-30%): (18-26%): (12-29%): (9-30%).

[0021] The present invention discloses a method for preparing a biochemical system sludge reduction microbial composite bacteria, comprising:

[0022] Step 1: Add sterile water into a sterile bottle to soak the soil material, mix the soil material and the sterile water thoroughly, and then let it settle; the soil material is a surface moist soil or moist silt with obvious humus on the riverside that is more than 20 years old;

[0023] Step 2: Take the supernatant and dilute the supernatant in a gradient manner using the dilution coating method. Take a suspension of each gradient dilution supernatant and evenly spread it on NB solid medium, LB solid medium, NA solid medium, TSA solid medium, and PDA solid medium, and place it in an incubator at a constant temperature of 40°C; take the undiluted supernatant and evenly spread it on NB solid medium, LB solid medium, NA solid medium, TSA solid medium, and PDA solid medium as a control and place it in an incubator at a constant temperature of 37°C;

[0024] Step 3: After a single colony grows, observe the bacterial separation on all dilution gradient plates, and visually select a target gradient whose separation effect meets the task requirements; screen out colonies with different apparent morphology in the target gradient and pick single colonies. Use the dilution coating method to dilute the single colony gradient, evenly spread it on the corresponding culture medium, and place it in an incubator at a constant temperature of 35°C for incubation; evenly spread the undiluted single colony on the corresponding culture medium as a control, and place it in an incubator at a constant temperature of 35°C for incubation. The incubator temperature is increased by 1°C, and this step is repeated until the incubator temperature reaches 45°C; repeat this step again until the colony morphology of each single colony is consistent;

[0025] Step 4: Pick the target single colony from the single colonies with consistent colony morphology, purify the endophytic bacteria by the partitioning and streaking method to obtain a monoclonal colony, and obtain the biochemical system sludge reduction microbial composite bacteria after 16S rDNA gene sequencing.

[0026] In step 1, the soil material is preferably: riparian wetland soil in humus alluvial soil or organic latent soil.

[0027] In step 3, the corresponding culture medium formula includes:

[0028] The formula of the culture medium of Bacillus smithii is:

[0029] LB solid medium: peptone 10 g / L, yeast powder 5 g / L, sodium chloride 5 g / L, and agar powder 20 g / L;

[0030] LB liquid medium: peptone 10 g / L, yeast powder 5 g / L, and sodium chloride 5 g / L;

[0031] After preparation, autoclave at 121°C for 20 minutes;

[0032] Heyndrickxia coagulans culture medium recipe:

[0033] NB solid medium: peptone 20 g / L, beef extract 3 g / L, and agar powder 20 g / L;

[0034] NB liquid medium: peptone 20 g / L and beef extract 3 g / L;

[0035] After preparation, autoclave at 121°C for 20 minutes;

[0036] The formula of the culture medium of Parageobacillus caldoxylosilyticus is:

[0037] LB solid medium: peptone 10 g / L, yeast powder 5 g / L, sodium chloride 5 g / L, and agar powder 20 g / L;

[0038] LB liquid medium: peptone 10 g / L, yeast powder 5 g / L and sodium chloride 5 g / L;

[0039] After preparation, autoclave at 121°C for 20 minutes;

[0040] Aeribacillus pallidus culture medium recipe:

[0041] LB solid medium: peptone 10 g / L, yeast powder 5 g / L, sodium chloride 5 g / L, and agar powder 20 g / L;

[0042] LB liquid medium: peptone 10 g / L, yeast powder 5 g / L and sodium chloride 5 g / L;

[0043] After preparation, autoclave at 121°C for 20 minutes;

[0044] The formula of the culture medium of Pallidibacillus thermolactis is as follows:

[0045] LB solid medium: peptone 10 g / L, yeast powder 5 g / L, sodium chloride 5 g / L, and agar powder 20 g / L;

[0046] LB liquid medium: peptone 10 g / L, yeast powder 5 g / L and sodium chloride 5 g / L;

[0047] After preparation, sterilize at 121℃ for 20 minutes.

[0048] The invention discloses a biochemical system sludge reduction microbial agent, comprising Bacillus smithii, Heyndrickxia coagulans, Parageobacillus caldoxylosilyticus, Aeribacillus pallidus, Pallidibacillus thermolactis, a biochemical system sludge reduction microbial composite bacteria or a biochemical system sludge reduction microbial composite bacteria obtained by a preparation method.

[0049] The present invention discloses Bacillus smithii, Heyndrickxiacoagulans, Parageobacillus caldoxylosilyticus, Aeribacillus pallidus, Pallidibacillus thermolactis, a biochemical system sludge reduction microbial composite bacteria, a biochemical system sludge reduction microbial composite bacteria obtained by a preparation method, or a biochemical system sludge reduction microbial agent for biochemical system sludge reduction, and use thereof in biochemical system sludge reduction.

[0050] The present invention discloses a biochemical system sludge reduction microbial composite bacteria, a preparation method thereof, and a method for using the biochemical system sludge reduction microbial composite bacteria or a biochemical system sludge reduction microbial agent in biochemical system sludge reduction, comprising:

[0051] Take the biochemical system sludge reduction microbial composite bacteria or the biochemical system sludge reduction microbial agent and add it into the biochemical system and mix it with the sludge.

[0052] Among them, the method of adding includes:

[0053] The biochemical system temperature should be between 25°C and 45°C, the DO value should be between 1.0mg / L and 4.0mg / L, and the pH value should be between 6 and 9.

[0054] The dosing points are: the aerobic tank inlet end of the biochemical system and the sludge return point of the secondary sedimentation tank;

[0055] For a biochemical system that reduces 1.0 ton of absolute dry sludge, the dosage of the biochemical system sludge reduction microbial composite bacteria or biochemical system sludge reduction microbial agent is 2.5-5 kg;

[0056] The daily dosage of biochemical system sludge reduction microbial composite bacteria or biochemical system sludge reduction microbial agent is determined based on the residual sludge output obtained from the daily water inlet load and output of the biochemical system.

[0057] Among them, the temperature at which the microbial activity of the biochemical system sludge reduction microbial composite bacteria or the biochemical system sludge reduction microbial agent is strongest is 35°C to 45°C, and the biochemical system temperature is preferably 35°C to 45°C.

[0058] The beneficial effects of the present invention are:

[0059] The ability of the microbial strains in the biochemical system sludge reduction microbial composite bacteria or the biochemical system sludge reduction microbial agent disclosed in the present invention to secrete different enzymes under specific environments has been verified through a large number of experiments as follows:

[0060] Bacillus smithii secretes proteases and amylases;

[0061] Heyndrickxia coagulans secretes L-lactate dehydrogenase, amylase, and protease;

[0062] Parageobacillus caldoxylosilyticus secretes xylanases and cellulases;

[0063] Aeribacillus pallidus secretes 4-α-glycosyltransferase, amylase, and protease;

[0064] Pallidibacillus thermolactis secretes glycolytic enzymes and lactate dehydrogenase.

[0065] The aforementioned microbial strains and their enzymes decompose the various components of aging or dead microorganisms in the biochemical system, providing energy for the microbial complex / microbial agent. The decomposed organic matrix in the sludge produces carbon dioxide and water, or decomposes some of the adsorbed organic matrix in the sludge into carbon dioxide and water, thereby reducing sludge volume. At the same time, competition among microorganisms inhibits the growth of some other bacteria, such as silk-producing microorganisms.

[0066] The present invention reduces the amount of excess sludge produced by adding the biochemical system sludge reduction microbial composite bacteria or the biochemical system sludge reduction microbial agent disclosed in the present invention to the biochemical system, thereby achieving source control of sludge reduction. The technical solution disclosed in the present invention has the following advantages:

[0067] 1. No hardware investment, extremely low energy consumption, and relatively low investment and operating costs;

[0068] 2. Reduce the amount of excess sludge in the biochemical system by 30% to 80%;

[0069] 3. For systems that need to supplement carbon sources, the amount of carbon source supplement can be reduced;

[0070] 4. The sludge after reduction is easier to dewater or dispose of.

[0071] The technical solution disclosed in the present invention is suitable for sewage treatment plants with substances with high organic content and no obvious toxicity. It is also used for centralized disposal of multiple sewage treatment plants and coordinated treatment of various organic wastes. It has the advantages of high activity, low investment, low energy consumption, easy operation, and no secondary pollution.

[0072] Biological Deposit Description:

[0073] (1) Biological material: SW-B101, classification name: Bacillus smithii, deposit number: CGMCC No. 31841, deposit date: September 3, 2024, depositor name: General Microbiology Center of China Culture Collection Administration (CGMCC), address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0074] (2) Biological material: SW-B102, classification name: Heyndrickxia coagulans, deposit number: CGMCC No. 31842; deposit date: September 3, 2024, depositor name: General Microbiology Center of China Culture Collection Administration (CGMCC), address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0075] (3) Biological material: SW-B106, classification name: Parageobacillus caldoxylosilyticus, the deposit number is: CGMCC No. 31844; the deposit date is: September 3, 2024, the depositor is: General Microbiology Center of China Culture Collection Administration (CGMCC), the address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0076] (4) Biological material: SW-B107, classification name: Aeribacillus pallidus, deposit number: CGMCC No. 31845; deposit date: September 3, 2024, deposited by: General Microbiology Center of China Culture Collection Administration (CGMCC), address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0077] (5) Biological material: SW-B109, classification name: Pallidibacillus thermolactis, the deposit number is: CGMCC No. 31847; the deposit date is: September 3, 2024, the depositary name is: General Microbiology Center of China Culture Collection Administration (CGMCC), the address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing. BRIEF DESCRIPTION OF THE DRAWINGS

[0078] The present invention will be described in further detail below with reference to the accompanying drawings and examples.

[0079] Figure 1 This is a microscope photograph of Bacillus smithii provided by the present invention.

[0080] Figure 2 This is a microscope photograph of Heyndrickxia coagulans provided by the present invention.

[0081] Figure 3 This is a microscope photograph of the Parageobacillus caldoxylosilyticus provided by the present invention.

[0082] Figure 4 The present invention provides a microscope photograph of Aeribacillus pallidus.

[0083] Figure 5 The present invention provides a microscope photograph of Pallidibacillus thermolactis.

[0084] Figure 6 It is a schematic diagram of the activation culture process provided by the present invention.

[0085] Figure 7 This is a schematic diagram after enrichment culture provided by the present invention.

[0086] Figure 8 This is one of the line separation schematic diagrams provided by the present invention.

[0087] Figure 9 This is the second schematic diagram of line separation provided by the present invention.

[0088] Figure 10 It is a schematic diagram of bacterial morphology observed under a microscope provided by the present invention.

[0089] Figure 11 It is a schematic diagram of the simulation small-scale experimental process provided by the present invention.

[0090] Figure 12 This is a schematic diagram of sludge concentration tracking in a biochemical system provided in the first verification example of the present invention.

[0091] Figure 13 This is a schematic diagram of tracking the discharge of excess sludge from the biochemical system provided in the first verification example of the present invention.

[0092] Figure 14 This is a schematic diagram of on-site photos provided in Verification Example 1 of the present invention.

[0093] Figure 15 This is a schematic diagram of sludge concentration tracking in a biochemical system provided in the second verification example of the present invention.

[0094] Figure 16 This is a schematic diagram of tracking the discharge of excess sludge from the biochemical system provided in the second verification example of the present invention.

[0095] Figure 17 This is a schematic diagram of the on-site photo provided by the second verification example of the present invention. DETAILED DESCRIPTION

[0096] After a long period of research, the inventors screened, isolated and purified five highly efficient sludge reduction strains from the environment, and numbered them #HT1, #HT2, #HT3, #HT4 and #HT5. The 16S rDNA was sequenced and compared with the NCBI geneBank database and the 16S rDNA bacterial database (Maidak et al, 2001) based on the sequencing results. A phylogenetic tree was constructed using Mega software to determine the species of the bacteria, and analysis was performed to determine the classification of the bacteria.

[0097] Microscopic photos of the morphological characteristics of the strain numbered #HT1 are shown in Figure 1 16S rDNA sequencing and NCBI geneBank data comparison showed that the strain was Bacillus smithii, and the strain was subsequently deposited in the General Microbiology Center of the China Culture Collection Administration with the deposit number: CGMCC No. 31841.

[0098] The 16S rDNA sequencing results of this strain are shown in SEQ ID No: 1:

[0099]

[0100]

[0101] Microscopic photos of the morphological characteristics of the strain numbered #HT2 are shown in Figure 2 16S rDNA sequencing and NCBI geneBank data comparison showed that the strain was Heyndrickxia coagulans, and the strain was subsequently deposited in the General Microbiology Center of the China Culture Collection Administration of Microorganisms with the deposit number: CGMCC No. 31842.

[0102] The 16S rDNA sequencing results of this strain are shown in SEQ ID No: 2:

[0103]

[0104] Microscopic photos of the morphological characteristics of the strain numbered #HT3 are shown in Figure 3 16S rDNA sequencing and NCBI geneBank comparison showed that the strain was Parageobacillus caldoxylosilyticus, and the strain was subsequently deposited in the General Microbiology Center of the China Culture Collection Administration with the deposit number: CGMCC No. 31844.

[0105] The 16S rDNA sequencing results of this strain are shown in SEQ ID No: 3:

[0106]

[0107] Microscopic photos of the morphological characteristics of the strain numbered #HT4 are shown in Figure 4 After comparison with GeneBank, the strain was identified as Aeribacillus pallidus, and the strain was subsequently deposited in the General Microbiology Center of the China Culture Collection Administration with the deposit number: CGMCC No. 31845.

[0108] The 16S rDNA sequencing results of this strain are shown in SEQ ID No: 4:

[0109]

[0110]

[0111] Microscopic photos of the morphological characteristics of the strain numbered #HT5 are shown in Figure 5 16S rDNA sequencing and NCBI geneBank comparison showed that the strain was Pallidibacillus thermolactis, and the strain was subsequently deposited in the General Microbiology Center of the China Culture Collection Administration of Microorganisms with the deposit number: CGMCC No. 31847.

[0112] The 16S rDNA sequencing results of this strain are shown in SEQ ID No: 5:

[0113]

[0114] Using the above five strains, with sludge reduction as the main evaluation indicator, five strain compound verifications were carried out respectively, and finally a biochemical system sludge reduction microbial composite bacteria was invented. The biochemical system sludge reduction microbial composite bacteria includes the above-mentioned Bacillus smithii, Heyndrickxia coagulans, Parageobacillus caldoxylosilyticus, Aeribacillus pallidus and Pallidibacillus thermolactis. According to the ratio of effective viable bacteria, the final ratio is: (10-25%): (25-30%): (18-26%): (12-29%): (9-30%).

[0115] Adding the above-mentioned biochemical system sludge reduction microbial composite bacteria into the biochemical system in a certain proportion can reduce the generation of residual sludge in the biochemical system; the biochemical system sludge reduction microbial composite bacteria decomposes different biological enzymes and the synergistic metabolism between strains to decompose the aged or dead sludge in the biochemical system to produce carbon dioxide and water, and at the same time inhibits the growth of some miscellaneous bacteria such as the growth of silk-producing microorganisms through competition between microorganisms, thereby achieving the purpose of sludge reduction.

[0116] The embodiment of the present invention provides a method for preparing a biochemical system sludge reduction microbial composite bacteria, comprising:

[0117] Step 1: Add sterile water into a sterile bottle to soak the soil material, mix the soil material and the sterile water thoroughly, and then let it settle; the soil material is surface moist soil or moist silt with obvious humus on the riverside for more than 20 years; for example, riparian wetland soil in humus alluvial soil or organic latent soil.

[0118] Step 2: Take the supernatant and dilute it by a dilution coating method to reduce the background bacterial concentration; take a suspension of each gradient dilution supernatant and evenly spread it on NB solid medium, LB solid medium, NA solid medium, TSA solid medium and PDA solid medium, and place it in an incubator at a constant temperature of 40°C; take the undiluted supernatant and evenly spread it on NB solid medium, LB solid medium, NA solid medium, TSA solid medium and PDA solid medium as a control and place it in an incubator at a constant temperature of 37°C;

[0119] Step 3: After the single colony grows, observe the bacterial separation on all dilution gradient plates, and visually observe and select a target gradient whose separation effect meets the task requirements; screen out colonies with different apparent morphology in the target gradient and pick single colonies, dilute the single colony gradient using the dilution coating method, evenly spread them on the corresponding culture medium, and place them in an incubator for constant temperature culture at 35°C; undiluted single colonies are evenly spread on the corresponding culture medium as a control, and placed in an incubator for constant temperature culture at 35°C. The incubator temperature is increased by 1°C, and this step is repeated until the incubator temperature reaches 45°C; this step is repeated again until the colony morphology of each single colony is consistent; wherein the apparent morphology is preferably the color, size and shape of the colony; in this step, the culture is cultured in the temperature range of 35°C-45°C where microbial activity is strongest, and the method of increasing temperature culture is to distinguish the growth rates of different single colonies.

[0120] Step 4: Select target colonies from among those with consistent morphology and purify the endophytic bacteria using the zoning and streaking method to obtain monoclonal colonies. After 16S rDNA gene sequencing, the biochemical system sludge reduction microbial complex is obtained. This step performs a secondary purification of the diluted and plated colonies to eliminate contamination from other bacteria and is suitable for the preparation of high-purity strains.

[0121] In step 3, the corresponding culture medium formula includes:

[0122] The formula of the culture medium of Bacillus smithii is:

[0123] LB solid medium: peptone 10 g / L, yeast powder 5 g / L, sodium chloride 5 g / L, and agar powder 20 g / L;

[0124] LB liquid medium: peptone 10 g / L, yeast powder 5 g / L, and sodium chloride 5 g / L;

[0125] After preparation, autoclave at 121°C for 20 minutes;

[0126] Heyndrickxia coagulans culture medium recipe:

[0127] NB solid medium: peptone 20 g / L, beef extract 3 g / L, and agar powder 20 g / L;

[0128] NB liquid medium: peptone 20 g / L and beef extract 3 g / L;

[0129] After preparation, autoclave at 121°C for 20 minutes;

[0130] The formula of the culture medium of Parageobacillus caldoxylosilyticus is:

[0131] LB solid medium: peptone 10 g / L, yeast powder 5 g / L, sodium chloride 5 g / L, and agar powder 20 g / L;

[0132] LB liquid medium: peptone 10 g / L, yeast powder 5 g / L and sodium chloride 5 g / L;

[0133] After preparation, autoclave at 121°C for 20 minutes;

[0134] Aeribacillus pallidus culture medium recipe:

[0135] LB solid medium: peptone 10 g / L, yeast powder 5 g / L, sodium chloride 5 g / L, and agar powder 20 g / L;

[0136] LB liquid medium: peptone 10 g / L, yeast powder 5 g / L and sodium chloride 5 g / L;

[0137] After preparation, autoclave at 121°C for 20 minutes;

[0138] The formula of the culture medium of Pallidibacillus thermolactis is as follows:

[0139] LB solid medium: peptone 10 g / L, yeast powder 5 g / L, sodium chloride 5 g / L, and agar powder 20 g / L;

[0140] LB liquid medium: peptone 10 g / L, yeast powder 5 g / L and sodium chloride 5 g / L;

[0141] After preparation, sterilize at 121℃ for 20 minutes.

[0142] The experimental process in this embodiment is as follows:

[0143] 1. Preparation of materials:

[0144] 1. Prepare the corresponding number of sterile 10ml centrifuge tubes according to needs;

[0145] 2. Sterilize a bottle of sterile water and divide it into 10ml sterile centrifuge tubes. Preferably, sterilize a 500ml bottle of sterile water and add 5ml sterile water to each centrifuge tube.

[0146] 3. Prepare a sterilized spreading rod or yellow sterile pipette tip and three sterilized 100ml conical flasks for dilution;

[0147] 4. Prepare a constant temperature incubator and a constant temperature shaker. The operating temperature range of the shaker and constant temperature incubator can reach 45°C.

[0148] 2. The preparation method includes:

[0149] Use a sterile conical flask to soak the soil material with sterile water, mix the soil material and sterile water thoroughly and let it stand for precipitation, take the supernatant and dilute it to 10 by 10 times. -1 , 10 -2 and 10 -3 , take 200 μl of undiluted supernatant or three dilution gradient suspensions and evenly spread them on NB solid medium, LB solid medium, NA solid medium, TSA solid medium, and PDA solid medium.

[0150] Place the plate in an incubator at 40°C for constant temperature culture, and place the control plate in a 37°C incubator. After 2-3 days, when monoclonal colonies grow, observe the bacterial separation on the four dilution gradient plates. Visually observe and select the gradient with the best separation effect for the subsequent separation of endogenous bacteria in the sample.

[0151] According to the color, size and shape of the colonies, strains with different apparent morphologies were selected, single clones were cultured and numbered.

[0152] Dilute and spread in the corresponding culture medium, place the corresponding culture medium in a 35℃-45℃ incubator for culture, and culture in the incubator at 35℃-45℃ until the colony morphology is consistent. Use the partitioning and streaking method to purify the endophytic bacteria to obtain monoclonal colonies, and perform 16S rDNA gene sequencing.

[0153] The present invention also provides a biochemical system sludge reduction microbial agent, including Bacillus smithii, Heyndrickxia coagulans, Parageobacillus caldoxylosilyticus, Aeribacillus pallidus, Pallidibacillus thermolactis, a biochemical system sludge reduction microbial composite bacteria or a biochemical system sludge reduction microbial composite bacteria obtained by a preparation method.

[0154] The present invention also provides Bacillus smithii, Heyndrickxia coagulans, Parageobacillus caldoxylosilyticus, Aeribacillus pallidus, Pallidibacillus thermolactis, a biochemical system sludge reduction microbial composite bacteria, a biochemical system sludge reduction microbial composite bacteria obtained by a preparation method, or a biochemical system sludge reduction microbial agent for biochemical system sludge reduction, and their use in biochemical system sludge reduction.

[0155] The present invention also provides a biochemical system sludge reduction microbial composite bacteria, a biochemical system sludge reduction microbial composite bacteria obtained by a preparation method, or a biochemical system sludge reduction microbial agent for biochemical system sludge reduction, and a method for using the same in biochemical system sludge reduction, including:

[0156] Take the biochemical system sludge reduction microbial composite bacteria or the biochemical system sludge reduction microbial agent and add it into the biochemical system and mix it with the sludge.

[0157] Among them, the method of adding includes:

[0158] The temperature of the biochemical system should be between 25°C and 45°C, which is suitable for the actual ambient temperature range of the biochemical system in all seasons, and preferably between 35°C and 45°C. Under this temperature, the microbial activity of the biochemical system sludge reduction microbial complex or the biochemical system sludge reduction microbial agent is the strongest. The DO value should be between 1.0mg / L and 4mg / L. Within this DO value range, anaerobic bacteria are predominant in the surface and inner layers. The pH value should be between 6 and 9. If the pH value exceeds this range, the biochemical system sludge reduction microbial complex or the biochemical system sludge reduction microbial agent will be inactivated.

[0159] The dosing points are: the aerobic tank inlet end of the biochemical system and the sludge return point of the secondary sedimentation tank;

[0160] For a biochemical system that reduces 1.0 ton of absolute dry sludge, the dosage of the biochemical system sludge reduction microbial composite bacteria or biochemical system sludge reduction microbial agent is 2.5-5 kg;

[0161] The daily dosage of biochemical system sludge reduction microbial composite bacteria or biochemical system sludge reduction microbial agent is determined based on the residual sludge output obtained from the daily water inlet load and output of the biochemical system.

[0162] Among them, the temperature at which the microbial activity of the biochemical system sludge reduction microbial composite bacteria or the biochemical system sludge reduction microbial agent is strongest is 35°C to 45°C.

[0163] like Figures 6 to 11 As shown, in order to conduct on-site verification of the technical solution of the present invention, two specific verification examples are provided:

[0164] First, prepare as follows:

[0165] 1. Analysis of excess sludge production;

[0166] 1. Preparation of biochemical system sludge reduction microbial composite bacteria or biochemical system sludge reduction microbial agent;

[0167] 2. Determine the best dosage according to the mud quality;

[0168] 3. Optimal abatement environment and effect tracking;

[0169] 4. The project implementation will achieve sludge reduction.

[0170] Verification Example 1: A municipal sewage company:

[0171] 1.1. Preliminary preparation: Based on the sludge performance analysis of the biochemical system, its MLSS concentration is 11,000 mg / L, MLVSS is 7,150 mg / L, and VSS / TSS ratio is 0.625; the biochemical system discharges about 15 tons of sludge per day, with a moisture content of about 85%.

[0172] 1.2. Preparation of composite bacterial strains:

[0173] Preparation of experimental bacterial solution: scrape 1 inoculum loop of bacteria from the slant, inoculate it into sterilized LB medium, and culture on a shaking platform for 24 hours.

[0174] Experimental instruments: shaking table, small-scale aeration device, dissolved oxygen detector, experimental containers, index detection instruments, activated sludge, and the enterprise's sewage were used to test the effect of different bacterial strains on sludge reduction.

[0175] 1.3 Experimental steps:

[0176] 1000ml of aerobic activated sludge was placed in a small-scale experimental device, 2000ml of mud-water mixture was added, 10ml of cultured bacterial solution was added, and an aeration device was installed;

[0177] Start the aeration device and maintain the dissolved oxygen concentration at around 2-5 mg / l;

[0178] During the continuous aeration reaction, the mud-water mixture was collected and the decrease of MLSS index was measured;

[0179] Experiments with different strains were performed simultaneously under the same conditions.

[0180] According to the test results, 80kg of compound bacteria was added every day, and the data changes were tracked and analyzed on site. Figures 12 to 13 shown.

[0181] like Figure 14 As shown in Table 1, the sewage treatment plant of this municipal sewage enterprise has a water volume of about 50,000 tons per day and adopts the A2 / O biochemical treatment process. The sludge production of its biochemical system is about 21 tons (85% water content) per 10,000 tons of water. The test results are shown in Table 1.

[0182] Table 1

[0183] Experimental groups Strain ratio MLSS concentration data Effect 1 20％：20％：20％：20％：20％ 9500 +++ 2 20％：25％：20％：15％：20％ 8050 +++++ 3 25％：25％：25％：10％：15％ 8250 ++++ 4 10％：30％：10％：25％：25％ 9000 + 5 20％：20％：25％：20％：15％ 8350 +++

[0184] Note: The order of strains is: Bacillus smithii, Heyndrickxia coagulans, Parageobacillus caldoxylosilyticus, Aeribacillus pallidus and Pallidibacillus thermolactis.

[0185] Experimental results and analysis:

[0186] A sludge reduction test was conducted by adding Bacillus smithii, Heyndrickxiacoagulans, Parageobacillus caldoxylosilyticus, Aeribacillus pallidus and Pallidibacillus thermolactis. After 24 hours of aeration, the optimal strain ratio was determined to be (20%): (25%): (20%): (15%): (20%) through MLSS and MLVSS analysis.

[0187] Verification Example 2: Industrial Wastewater Sludge Reduction Case:

[0188] 2.1: On-site situation collection: Preliminary preparation: Combined with the sludge performance analysis of the system, its MLSS concentration is 9000mg / L, MLVSS is 5400mg / L, and VSS / TSS ratio is 0.6; the biochemical system discharges about 18 tons of sludge per day, with a moisture content of about 80%.

[0189] 2.2: Experimental Preparation

[0190] Preparation of experimental bacterial solution: scrape 1 inoculum loop of bacteria from the slant, inoculate it into sterilized LB medium, and culture on a shaking platform for 24 hours.

[0191] Experimental instruments: shaking table, small-scale aeration device, dissolved oxygen detector, experimental containers, index detection instruments, activated sludge, and industrial wastewater of the project for experimental use.

[0192] 2.3: Experimental steps: 1000ml of aerobic activated sludge was placed in a small-scale experimental device, 2000ml of the corresponding industrial wastewater was added, 10ml of the cultured bacterial solution was added, and an aeration device was installed;

[0193] Start the aeration device and maintain the dissolved oxygen concentration at around 2-5 mg / l;

[0194] Continuous aeration reaction, taking mud-water mixture in the middle and measuring mlss index;

[0195] At the same time, experiments with different proportions of strains were performed under the same conditions.

[0196] According to the test results, 5kg of compound bacteria was added every day, and the data changes were tracked and analyzed on site. Figures 15 and 16 shown.

[0197] like Figure 17 As shown in the figure, this industrial wastewater plant handles approximately 20,000 tons of water per day. The biochemical system uses a SBR treatment process, generating approximately 1.0 ton of dry excess sludge per day. Approximately 5 tons of 80% water sludge are produced per day. The comprehensive treatment cost for this 80% water sludge is approximately 800 yuan per ton, or 4,000 yuan per day. The combined bacterial strain was added at a dosage of 5 kg per day. The test results are shown in Table 2.

[0198] Table 2

[0199] Experimental groups Strain ratio MLSS concentration data Effect 1 20％：20％：20％：20％：20％ 7900 +++ 2 20％：25％：25％：10％：20％ 8100 + 3 20％：25％：25％：15％：15％ 7250 ++++ 4 10％：30％：10％：25％：25％ 8000 ++ 5 20％：20％：25％：20％：15％ 7150 +++++

[0200] Note: The order of strains is: Bacillus smithii, Heyndrickxia coagulans, Parageobacillus caldoxylosilyticus, Aeribacillus pallidus and Pallidibacillus thermolactis.

[0201] Experimental results and analysis:

[0202] A sludge reduction test was conducted by adding Bacillus smithii, Heyndrickxiacoagulans, Parageobacillus caldoxylosilyticus, Aeribacillus pallidus and Pallidibacillus thermolactis. After 24 hours of aeration, the optimal strain ratio was determined to be (20%): (20%): (25%): (20%): (15%) through MLSS and MLVSS analysis.

[0203] In summary, the beneficial effects of the embodiments of the present invention are:

[0204] The ability of the microbial strains in the biochemical system sludge reduction microbial composite bacteria or the biochemical system sludge reduction microbial agent disclosed in the present invention to secrete different enzymes under specific environments has been verified through a large number of experiments as follows:

[0205] Bacillus smithii secretes proteases and amylases;

[0206] Heyndrickxia coagulans secretes L-lactate dehydrogenase, amylase, and protease;

[0207] Parageobacillus caldoxylosilyticus secretes xylanases and cellulases;

[0208] Aeribacillus pallidus secretes 4-α-glycosyltransferase, amylase, and protease;

[0209] Pallidibacillus thermolactis secretes glycolytic enzymes and lactate dehydrogenase.

[0210] The aforementioned microbial strains and their enzymes decompose the various components of aging or dead microorganisms in the biochemical system, providing energy for the microbial complex / microbial agent. The decomposed organic matrix in the sludge produces carbon dioxide and water, or decomposes some of the adsorbed organic matrix in the sludge into carbon dioxide and water, thereby reducing sludge volume. At the same time, competition among microorganisms inhibits the growth of some other bacteria, such as silk-producing microorganisms.

[0211] The present invention reduces the amount of excess sludge produced by adding the biochemical system sludge reduction microbial composite bacteria or the biochemical system sludge reduction microbial agent disclosed in the present invention to the biochemical system, thereby achieving source control of sludge reduction. The technical solution disclosed in the present invention has the following advantages:

[0212] 1. No hardware investment, extremely low energy consumption, and relatively low investment and operating costs;

[0213] 2. Reduce the amount of excess sludge in the biochemical system by 30% to 80%;

[0214] 3. For systems that need to supplement carbon sources, the amount of carbon source supplement can be reduced;

[0215] 4. The sludge after reduction is easier to dewater or dispose of.

[0216] The technical solution disclosed in the present invention is suitable for sewage treatment plants with substances with high organic content and no obvious toxicity. It is also used for centralized disposal of multiple sewage treatment plants and coordinated treatment of various organic wastes. It has the advantages of high activity, low investment, low energy consumption, easy operation, and no secondary pollution.

[0217] Compared with the prior art, the advantages of the present invention are shown in Table 3:

[0218] Table 3

[0219] Patent for this invention Existing technology Sludge reduction between 30% and 80% Just reduce the sludge moisture content No hardware investment Large hardware investment Operating cost: RMB 500 / ton of absolute dry sludge The cost of one ton of absolutely dry sludge is more than 1,500 yuan Extremely low energy consumption High energy consumption

[0220] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications or substitutions that can be easily conceived by a person skilled in the art within the technical scope disclosed in the present invention should be included in the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be based on the scope of protection of the claims.

Claims

1. A biochemical system sludge reduction microbial composite bacteria, characterized in that: include: Bacillus smithii, deposit number: CGMCC No. 31841; Heyndrickxia coagulans, deposit number: CGMCC No. 31842; Parageobacillus caldoxylosilyticus, deposit number: CGMCC No. 31844; Aeribacillus pallidus, deposit number: CGMCC No. 31845; Pallidibacillus thermolactis, deposit number: CGMCC No. 31847; the deposit date is September 3, 2024; the depository is China General Microbiology Center (CGMCC); the address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing; among them, The ratio of the effective viable bacteria count of Bacillus smithii: Heyndrickxia coagulans: Parageobacillus caldoxylosilyticus: Aeribacillus pallidus: Pallidibacillus thermolactis is: (10~25%): (20~30%): (10~25%): (10~25%): (15~25%).

2. A biochemical system sludge reduction microbial composite bacteria according to claim 1, characterized in that: The optimal strain ratio of Bacillus smithii: Heyndrickxia coagulans: Parageobacillus caldoxylosilyticus: Aeribacillus pallidus: Pallidibacillus thermolactis is: 20%: 25%: 20%: 15%: 20%.

3. The biochemical system sludge reduction microbial composite bacteria according to claim 1, characterized in that: The optimal strain ratio of Bacillus smithii: Heyndrickxia coagulans: Parageobacillus caldoxylosilyticus: Aeribacillus pallidus: Pallidibacillus thermolactis is: 20%: 20%: 25%: 20%: 15%.

4. A biochemical system sludge reduction microbial agent, characterized in that: The invention comprises a biochemical system sludge reduction microbial composite bacteria as described in any one of claims 1 to 3.

5. Use of a biochemical system sludge reduction microbial composite bacteria according to any one of claims 1 to 3 or a biochemical system sludge reduction microbial agent according to claim 4 in biochemical system sludge reduction.

6. A method for using the biochemical system sludge reduction microbial composite bacteria according to any one of claims 1 to 3 or the biochemical system sludge reduction microbial agent according to claim 4 in biochemical system sludge reduction, characterized in that: include: Take the biochemical system sludge reduction microbial composite bacteria or the biochemical system sludge reduction microbial agent and add it into the biochemical system and mix it with the sludge.

7. The method of use according to claim 6, wherein: Dosing methods include: The biochemical system temperature should be between 25°C and 45°C, the DO value should be between 1.0mg / L and 4.0mg / L, and the pH value should be between 6 and 9. The dosing points are: the aerobic tank inlet end of the biochemical system and the sludge return point of the secondary sedimentation tank; For a biochemical system that reduces 1.0 ton of absolute dry sludge, the dosage of the biochemical system sludge reduction microbial composite bacteria or biochemical system sludge reduction microbial agent is 2.5~5kg; The daily dosage of biochemical system sludge reduction microbial composite bacteria or biochemical system sludge reduction microbial agent is determined based on the residual sludge output obtained from the daily water inlet load and output of the biochemical system.

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