Preparation method of mineralizing microbial inoculum and application of mineralizing microbial inoculum in cadmium mineralization

Abstract

The invention discloses a preparation method of a mineralized microbial inoculum, which specifically comprises the following steps: inoculating sulfur-producing bacteria into a liquid anaerobic basic culture medium, and culturing for 24-48 hours under the conditions of 30-37 DEG C, pH 6.8-7.2 and anaerobic or micro-aerobic conditions to obtain a bacterial suspension with the viable bacterium concentration of 107-109 CFU / mL; domesticating the strain by adopting Cd (NO3) 2 to obtain a mineralized microbial inoculum; or mixing the domesticated bacterial liquid with a carrier, uniformly stirring, and drying or embedding to obtain a mineralized bacterial agent; or co-culturing the activated sulfur-producing bacteria and iron-reducing bacteria to prepare a mineralized microbial inoculum; after the Cd-polluted sludge is treated by the mineralization microbial agent, the exchangeable Cd proportion is reduced to be below 7% from original 30-50%, the Cd leaching concentration in a TCLP test is below 0.3 mg / L. The mineralization microbial agent is wide in strain source, stable in preparation, simple and convenient in process, low in cost and suitable for the green low-carbon treatment process of municipal sludge and Cd-containing industrial sludge.

Description

Technical Field

[0001] The present invention belongs to the field of environmental pollution control and solid waste resource utilization, and particularly relates to a preparation method of a mineralizing bacterial agent and application of the mineralizing bacterial agent in the stabilization treatment of heavy metal cadmium (Cd) in municipal or industrial sludge. Background Art

[0002] Cd, a typical highly toxic and highly mobile heavy metal, exists in sludge in exchangeable and carbonate-bound forms. It is easily released during landfilling, agricultural use, or construction material utilization, posing a risk of secondary pollution. Therefore, developing efficient, green, and low-cost Cd stabilization technologies has become a key issue that needs to be addressed in the resource utilization of sludge.

[0003] Existing methods for treating heavy metals in sludge primarily include chemical passivation (such as the addition of CaO, iron salts, and phosphates), thermal treatment (such as incineration and vitrification), biological adsorption, and biochar fixation. While chemical methods are relatively effective, they pose challenges such as high cost, drastic pH fluctuations, large amounts of additives, and unstable heavy metal conversion states. Thermal treatment can completely remove Cd, but due to high energy consumption and complex equipment, it is unsuitable for large-scale deployment in conventional sludge treatment plants. While biochar materials have a certain Cd adsorption capacity, their interaction with Cd is primarily physical adsorption and weak complexation, resulting in insufficient long-term stability. Furthermore, the preparation process still relies on high-temperature carbonization, resulting in high carbon emissions and a negative impact on green transformation goals.

[0004] In recent years, some studies have explored the in situ precipitation of heavy metals through microbial-driven heavy metal mineralization mechanisms. Sulfur-producing bacteria (such as Desulfovibrio) can produce hydrogen sulfide (H2S) through sulfate reduction metabolism, which in turn reacts with Cd 2+ Cadmium sulfide (CdS) precipitates are formed. CdS is an insoluble yellow-brown solid with high thermodynamic stability and water insolubility, making it an ideal Cd fixation form. However, currently this type of "microbial agent-driven Cd mineralization" technology is mostly concentrated in heavy metal wastewater treatment, and is rarely used in municipal sludge systems containing high concentrations of organic matter, strong toxicity inhibition, and weak pH buffering capacity. There are technical bottlenecks such as poor bacterial stability, low mineralization efficiency, and easy inactivation of bacterial strains. In addition, there is still a lack of systematic solutions for the storage, addition method, and scene adaptability of microbial agents, which seriously limits the promotion and engineering application of this type of microbial agent in the treatment of heavy metal pollution in sludge.

[0005] Therefore, there is an urgent need to develop a composite mineralizing agent with high Cd tolerance, high H2S production capacity, and sulfur-producing bacteria as the core that can grow stably in a sludge environment, and to construct a sludge treatment process path suitable for Cd stabilization to achieve efficient, long-lasting, and eco-friendly treatment of Cd in sludge. Summary of the Invention

[0006] The present invention aims to solve the problems of high Cd mobility, poor stability of traditional passivators, and unsustainable biological adsorption pathways in the existing sludge Cd pollution treatment, and provide a method for efficiently and stably fixing Cd in complex environments of municipal or industrial sludge. 2+ The mineralizing agent is a sulfur-producing bacteria. The mineralizing agent uses sulfur-producing bacteria as the dominant functional bacteria group, produces hydrogen sulfide (H2S) through microbial metabolism, and realizes the reduction of Cd 2+ In-situ sulfide precipitation is carried out to generate stable, insoluble CdS minerals with low migration risk, thereby achieving green and low-carbon treatment of heavy metal Cd in sludge.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions: 1. Preparation of mineralizing agent (1) Inoculate sulfur-producing bacteria into liquid anaerobic basal culture medium and culture at 30-37°C and pH 6.8-7.2 for 24-48 hours to obtain a bacterial concentration of 10 7 -10 9 CFU / mL of bacterial suspension; The sulfur-producing bacteria are selected from Desulfovibrio ( Desulfovibrio desulfuricans )ATCC 27774, sulfate-reducing bacteria ( Desulfovibrio vulgaris ) DSM 644, purchased from Beijing Biobowei Biotechnology Co., Ltd.

[0008] (2) Using Cd(NO3)2 to acclimate the bacteria to obtain a mineralizing agent; The mineralizing agent can also be prepared by co-culturing activated sulfur-producing bacteria and iron-reducing bacteria, wherein the iron-reducing bacteria is Shewanella putrefaciens ( Shewanella putrefaciens ), purchased from Beijing Biobowei Biotechnology Co., Ltd.

[0009] The mineralizing agent can also be prepared by mixing the acclimated bacterial solution with a carrier at a mass ratio of 1: (1-5), stirring evenly, and then drying or embedding to obtain the mineralizing agent; The carrier is selected from vermiculite powder, white mud powder, zeolite powder, straw powder, sodium alginate, calcium alginate, and polyvinyl alcohol (PVA). After mixing, the mixture is freeze-dried or spray-dried to obtain the microbial agent, or the microbial agent is obtained by embedding using a conventional embedding method.

[0010] 2. Cadmium stabilization treatment Add the mineralizing agent to the cadmium-contaminated sludge at an inoculum rate of 1-10%, mix well, and let it stand or stir at 15-35°C for 5-10 days to react with H2S produced by the agent and Cd in the sludge. 2+ The reaction generates CdS precipitate, realizing the mineralization and fixation of heavy metal cadmium. After the reaction is completed, it is mechanically dehydrated to obtain a cadmium-stabilized sludge product, which can be used in scenarios such as garden soil matrix and covering material.

[0011] Compared with the prior art, the present invention has the following significant advantages: (1) The passivation mechanism is novel and stable: based on the production of H2S by microbial metabolism to drive Cd 2+ Mineralization into CdS crystals, forming an insoluble residue with high thermodynamic stability, avoiding secondary release; (2) Accurate strain domestication and strong adaptability: The strains used were conditioned by high concentrations of Cd 2+ Step-by-step induction screening can survive and maintain activity in high organic matter, weakly acidic, and heavy metal toxic environments, and is particularly suitable for sludge systems; (3) Flexible preparation forms and diverse application methods: The microbial agent can be made into various forms such as liquid, freeze-dried powder, and embedded particles. It is suitable for various scenarios such as static stacking, dynamic stirring, and combined fermentation, and is easy to add, store, and transport; (4) High Cd fixation efficiency and significantly reduced mobility: the proportion of exchangeable Cd in the treated sludge is reduced to <5%, and the TCLP leaching concentration is lower than 0.3 mg / L, which meets the limit requirements of the "Hazardous Waste Identification Standard (GB 5085.7-2019)"; (5) Green and environmentally friendly, simple process, and controllable cost: no need for additional high-energy heat treatment, non-toxic and harmless reaction products, and environmentally friendly preparation process, suitable for large-scale promotion. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 The X-ray diffraction analysis results of the sludge after the reaction in Example 1 are as follows; Figure 2 The X-ray diffraction analysis results of the sludge after the reaction in Example 2; Figure 3 The SEM-EDS analysis results of the sludge after the reaction in Example 2; Figure 4 This is the XRD pattern of the sludge after the reaction in Example 4; Figure 5 The following are the SEM images and EDS analysis results of the sludge after the reaction in Example 4. DETAILED DESCRIPTION

[0013] The present invention is further described in detail below by way of examples, but the protection scope of the present invention is not limited to the contents described above.

[0014] Example 1: Using Desulfovibrio desulfuricans ATCC 27774 was used as the main functional bacteria to construct a liquid sulfur-producing agent and applied it to the in-situ mineralization treatment of low-concentration Cd-contaminated sludge. 1. Preparation of microbial agents The strain was inoculated into anaerobic liquid basal medium (containing 2.0 g of MgSO4·7H2O, 2.0 g of Na2SO4, 1.0 g of yeast powder, 2.0 g of sodium acetate, 1.0 g of NaHCO3, 0.5 g of L-cysteine, and 0.2 g of Na2S·9H2O per liter of water, pH 7.0) and cultured at 37°C under anaerobic conditions for 48 h to obtain a viable cell count of 10 7 CFU / mL bacterial suspension; then Cd 2+ The concentration step-by-step induction method was used for acclimation. Cd(NO3)2 was added in sequence to make the metal concentration in the bacterial solution reach 25, 50, and 75 mg / L. Each stage was cultured for 48 hours to obtain a bacterial solution with stable H2S production ability and strong Cd tolerance. After acclimation, the concentration of viable bacteria in the bacterial solution was maintained at 8×10 7 CFU / mL, the sulfur production capacity did not decrease significantly, and the electronic structure of the extracellular Fe-S clusters determined by EPR did not undergo passivation, indicating that the bacterial metabolic system was stable; 2. Add 500g of municipal sludge with a moisture content of 80% (Cd content of 400mg / L) to a 1L wide-mouth sealed reaction bottle, add 50g of the above-mentioned liquid bacterial agent (accounting for 10wt% of the wet weight of the sludge), seal the bottle, and place it in a 35℃ constant temperature box for anaerobic culture for 7 days. During the reaction process, open the bottle for 1 minute every day to simulate the periodic ventilation operation under on-site construction conditions. After the reaction, immediately analyze the physical and chemical indicators of the sludge sample; The results showed that the proportion of exchangeable Cd decreased from 42.5% before treatment to 5.3%, while the residual Cd increased significantly to 52.1%, indicating that Cd was successfully transformed from an easily mobile form to a stable precipitated form. The Cd leaching concentration in the TCLP test was 0.23 mg / L, significantly lower than the hazardous waste limit (1.0 mg / L). In addition, X-ray diffraction analysis was used to detect the change in the Cd mineral phase in the sludge after the reaction. The characteristic peaks of hexagonal CdS appeared at 2θ of 26.5° and 43.8°, respectively, indicating that Cd was precipitated and mineralized in the form of CdS ( Figure 1 ), with thermodynamic stability and leaching resistance, the treated sludge is safe for transport or reuse. The liquid inoculant in this embodiment is simple to prepare, features flexible inoculation methods, a short reaction cycle, and excellent fixation effects, making it suitable for rapid in-situ treatment of municipal sludge and subsequent pre-treatment for ecological restoration.

[0015] Example 2: Using Desulfovibrio vulgaris (DSM 644) is a strain of bacteria that is freeze-dried to prepare a mineralizing agent that is easy to transport and store for a long time. It is also used to treat sludge contaminated with high Cd concentration (100 mg / kg). 1. Inoculate the strain into anaerobic liquid basal medium (containing 1.5g MgCl2·6H2O, 2.0g Na2SO3, 1.2g NaHCO2, 1.0g NH2Cl, 1.0g yeast extract powder, 2.5g sodium acetate, 0.1g Na2S·9H2O per liter of water, pH 7.0) and culture at 30°C under anaerobic conditions for 48h to obtain a viable cell count of 1.0×10 8 CFU / mL of bacterial suspension (OD 600 =1.2); then metal adaptation was adopted to add Cd(NO3)2 to make the Cd 2+ The concentration increased from 25 mg / L, 50, 75 to 100 mg / L, and each stage was cultured for 48 hours. The metabolic activity of the bacterial community remained stable, and the sulfur production capacity decreased by no more than 12%. It was identified as a Cd-resistant and stable sulfur-producing bacterial strain.

[0016] 2. Mix the bacterial solution with 100g / L trehalose solution at a volume ratio of 1:0.2, pre-cool at -80°C for 12 hours, and then sublimate and dry in a vacuum freeze dryer for 48 hours to obtain freeze-dried bacterial agent powder. The dry powder has a moisture content of 6%, a bacterial survival rate of 78%, and the sulfur production function is basically retained. The freeze-dried bacterial agent can be stored in a dry bottle at 4°C for 60 days before use, and it can still recover to OD within 72 hours. 600 About 0.85, and maintain the H2S production rate above 90%, with stable addition capability on the engineering site.

[0017] 3. Add the freeze-dried bacterial agent at a ratio of 7 wt% directly to 1 kg of municipal sludge with a moisture content of 80%, place it in a 1.5 L sealed reaction bottle, and let it react at room temperature (25±2°C) for 10 days; no ventilation is allowed during the reaction, and only a slight stirring is performed once a day to ensure sufficient internal contact. After the reaction is completed, the changes in heavy metal morphology and leaching toxicity before and after are compared. The results show that the exchangeable Cd decreases from the initial 47.6% to 6.1%, and the residual Cd increases significantly from 14.8% to 51.7%. In the TCLP test, the Cd leaching concentration decreases from 2.10 mg / L to 0.27 mg / L, meeting the target of hazardous waste detoxification treatment. After freeze-drying in step 2, the freeze-dried bacterial agent is clearly detected by XRD to show CdS diffraction peaks at 2θ=26.5° and 43.9° ( Figure 2 ), further analyzed by SEM-EDS ( Figure 3 ) confirmed the presence of crystalline Cd-S co-precipitates on the surface of sludge particles, and the mineralization morphology was mainly pyrite-like structure. Combined with the thermodynamic deduction results (ΔG° ≈ −180kJ / mol), it showed that CdS precipitation was a spontaneous reaction process.

[0018] The freeze-dried bacterial agent used in this embodiment is convenient for long-distance transportation, storage and quantitative addition on site, and is suitable for off-site treatment projects of large-scale heavy metal sludge. It is particularly suitable for centralized sludge conditioning centers and the pre-treatment stage of Cd-containing sludge in mining areas, and can achieve stable, efficient and safe heavy metal passivation in high Cd concentration environments.

[0019] Example 3: This example uses immobilization and embedding technology to prepare a mechanically stable granular sulfur-producing bacteria agent, which is then used for in-situ passivation treatment of sludge containing 50 mg / kg Cd under aeration tank conditions. 1. The strain is Desulfovibrio desulfuricans ATCC 27774 was cultured in the same medium as in Example 1 at 37°C for 48 hours to obtain a viable bacterial concentration of about 9 × 10 7 CFU / mL of sulfur-producing bacteria solution (OD 600 is 1.05), Cd(NO3)2 (final concentration is 50 mg / L) was added to the bacterial solution after acclimation for 48 hours and then used directly for particle embedding operation; 2. The embedding material is a carrier base liquid prepared by mixing 2.5wt% sodium alginate solution and vermiculite powder (particle size <100μm) in a mass ratio of 2:1. The bacterial suspension is added to the carrier base liquid in a volume ratio of 1:1 to form a uniform suspension. The suspension is slowly added dropwise to an aqueous solution containing 2.0% CaCl2 using a syringe pump. Cross-linking is carried out at room temperature for 30 minutes to obtain round biological agent particles with a diameter of approximately 3mm. The wet weight density of the obtained particles is approximately 0.92g / cm 3 The moisture content was 68% by the loss on drying method, and the number of viable bacteria per gram of dry granules was 1.6×10 7 The CFU activity retention rate reached 84% after 60 days of storage at room temperature. Scanning electron microscopy revealed that the granules had a rough and porous surface, which facilitated the release of H2S and contact with sludge, preventing the embedded bacteria from leaking out.

[0020] 3. A 1.0 kg sludge sample was placed in a 2 L glass aeration reactor. 5 wt% (wet basis) of the prepared immobilized bacterial agent granules were added. Low-speed stirring (50 rpm) and minimal aeration (0.3 L / min) were initiated for 7 days, maintaining the temperature between 28 and 30°C. Post-reaction sampling revealed that the exchangeable Cd content decreased from 41.3% before treatment to 4.5%, while the residual content increased to 54.8%. The Cd leaching concentration in the TCLP test decreased to 0.19 mg / L. XRD analysis of the reaction sludge revealed typical diffraction peaks for CdS crystals. SEM-EDS confirmed the presence of Cd-S aggregates on the surface of the sludge granules, showing irregular clustered deposits. The S / Cd atomic ratio in the EDS was approximately 1.02, suggesting the formation of CdS precipitates. Due to the excellent mechanical strength and structural stability of the immobilized granules under aeration, no granule disintegration or bacterial liquid leakage was observed, indicating stable Cd immobilization and well-maintained sludge structure.

[0021] The granular bacterial agent in this embodiment is particularly suitable for use in aeration environments such as urban sewage treatment plant conditioning tanks, aerobic / facultative aerobic reactors, etc. It has the advantages of strong positioning, controllable release, and high bacterial stability. It can be expanded to be applied to the combined mineralization treatment system of sludge where Cd and other heavy metals coexist.

[0022] Example 4: This example constructs sulfur-producing bacteria ( Desulfovibrio vulgaris ) and iron-reducing bacteria ( Shewanella putrefaciens ) Co-cultured bacterial agent, which improves the mineralization efficiency of Cd and the controllability of CdS precipitation morphology through microecological coupling strategy, and is used for the ecological safety and stabilization treatment of high Cd concentration sludge 1. The two strains were pre-cultured under anaerobic conditions: the sulfur-producing bacteria were cultured at 37°C for 48 h in the same culture medium as in Example 1; the iron-reducing bacteria were cultured at 30°C for 36 h in a culture medium containing 5 g / L sodium lactate, 3 mmol / L iron citrate complex, and 2 g / L yeast powder; the two bacterial suspensions were then mixed at a volume ratio of 1:1 and cultured for 24 h to form a stable mutually promoting symbiotic system. The OD value of the bacterial suspension was measured. 600 The value is 1.15, and the total CFU concentration is about 1.2×10 8 CFU / mL, H2S and Fe 2+ The synergistic release ability was good. Transmission electron microscopy (TEM) observation revealed that the cells were surrounded by Fe / Cd / S co-precipitation membrane structures, which initially showed a synergistic mineralization tendency.

[0023] 2. Add FeCl3 solution to the sludge (to make Fe 3+ The content reached 0.3wt%), the moisture content was adjusted to 75%, and the pH was 6.8; 1.0kg of treated sludge was placed in a 2.5L reaction barrel, and 60mL of co-cultured bacterial solution (accounting for 6wt% of the wet weight of the sludge) was added. The reaction system was sealed and maintained at 32℃ for 10 days. In order to simulate the actual engineering conditions, stirring was started for 15 minutes every two days (speed 30rpm), without additional ventilation, relying on the self-stable anaerobic microenvironment of the system. After the reaction, the sample was treated by frozen centrifugation and dried to constant weight. The BCR method was used to analyze the changes in heavy metal morphology: the exchangeable Cd decreased from 45.7% to 3.4%, and the proportion of residual state increased to 60.2%; the TCLP leaching concentration was 0.17mg / L. Compared with the control group using only sulfur-producing bacteria, the amount of CdS precipitation increased by about 21%, the crystal particles were more dense and uniform in morphology, and the CdS peak intensity of the crystal face index (111), (220) and (311) in the XRD spectrum increased ( Figure 4 ), SEM images show that a dense pyrite-like coating is formed on the sludge surface, which effectively inhibits Cd migration ( Figure 5EDS analysis showed that the atomic ratio of Cd / S / Fe was approximately 1:1.1:0.6. It is speculated that the co-culture process generated some CdFeS2-like composite precipitates, which helped to further enhance the stability and structural strength of the precipitates ( Figure 5 ).

[0024] The co-culture bacterial agent used in this embodiment is suitable for complex sludge environments containing high concentrations of Cd and Fe-type synergistic pollution. It has strong adaptability and stable metabolic capacity. It can be applied to heavy metal-iron complex pollution systems such as metallurgical sludge and mineral processing waste mud to achieve efficient, low-carbon and safe heavy metal biomineralization and ecological sequestration.

Claims

1. A method for preparing a mineralizing agent, characterized in that: Inoculate sulfur-producing bacteria into liquid anaerobic basal culture medium and culture at 30-37°C, pH 6.8-7.2, anaerobic or microaerobic conditions for 24-48 hours to obtain a viable bacteria concentration of 10 7 -10 9 CFU / mL bacterial suspension; the bacteria were domesticated with Cd(NO3)2 to obtain a mineralized bacterial agent.

2. The method for preparing the mineralizing agent according to claim 1, wherein: The acclimated bacterial solution is mixed with the carrier in a mass ratio of 1: (1-5), stirred evenly, and then dried or embedded to obtain a mineralizing bacterial agent.

3. The method for preparing the mineralizing agent according to claim 2, wherein: The carrier is selected from vermiculite powder, white mud powder, zeolite powder, straw powder, sodium alginate, calcium alginate and polyvinyl alcohol.

4. A method for preparing a mineralizing bacterial agent, characterized in that: The activated sulfur-producing bacteria and iron-reducing bacteria are co-cultured to prepare a mineralizing bacterial agent.

5. The method for preparing the mineralizing bacterial agent according to claim 1 or 4, characterized in that: Sulfur-producing bacteria are selected from Desulfovibrio ( Desulfovibrio desulfuricans ), sulfate-reducing bacteria ( Desulfovibrio vulgaris ).

6. The method for preparing the mineralizing bacterial agent according to claim 4, characterized in that: Iron-reducing bacteria are Shewanella putrefaciens ( Shewanella putrefaciens ).

7. Use of the mineralizing agent prepared by the preparation method of the mineralizing agent according to claim 1, 2 or 4 in cadmium stabilization treatment.

8. The use according to claim 7, characterized in that: Add the mineralizing bacteria agent to the cadmium-contaminated sludge at an inoculation rate of 1-10%, mix well, and let it stand or stir for 5-10 days at 15-35°C under anaerobic conditions to achieve the mineralization and fixation of heavy metal cadmium. After the reaction is completed, it is mechanically dehydrated to obtain the cadmium-stabilized sludge product.

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