Biochar immobilized microbial composite material as well as preparation method and application thereof

Abstract

The embodiment of the invention discloses a biochar immobilized microorganism composite material as well as a preparation method and application thereof. The method comprises the following steps: activating a bacterial strain, centrifugally collecting thalli, and preparing a bacterial suspension with normal saline, with the preservation number of the bacterial strain being CGMCC No.31176; and fixing the bacterial suspension and the biochar under an oscillation condition. The biochar immobilized microbial composite material provided by the invention has relatively high microbial immobilization efficiency, is beneficial to attachment and survival of more DT-6 bacteria, has relatively high remediation efficiency on TCEP, TCIPP and TDCPP contained in soil, and has a good application prospect in the aspect of remediation of soil chlorinated organophosphate pollution.

Description

Technical Field

[0001] The embodiments of the present invention relate to the technical field of soil remediation, and specifically to a biochar-immobilized microbial composite material and a preparation method and application thereof. Background Art

[0002] Chlorinated organophosphates are an important class of organophosphate flame retardants / plasticizers (OPEs). They are widely used as flame retardants in paints, building materials, furniture, plastics and other products. They mainly include tris-(2-chloroethyl) phosphate (TCEP), tris-(1-chloro-2-propyl) phosphate (TCIPP) and tris[2-chloro-1-(chloromethyl)ethyl]phosphate (TDCPP). Their consumption accounts for about 55% of the total consumption of OPEs. Chlorinated organophosphates are mainly added to the final product by addition rather than chemical bonding, making them very easy to release into the surrounding environment. More importantly, chlorinated organophosphates have toxic effects such as carcinogenicity, teratogenicity, neurotoxicity, developmental toxicity, reproductive toxicity and endocrine disruption, which are very harmful to the ecosystem and human health. At present, chlorinated organophosphates are the main OPEs detected in various environmental media such as water, soil, and air. In view of the production and use of chlorinated organophosphates, their toxic effects, and environmental occurrence, it is urgent to carry out research on environmental remediation technologies for chlorinated organophosphates.

[0003] The removal technologies of chlorinated organophosphates mainly include hydrolysis, microbial degradation and photodegradation. Among them, photodegradation methods such as ultraviolet (UV) / H2O2, UV / photocatalyst, UV / persulfate and visible light / photocatalyst are all carried out under pure systems, and their effects on the reduction of OPEs in actual environments remain to be investigated. Microbial degradation is considered to be the most promising technology for the removal of organic pollutants, but the microbial degradation of OPEs is mainly focused on tributyl phosphate (TBP) and triphenyl phosphate (TPhP). There are also some studies on the microbial degradation of TCEP and TDCPP, but the degradation effects are not ideal, and there are few reports that can simultaneously degrade the three chlorinated organophosphates TCEP, TCIPP and TDCPP. In addition, for soil chlorinated organophosphate pollution, the adaptability of strategies such as hydrolysis and photodegradation is poor. Therefore, it is urgent to develop microbial degradation technology for chlorinated organophosphates.

[0004] When microbial agents are applied to the soil alone, they may face competition from other microorganisms in the soil and find it difficult to survive or reproduce stably in the soil. They may also be unable to adapt to the specific soil environment, resulting in limited growth or death, thereby reducing the efficiency of microbial remediation. The biochar-immobilized microbial technology can solve the above problems well, by enhancing the adaptability of microbial agents in the soil, thereby improving the remediation efficiency. In addition, biochar itself has the ability to improve soil and soil microenvironment. Therefore, it is crucial to prepare biochar-immobilized microbial composite materials based on efficient chlorinated organophosphate-degrading bacteria and apply them to the remediation of chlorinated organophosphate-contaminated soil for the treatment of soil chlorinated organophosphate pollution. Summary of the invention

[0005] To this end, an embodiment of the present invention provides a biochar-immobilized microbial composite material and a preparation method and application thereof.

[0006] In order to achieve the above purpose, the embodiment of the present invention provides the following technical solutions:

[0007] According to a first aspect of an embodiment of the present invention, the present invention provides a method for preparing a biochar-immobilized microbial composite material, the method comprising:

[0008] The strain was activated, the bacterial cells were collected by centrifugation, and a bacterial suspension was prepared with physiological saline. The deposit number of the strain is CGMCC No.31176;

[0009] The bacterial suspension was fixed with biochar under shaking conditions.

[0010] Furthermore, the OD of the bacterial suspension 600 It is 0.8-1.

[0011] Furthermore, the activation method is: inoculating the strain into an inorganic salt liquid culture medium with a TCIPP concentration of 50 mg / L for activation for 20 hours; wherein the composition of the inorganic salt liquid culture medium is: MgSO40.20 g, FeCl30.005 g, CaCl20.01 g, KH2PO41.50 g, NaNO34.00 g, Na2HPO40.50 g, pH=7.0, diluted to 1000 mL with pure water, and sterilized at 121°C for 20 minutes.

[0012] Furthermore, the preparation method of the biochar is: crushing the leaves of Larix olgensis and putting them into a crucible, placing it in a muffle furnace, and carbonizing them at 500-700°C for 4-6 hours in anoxic conditions, and after the temperature of the muffle furnace drops to room temperature, taking out the black residue, grinding it, and passing it through a 100-mesh sieve to obtain the biochar.

[0013] Furthermore, the volume-to-weight ratio of the bacterial suspension to the biochar is (15-20):1.

[0014] Furthermore, the shaking conditions are: 25-30°C, 150-180rpm, 4-6h.

[0015] According to a second aspect of the embodiments of the present invention, the present invention provides a biochar-immobilized microorganism composite material, which is made by the method described in any one of the above items.

[0016] According to a third aspect of an embodiment of the present invention, the present invention provides use of the biochar-immobilized microbial composite material as described above in remediating soil contaminated by chlorinated organophosphates.

[0017] According to a fourth aspect of an embodiment of the present invention, the present invention provides a method for remediating soil contaminated by chlorinated organophosphate, the method comprising:

[0018] The chlorinated organic phosphate contaminated soil and the biochar-immobilized microbial composite material as described above are mixed evenly in a mass ratio of 1:(0.02-0.1), the soil moisture content is adjusted to 50-60% of the field water holding capacity, and static culture is carried out at 20-30° C. for 30-40 days.

[0019] Furthermore, in the soil contaminated with chlorinated organophosphate, the concentrations of TCEP, TCIPP and TDCPP are 5-50 mg / kg, 5-50 mg / kg and 5-50 mg / kg, respectively.

[0020] The embodiments of the present invention have the following advantages:

[0021] The biochar-immobilized microbial composite material provided by the present invention has a high microbial immobilization efficiency, is conducive to the attachment and survival of more DT-6 bacteria, has a high remediation efficiency for TCEP, TCIPP and TDCPP contained in the soil, and has good application prospects in the remediation of soil chlorinated organophosphate pollution. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the implementation methods of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for the implementation methods or the description of the prior art. Obviously, the drawings in the following description are only exemplary, and for ordinary technicians in this field, other implementation drawings can be derived from the provided drawings without creative work.

[0023] Figure 1 The phylogenetic tree of the strain DT-6 provided by the present invention. DETAILED DESCRIPTION

[0024] The following is a description of the implementation of the present invention by specific embodiments. People familiar with the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0025] In the following content, the composition of the inorganic salt liquid culture medium is: MgSO40.20 g, FeCl30.005 g, CaCl20.01 g, KH2PO41.50 g, NaNO34.00 g, Na2HPO40.50 g, pH=7.0, diluted to 1000 mL with pure water, and sterilized at 121℃ for 20 min.

[0026] Inorganic salt solid culture medium: Add 18g / L agar to the inorganic salt liquid culture medium.

[0027] Screening and isolation of strains

[0028] Weigh 2 g of soil sample (soil from a certain area in Shenyang that had been contaminated with TCIPP for a long time and had a high pollution concentration) and add it to 100 mL of inorganic salt liquid culture medium with a TCIPP concentration of 10 mg / L. Culture it at 35°C and 160 r / min for 7 days with constant temperature shaking. Then, transfer 10 mL of the bacterial solution to 90 mL of inorganic salt liquid culture medium with a TCIPP concentration of 20 mg / L. Repeat the above steps to increase the TCIPP concentration to 30, 40, and 50 mg / L, respectively.

[0029] Take 1 mL of the suspension from the last culture cycle and dilute it with sterile water for 10 6 -10 8 After the bacteria have grown to a moderate size, they are isolated by streaking on the plate multiple times to obtain a single TCIPP-degrading bacterium.

[0030] After enrichment, acclimatization, separation and purification, 8 strains with good growth and different colony morphology were obtained, which were numbered as DT-1, DT-2, DT-3, DT-4, DT-5, DT-6, DT-7 and DT-8. After 7 days of cultivation in inorganic liquid medium with TCIPP (10 mg / L) as the only carbon source, the growth of each strain and its degradation of TCIPP were measured. Among them, strain DT-6 had the best growth, and OD600 was significantly higher than that of other strains; and strain DT-6 also had the strongest ability to degrade TCIPP, with a degradation rate of 29.1%, which was significantly higher than that of other strains.

[0031] 16S rDNA molecular identification: Total bacterial DNA was extracted and PCR amplified using bacterial 16S rDNA universal primers and 2×TsingKEMaster Mix (Code No.: TSE003). The PCR product sequencing was completed by Beijing Aoweisen Technology, and the sequencing results are shown in SEQ ID NO: 1.

[0032] The sequencing results were compared with the 16S rDNA sequences of the reference strains in the GenBank database, and the sequences with similarity greater than 97% were selected to construct a phylogenetic tree using the neighbor-joining method using MEGA 11.0 software ( Figure 1 The results showed that the 16S rRNA gene sequence of strain DT-6 was highly similar to that of Ochrobactrum sp. (the highest sequence identity was 99.83%), and it belonged to Ochrobactrum sp. in molecular phylogenetic classification.

[0033] The inventors deposited strain DT-6, and the classification name of DT-6 is Ochrobactrum sp. The deposit address is General Microbiology Center of China Microbiological Culture Collection Administration, the deposit date is July 4, 2024, and the deposit number is CGMCC No.31176.

[0034] Example 1

[0035] This embodiment provides a biochar-immobilized microbial composite material, and the preparation method thereof comprises the following steps:

[0036] (1) Preparation of biochar:

[0037] The leaves of Larix olgensis were crushed and put into a crucible, which was then placed in a muffle furnace and carbonized at 500°C for 6 hours without oxygen. After the temperature of the muffle furnace dropped to room temperature, the black residue was taken out, ground, and passed through a 100-mesh sieve to obtain biochar.

[0038] (2) Preparation of bacterial suspension:

[0039] The DT-6 bacteria were inoculated into an inorganic salt liquid medium with a TCIPP concentration of 50 mg / L and activated for 20 h. The activated DT-6 bacterial liquid was centrifuged at 3000 rpm for 10 min, the supernatant was discarded, and the OD value was prepared using physiological saline. 600 =1 bacterial suspension.

[0040] (3) Preparation of composite materials:

[0041] The biochar of step (1) and the bacterial suspension of step (2) were mixed at a mass volume ratio of 1 g:20 mL, adsorbed and oscillated for 4 h at 30° C. and 180 rpm, centrifuged at 1000 rpm for 10 min, and the supernatant was discarded to obtain a biochar-immobilized microbial composite material.

[0042] Example 2

[0043] This embodiment provides a biochar-immobilized microbial composite material, and the preparation method thereof comprises the following steps:

[0044] (1) Preparation of biochar:

[0045] The leaves of Larix olgensis were crushed and put into a crucible, which was then placed in a muffle furnace and carbonized at 700°C for 4 h in the absence of oxygen. After the temperature of the muffle furnace dropped to room temperature, the black residue was taken out, ground, and passed through a 100-mesh sieve to obtain biochar.

[0046] (2) Preparation of bacterial suspension:

[0047] The DT-6 bacteria were inoculated into an inorganic salt liquid medium with a TCIPP concentration of 50 mg / L and activated for 20 h. The activated DT-6 bacterial solution was centrifuged at 3000 rpm for 10 min, the supernatant was discarded, and the OD value was prepared using physiological saline. 600 =1 bacterial suspension.

[0048] (3) Preparation of composite materials:

[0049] The biochar of step (1) and the bacterial suspension of step (2) were mixed at a volume ratio of 1 g:15 mL, adsorbed and oscillated for 5 h at 25° C. and 160 rpm, centrifuged at 1000 rpm for 10 min, and the supernatant was discarded to obtain a biochar-immobilized microbial composite material.

[0050] Example 3

[0051] This embodiment provides a biochar-immobilized microbial composite material, and the preparation method thereof comprises the following steps:

[0052] (1) Preparation of biochar:

[0053] The leaves of Larix olgensis were crushed and placed in crucibles, which were placed in a muffle furnace and carbonized at 600°C for 5 h in the absence of oxygen. After the temperature of the muffle furnace dropped to room temperature, the black residue was taken out, ground, and passed through a 100-mesh sieve to obtain biochar.

[0054] (2) Preparation of bacterial suspension:

[0055] The DT-6 bacteria were inoculated into an inorganic salt liquid medium with a TCIPP concentration of 50 mg / L and activated for 20 h. The activated DT-6 bacterial liquid was centrifuged at 3000 rpm for 10 min, and then the supernatant was discarded and the OD was prepared with physiological saline. 600 =1 bacterial suspension.

[0056] (3) Preparation of composite materials:

[0057] The biochar of step (1) and the bacterial suspension of step (2) were mixed at a volume ratio of 1 g:18 mL, adsorbed and oscillated for 6 h at 30° C. and 150 rpm, centrifuged at 1000 rpm for 10 min, and the supernatant was discarded to obtain a biochar-immobilized microbial composite material.

[0058] Comparative Example 1

[0059] This comparative example provides a biochar-immobilized microorganism composite material, which differs from Example 1 only in that in step (1), larch branches are used to prepare biochar. The specific contents are as follows:

[0060] The branches of Larix gmelinii were crushed and put into a crucible, which was then placed in a muffle furnace and carbonized at 700°C for 4 h in the absence of oxygen. After the temperature of the muffle furnace dropped to room temperature, the black residue was taken out, ground, and passed through a 100-mesh sieve to obtain biochar.

[0061] Comparative Example 2

[0062] This comparative example provides a biochar-immobilized microorganism composite material, which differs from Example 1 only in that in step (1), corn stalks are used to prepare biochar. The specific contents are as follows:

[0063] The corn stalks were crushed and put into a crucible, which was then placed in a muffle furnace and carbonized at 500°C for 6 h in the absence of oxygen. After the temperature of the muffle furnace dropped to room temperature, the black residue was taken out, ground, and passed through a 100-mesh sieve to obtain biochar.

[0064] Comparative Example 3

[0065] This comparative example provides a biochar-immobilized microorganism composite material, which differs from Example 1 only in that in step (1), municipal sludge is used to prepare biochar. The specific contents are as follows:

[0066] The municipal sludge was dried and crushed, then put into a crucible, placed in a muffle furnace, and carbonized at 500°C for 2 hours in the absence of oxygen. After the temperature of the muffle furnace dropped to room temperature, the black residue was taken out, ground, and passed through a 100-mesh sieve to obtain biochar.

[0067] Comparative Example 4

[0068] This comparative example provides a biochar-immobilized microorganism composite material, which differs from Example 1 only in that in step (1), cow dung is used to prepare biochar. The specific contents are as follows:

[0069] The cow dung was dried and crushed, then put into a crucible, placed in a muffle furnace, and carbonized at 450°C for 4 hours without oxygen. After the temperature of the muffle furnace dropped to room temperature, the black residue was taken out, ground, and passed through a 100-mesh sieve to obtain biochar.

[0070] Test Example 1

[0071] Immobilization rate test of biochar-immobilized microbial composites

[0072] Test method: The supernatant removed in step (3) of each embodiment and comparative example was transferred to a 50 ml volumetric flask, and the OD of the supernatant was measured. 600 The value is recorded as P1, and the amount of bacteria absorbed is recorded as OD 600 The values ​​were expressed indirectly, and the immobilization rate of the composite material was obtained by subtraction.

[0073] The calculation formula of the immobilization rate is as follows:

[0074] Immobilization rate (%) = (P0-P1) / P0×100% (P0=1)

[0075] The test results of the immobilization rates of the biochar-immobilized microbial composite materials of Examples 1-3 and Comparative Examples 1-4 are shown in Table 1.

[0076] Table 1

[0077] sample Immobilization rate Example 1 87.4％ Example 2 89.5％ Example 3 85.2％ Comparative Example 1 63.2％ Comparative Example 2 58.6％ Comparative Example 3 42.1％ Comparative Example 4 60.7％

[0078] The results showed that the biochar prepared using larch leaves as raw materials in the embodiment of the present invention had a rough surface structure, a large specific surface area and a large porosity, and exhibited excellent adsorption affinity to DT-6 bacteria. The prepared composite material had more microbial attachment and survival.

[0079] Test Example 2

[0080] Remediation of chlorinated organophosphate-contaminated soil by biochar-immobilized microbial composites

[0081] The chlorinated organophosphate contaminated soil came from TCEP, TCIPP and TDCPP contaminated brown soil that was artificially prepared in the laboratory and aged for 180 days. After testing, the concentrations of TCEP, TCIPP and TDCPP in the soil were 25.4 mg / kg, 23.8 mg / kg and 24.6 mg / kg, respectively.

[0082] Soil contaminated with chlorinated organophosphates and biochar-immobilized microbial composite materials were evenly mixed in a mass ratio of 1g:0.05g, and the soil moisture content was adjusted to 60% of the field water holding capacity. The soil was statically cultured at 25°C for 30 days, and the remediation efficiency of each pollutant was tested.

[0083] The test method for the remediation efficiency is as follows: After the remediation is completed, the concentrations of TCEP, TCIPP and TDCPP in the soil are analyzed using accelerated solvent extraction-gas chromatography-mass spectrometry.

[0084] Remediation efficiency = (concentration of chlorinated organophosphates in soil before remediation - concentration of chlorinated organophosphates in soil after remediation) / concentration of chlorinated organophosphates in soil before remediation × 100%

[0085] The test results of the remediation efficiency of the composite materials of Examples 1-3 and Comparative Examples 1-4 for various pollutants in the contaminated soil are shown in Table 2. At the same time, a comparison was made with microorganisms that were not solidified with biochar, that is, the above contaminated soil and the bacterial suspension of Example 1 were cultured under the same conditions according to the same microbial feed ratio (see Comparative Example 5 for remediation efficiency).

[0086] Table 2

[0087]

[0088] The results show that the biochar-immobilized microbial composite material provided by the embodiment of the present invention has a high remediation efficiency for TCEP, TCIPP and TDCPP in contaminated soil, and has good application prospects in the remediation of soil chlorinated organophosphate pollution.

[0089] Although the present invention has been described in detail above by general description and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made to the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all belong to the scope of protection claimed by the present invention.

Claims

1. A method for preparing a biochar-immobilized microbial composite material, characterized in that: The method comprises: The strain was activated, the bacterial cells were collected by centrifugation, and a bacterial suspension was prepared with physiological saline. The deposit number of the strain is CGMCC No.31176; The bacterial suspension was fixed with biochar under shaking conditions.

2. The method for preparing the biochar-immobilized microbial composite material according to claim 1, characterized in that: The OD of the bacterial suspension 600 It is 0.8-1.

3. The method for preparing the biochar-immobilized microbial composite material according to claim 1, characterized in that: The activation method is as follows: inoculating the strain into an inorganic salt liquid culture medium with a TCIPP concentration of 50 mg / L for activation for 20 hours; wherein the inorganic salt liquid culture medium is composed of: MgSO4 0.20 g, FeCl3 0.005 g, CaCl2 0.01 g, KH2PO4 1.50 g, NaNO3 4.00 g, Na2HPO4 0.50 g, pH = 7.0, diluting to 1000 mL with pure water, and sterilizing at 121°C for 20 minutes.

4. The method for preparing the biochar-immobilized microbial composite material according to claim 1, characterized in that: The biochar preparation method comprises the following steps: crushing larch leaves and putting them into a crucible, placing the crucible in a muffle furnace, and performing anoxic carbonization at 500-700° C. for 4-6 hours, and after the temperature of the muffle furnace drops to room temperature, taking out the black residue, grinding it, and passing it through a 100-mesh sieve to obtain the biochar.

5. The method for preparing the biochar-immobilized microbial composite material according to claim 1, characterized in that: The volume weight ratio of the bacterial suspension to the biochar is (15-20):

1.

6. The method for preparing the biochar-immobilized microbial composite material according to claim 1, characterized in that: The shaking conditions are: 25-30°C, 150-180 rpm, 4-6 h.

7. A biochar-immobilized microbial composite material, characterized in that: The method is prepared by the method according to any one of claims 1 to 6.

8. Use of the biochar-immobilized microbial composite material according to claim 7 in remediation of soil contaminated by chlorinated organophosphates.

9. A method for remediating soil contaminated by chlorinated organophosphates, characterized in that: The method comprises: The chlorinated organic phosphate contaminated soil and the biochar-immobilized microbial composite material as claimed in claim 7 are mixed uniformly in a mass ratio of 1:(0.02-0.1), the soil moisture content is adjusted to 50-60% of the field water holding capacity, and statically cultured at 20-30° C. for 30-40 days.

10. The method for remediating soil contaminated by chlorinated organophosphate according to claim 9, characterized in that: In the soil contaminated by chlorinated organophosphate, the concentrations of TCEP, TCIPP and TDCPP are 5-50 mg / kg, 5-50 mg / kg and 5-50 mg / kg, respectively.

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