Iron carrier-3D printing microecological agent for compost, preparation and composting method

Abstract

The application discloses an iron carrier-3D printing micro-ecological bacterial agent for compost, a preparation method and a compost method, and belongs to the technical field of aerobic composting. The method mixes a non-biologically toxic fixing agent with multifunctional degradation bacterial mixed liquid, iron carrier enterobactin and iron carrier putrebactin, fills the mixed liquid into a 3D biological printing bin, adopts a non-biologically toxic crosslinking agent for 3D printing, and obtains the iron carrier-3D printing micro-ecological bacterial agent with a diameter of 0.2-2 mm. The ecological bacterial agent is added in the initial stage of composting and applied to the composting process, and the composting efficiency can be improved. The preparation method can be used for personalized preparation of the bacterial agent, the acquisition capacity of degradation bacterial strains for iron resources is improved, and directional strengthening is realized. The iron carrier-3D printing micro-ecological bacterial agent has the characteristics of high specificity, good stability and strong composting effect.

Description

Technical Field

[0001] This invention belongs to the field of aerobic composting technology, specifically relating to a composting iron carrier-3D printed microbial agent, its preparation and composting method. Background Technology

[0002] Composting is a green, economical, and simple technology for the rapid, harmless, volume-reducing, and resource-oriented treatment of organic solid waste. It first utilizes the hydrolysis of aerobic microorganisms to break down macromolecular organic matter such as starch, protein, lipids, and lignocellulose in the initial raw materials into secondary metabolites such as polysaccharides, polypeptides, and fatty acids. Further, through microbial synthesis and metabolism, as well as physical processes such as uronic acid condensation and Maillard reactions, a stable and complex humic substance rich in redox groups such as phenols and quinones is formed. The resulting humic substance can be used as a heavy metal remediation agent and a soil conditioner.

[0003] The microbial community structure in composting is complex, making targeted bio-initiative control difficult. Driven by ambient temperature, microorganisms such as lactobacilli experience decreased activity at high temperatures, while thermotolerant microorganisms such as Bacillus and sucralospores proliferate rapidly. After the compost pile cools, some actinomycetes and proteobacteria gain ecological dominance, further degrading recalcitrant organic matter. Composting involves complex synergistic processes of microbial communities, but the current bioconversion efficiency for organic matter remains below 50%, resulting in a low level of resource utilization. Traditional bio-initiatives often involve the isolated addition of functional microorganisms, but the effects are unstable after adding the initiator, and different initiators have varying applicability to substrate components, making it difficult to find targeted bioconversion initiators applicable to different compost substrates.

[0004] Furthermore, iron is a crucial substance for the growth and survival of microorganisms, participating in multiple stages such as nutrient uptake, biomass synthesis, and reproduction. Microorganisms can generally utilize ferrous iron (Fe2+), but in aerobic composting environments, iron often exists in its ferric form (Fe3+). Microorganisms typically synthesize siderophores to transport and utilize ferric iron. However, the biosynthesis of siderophores requires a large amount of energy, which is detrimental to the rapid proliferation of microorganisms. Therefore, there is an urgent need to develop a composting system bio-agent that incorporates siderophores. Summary of the Invention

[0005] The purpose of this invention is to overcome the problems of low activity and poor stability of traditional biological agents, improve the ability of organisms to utilize iron, and enhance the stability of the microbial community structure and function. Specifically, it provides a 3D-printed microbial agent with an iron carrier for composting, its preparation, and a composting method.

[0006] The specific technical solution adopted in this invention is as follows:

[0007] In a first aspect, the present invention provides a method for preparing a composting iron carrier-3D printed microbial agent, the specific method of which is as follows:

[0008] A non-biotoxic fixative is mixed with a multifunctional degrading bacterial solution, enterobactin, and putrebactin, and then filled into a 3D bioprinting chamber. 3D printing is performed using a non-biotoxic cross-linking agent to obtain an ferrocarrier-3D printing microecological agent. The multifunctional degrading bacteria are one or more of the following: lignocellulose-degrading bacteria, oil-degrading bacteria, starch-degrading bacteria, or protein-degrading bacteria capable of degrading compost substrates.

[0009] Preferably, the lignocellulose-degrading bacterium is *Thermobifida fusca*, accession number ATCC BAA-629, deposited at the American Center for Type Culture Collection (ACC); the oil-degrading bacterium is *Bacillus licheniformis*, accession number CGMCC 1.8791, deposited on October 21, 2008, at the China General Microbiological Culture Collection (CGC); the starch-degrading bacterium is *Bacillus stequilensis*, accession number CGMCC 1.763, deposited on March 1, 1972, at the CGC; and the protein-degrading bacterium is *Bacillus subtilis*, accession number CGMCC 1.9086, deposited on April 9, 2009, at the CGC.

[0010] Preferably, the volume ratio of the fixative, the multifunctional degrading bacterial mixture, the siderophore enterobactin, and the siderophore putrebactin is (100-120):(100-120):(0.5-1):(0.5-1).

[0011] Preferably, the fixative is a sodium alginate solution or a carboxymethyl cellulose solution.

[0012] Furthermore, the concentration of the sodium alginate solution is 1.5% to 4%.

[0013] Preferably, the crosslinking agent is a calcium chloride solution.

[0014] Furthermore, the crosslinking agent is a calcium chloride solution with a concentration of 1-5%.

[0015] Preferably, the 3D printing process parameters are set as follows: the voltage between the printing nozzle and the lower support layer is 10kV, the printing chamber driving pressure is 2MPa, and the nozzle extrusion pressure is 0.2MPa.

[0016] In a second aspect, the present invention provides a ferrocarrier-3D printing microbial agent prepared according to the method described in the first aspect.

[0017] Thirdly, the present invention provides a composting method using the iron carrier-3D printed microbial agent described in the second aspect, wherein the iron carrier-3D printed microbial agent is added at the initial stage of composting, wherein the mass ratio of the iron carrier-3D printed microbial agent to the initial composting material is 0.1% to 0.5%; the initial composting material needs to be pre-dehydrated, and the particle size range after dehydration is 2 to 5 cm, and the moisture content is 65% to 75%.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0019] The iron-supported 3D-printed microbial agent prepared in this invention, compared with traditional agents, enhances the iron resource acquisition ability of bacterial strains due to the addition of an iron support, thereby strengthening their degradation and transformation capabilities on compost substrates. Furthermore, the composting process involves a complex environment with rapid changes in substrate composition, ambient temperature, pH, and conductivity. This invention utilizes 3D printing technology to immobilize the microbial agent with sodium alginate and calcium chloride, protecting the functional bacterial strains from external environmental stressors, thus strengthening community structure and functional stability, and improving composting efficiency. Attached Figure Description

[0020] Figure 1 This is a graph showing the temperature change curves of the experimental and control groups in Example 2.

[0021] Figure 2 This is a graph showing the changes in oxygen concentration at the reactor outlet of the experimental and control groups in Example 2.

[0022] Figure 3 This is a graph showing the changes in organic matter content during the composting process of the control group, PU group, and EN group in Example 5.

[0023] Figure 4 The graph shows the changes in methane emissions during the composting process of the control group, PU group, and EN group in Example 5. Detailed Implementation

[0024] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0025] The multifunctional degrading bacteria used in the specific embodiments of this invention are as follows:

[0026] The lignocellulose-degrading bacterium is *Thermobifida fusca*, with accession number ATCCBAA-629, deposited at the American Center for Type Culture Collection (ACC) and the China General Microbiological Culture Collection (CGMCC). The lipid-degrading bacterium is *Bacillus licheniformis*, with accession number CGMCC 1.8791, deposited on October 21, 2008, also deposited at CGMCC. The starch-degrading bacterium is *Bacillus tequilensis*, with accession number CGMCC 1.763, deposited on March 1, 1972, also deposited at CGMCC. The protein-degrading bacterium is *Bacillus subtilis*, with accession number CGMCC. 1.9086, deposited on April 9, 2009, by the China General Microbiological Culture Collection Center.

[0027] Example 1

[0028] This embodiment provides a method for preparing a siderophore-based 3D printing microbial agent containing enterobactin and putrebactin, as detailed below:

[0029] (1) Preparation of mixed bacterial solution of multifunctional degrading bacteria.

[0030] Lignocellulose-degrading bacteria *Thermobifida fusca*, lipid-degrading bacteria *Bacillus licheniformis*, starch-degrading bacteria *Bacillus tequilensis*, and protein-degrading bacteria *Bacillus subtilis* were selected and added to LB liquid medium at an inoculation ratio of 0.5%. The cultures were incubated at 35℃ and 150 rpm for 1.5 days to allow the bacterial culture OD to adjust. 600 The value reached 1.0. Then, the above bacterial solutions were mixed in a volume ratio of 1:1:1:1 to prepare a multifunctional degrading bacterial mixed solution.

[0031] (2) Preparation of enterobactin as a siderophore.

[0032] In this embodiment, the siderophore enterobactin is mainly produced by a siderophore-producing bacterial strain obtained through genetic modification of Escherichia coli. Specifically, the following steps are taken: first, gene fragments at both ends of the fepA sequence are amplified; then, the amplified genes are ligated using fusion PCR; next, the fusion fragment is introduced into pHGMO to obtain a plasmid; subsequently, the plasmid is transferred to Escherichia coli (K-12MG1655) strain, and selection and culture are performed using CAS solid medium to obtain mutant strains capable of producing the siderophore enterobactin.

[0033] The mutant strains capable of producing enterobactin were inoculated into LB liquid medium and cultured at 35°C and 150 rpm for 3 days. When the bacterial culture OD... 600 The culture was stopped when the concentration reached 1.5. The bacterial culture was centrifuged at 8000 r / min and 25℃ for 10 min, and then the supernatant was passed through a 0.22 μm membrane to obtain the siderophore enterobactin.

[0034] (3) Preparation of putrebactin as a ferrocarrier.

[0035] In this embodiment, the siderophore putrebactin is mainly produced by genetically modifying Shewanella to obtain a siderophore-producing strain. Specifically, the gene fragments at both ends of the putA sequence are first amplified, then the amplified genes are ligated using fusion PCR, and the recombinant plasmid is introduced into Shewanella oneidensis MR-1 strain. Screening and culture are then performed using CAS solid medium to obtain mutant strains capable of producing the siderophore putrebactin.

[0036] The mutant strains capable of producing the siderophore putrebactin were inoculated into LB liquid medium and cultured at 35°C and 150 rpm for 2 days. When the bacterial culture OD... 600 The culture was stopped when the concentration reached 1.5. The bacterial culture was centrifuged at 8000 r / min and 25℃ for 10 min, and then the supernatant was passed through a 0.22 μm membrane to obtain the siderophore putrebactin.

[0037] The preparation method of CAS solid culture medium is as follows: Solution A: Dissolve 60.5 mg of Chromium Azuril S in 50 mL of deionized water; add 10 mL of ferric iron solution (1 mmol / L FeCl3·6H2O, 10 mmol / L hydrochloric acid as solvent); dissolve 72.9 mg of CTAB in 40 mL of deionized water. Mix the above three solutions and bring the volume to 100 mL, adjust the pH to neutral, and sterilize at 121℃ for 20 min. Solution B: Add 30.24 g of Pipes to 900 mL of LB medium, pH = 6.8, and sterilize at 121℃ for 20 min. Mix solutions A and B thoroughly and pour into plates to obtain CAS solid culture medium.

[0038] (4) Preparation of fixatives and crosslinking agents.

[0039] 2g of sodium alginate was dissolved in 98g of water and stirred with a rotor for 60 minutes until completely dissolved. The solution was then transferred to an Erlenmeyer flask, wrapped in kraft paper, and autoclaved to obtain a non-biotoxic fixative. 2g of calcium chloride was dissolved in 98g of water and stirred with a rotor for 60 minutes until completely dissolved. The solution was then transferred to a beaker to obtain a non-biotoxic crosslinking agent.

[0040] (5) 3D printing to prepare iron carrier-3D printing microbial agent.

[0041] Sterilized sodium alginate solution was mixed with a multifunctional degrading bacterial mixture, enterobactin, and putrebactin in a volume ratio of 100:100:1:1, and then filled into a 3D bioprinting chamber. The voltage between the print head and the lower support layer was adjusted to 10kV, the print chamber drive pressure was adjusted to 2MPa, and the print head extrusion pressure was 0.2MPa. Calcium chloride solution was placed below the print head, and the microsphere droplets ejected from the print head immersed in the calcium chloride solution to form solid spherical bacterial agents, resulting in an enterobactin and putrebactin-containing enterobactin-3D printing microecological bacterial agent. The diameter of the bacterial spheres was in the range of 0.2-2mm.

[0042] Example 2

[0043] This embodiment utilizes the iron carrier-3D printed microbial agent containing enterobactin and putrebactin prepared in Example 1 for composting. The specific process is as follows:

[0044] Kitchen waste, after being crushed and dehydrated, was collected from a kitchen waste transfer station. The kitchen waste was then mixed with rice husks at a mass ratio of 10:1, and the moisture content was adjusted to 60%–65%, with a particle size range of 2–5 cm, to obtain the initial composting material. A total of two reactors were set up, each with a volume of 20L.

[0045] The control group received no bacterial agent, while the experimental group received 0.5% by mass of the siderophore-3D printing microbial agent containing enterobactin and putrebactin prepared in Example 1, with a ventilation rate set to 0.2 L / min. -1 ·kg -1 DM. A thermometer was placed at the center of the reactor core to monitor the reactor core temperature in real time. The results are as follows: Figure 1 As shown. Oxygen concentration changes were monitored daily at the outlet, and the results are as follows. Figure 2 As shown.

[0046] The results showed that adding microbial agents to the experimental group could accelerate the start-up and heating of the reactor, extend the high-temperature period by 2 days, and accelerate the utilization rate of oxygen concentration in the reactor.

[0047] Example 3

[0048] This embodiment provides a method for preparing a siderophore-based 3D printing microbial agent containing enterobactin. The preparation of the multifunctional degrading bacterial mixture, the siderophore enterobactin, the cross-linking agent, and the fixative is the same as in Example 1.

[0049] Sterilized sodium alginate solution, multifunctional degrading bacteria mixture, and enterobactin (a siderophore) were mixed at a volume ratio of 100:100:1, and then filled into a 3D bioprinting chamber. The voltage between the print head and the lower support layer was adjusted to 10kV, the print chamber drive pressure was adjusted to 2MPa, and the print head extrusion pressure was 0.2MPa. Calcium chloride solution was placed below the print head, and the microsphere droplets ejected from the print head immersed in the calcium chloride solution to form solid spherical bacterial agents, thus obtaining an enterobactin-containing siderophore-3D printing microecological bacterial agent, named enterobactin-new ecological bacterial agent (EN).

[0050] Example 4

[0051] This embodiment provides a method for preparing a putrebactin-containing siderophore-based 3D printing microbial agent. The preparation of the multifunctional degrading bacterial mixture, the siderophore putrebactin, the cross-linking agent, and the fixative is the same as in Example 1.

[0052] Sterilized sodium alginate solution, multifunctional degrading bacteria mixture, and putrebactin (a siderophore) were mixed at a volume ratio of 100:100:1, and then filled into a 3D bioprinting chamber. The voltage between the print head and the lower support layer was adjusted to 10kV, the print chamber drive pressure was adjusted to 2MPa, and the print head extrusion pressure was 0.2MPa. Calcium chloride solution was placed below the print head, and the microsphere droplets ejected from the print head immersed in the calcium chloride solution to form solid spherical bacterial agents, thus obtaining a siderophore-3D printing microecological bacterial agent containing putrebactin, named putrebactin-new ecological bacterial agent (PU).

[0053] Example 5

[0054] This embodiment utilizes enterobactin-novel inoculant (EN) and putrebactin-novel inoculant (PU) prepared in Examples 3 and 4 for composting. The specific process is as follows:

[0055] Kitchen waste, after being crushed and dehydrated, was collected from a kitchen waste transfer station. The kitchen waste was then mixed with rice husks at a mass ratio of 10:1, and the moisture content was adjusted to 60%–65%, with a particle size range of 2–5 cm, to obtain the initial composting material. A total of three reactors were set up, each with a volume of 20L.

[0056] The control group (CT) received no bacterial agent, the EN group received 0.5% (by mass) of enterobactin-novel bacterial agent prepared in Example 3, and the PU group received 0.5% (by mass) of putrebactin-novel bacterial agent prepared in Example 4. The ventilation rate was set to 0.2 L / min. -1 ·kg -1 A 24-day composting experiment was conducted on DM, with daily temperature measurements and effluent gas collection. Samples were also taken on days 1, 5, 12, and 24 for physicochemical analysis based on temperature changes. The organic matter degradation rate was as follows: Figure 3 As shown. Emissions of carbon dioxide, methane, and nitrous oxide were determined by gas chromatography, with CH4 emission data as shown below. Figure 4 As shown.

[0057] The results showed that, at the end of composting, the addition of neobiotics significantly improved the organic matter degradation rate compared with the control group. The addition of neobiotics could reduce cumulative CH4 emissions by 10–20% within 24 days.

[0058] The microbial agent of this invention can be customized to achieve targeted enhancement by improving the ability of degrading strains to acquire iron resources. This iron carrier-3D printed microbial agent is characterized by high specificity, good stability, and strong composting effect.

[0059] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the invention. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the invention. Therefore, all technical solutions obtained through equivalent substitution or transformation fall within the protection scope of the present invention.

Claims

1. A method for preparing a composting iron carrier-3D printed microbial agent, characterized in that, The specific method is as follows: A non-biotoxic fixative is mixed with a multifunctional degrading bacterial solution, enterobactin, and putrebactin, and then filled into a 3D bioprinting chamber. A non-biotoxic cross-linking agent is used for 3D printing to obtain an enterobactin-3D printing microbial agent. The multifunctional degrading bacteria are one or more of the following: lignocellulose-degrading bacteria, oil-degrading bacteria, starch-degrading bacteria, or protein-degrading bacteria capable of degrading compost substrates. The fixative is a sodium alginate solution or a carboxymethyl cellulose solution; the volume ratio of the fixative, the multifunctional degrading bacterial mixture, the siderophore enterobactin, and the siderophore putrebactin is 100:100:1:

1.

2. The method for preparing the iron carrier-3D printing microbial agent for composting according to claim 1, characterized in that, The lignocellulose-degrading bacteria are *Schizophyllum commune* (brown thermophilic spores). Thermobifida fusca The accession number is ATCC BAA-629, and the depositary institution is the American Center for Type Culture Collection; the oil-degrading bacteria is Bacillus licheniformis (…). Bacillus licheniformis The accession number is CGMCC 1.8791, the accession date is October 21, 2008, and the depositary institution is the China General Microbiological Culture Collection Center; the starch-degrading bacterium is *Bacillus tekirae* (…). Bacillus tequilensis The accession number is CGMCC 1.763, the accession date is March 1, 1972, and the depositary institution is the China General Microbiological Culture Collection Center; the protein-degrading bacterium is Bacillus subtilis (…). Bacillus subtilis The accession number is CGMCC1.9086, the accession date is April 9, 2009, and the depositary institution is the China General Microbiological Culture Collection Center.

3. The method for preparing iron carrier-3D printing microbial agent for composting according to claim 1, characterized in that, The concentration of the sodium alginate solution is 1.5-4%.

4. The method for preparing iron carrier-3D printing microbial agent for composting according to claim 1, characterized in that, The crosslinking agent is a calcium chloride solution.

5. The method for preparing iron carrier-3D printing microbial agent for composting according to claim 4, characterized in that, The crosslinking agent is a calcium chloride solution with a concentration of 1-5%.

6. The method for preparing iron carrier-3D printing microbial agent for composting according to claim 1, characterized in that, The 3D printing process parameters are set as follows: the voltage between the printing nozzle and the lower support layer is 10kV, the printing chamber driving pressure is 2MPa, and the nozzle extrusion pressure is 0.2MPa.

7. A ferrocarrier-3D printing microbial agent prepared by the method according to any one of claims 1 to 6.

8. A composting method using the ferrocarrier-3D printed microbial agent as described in claim 7, characterized in that, The iron carrier-3D printing microbial agent is added at the initial stage of composting, wherein the mass ratio of the iron carrier-3D printing microbial agent to the initial compost material is 0.1%~0.5%; the initial compost material needs to be dehydrated in advance, and the particle size range after dehydration is 2~5cm, and the moisture content is 65~75%.

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