Method for inducing biofilm formation and strengthening medium-chain fatty acid production by adopting modified biochar
By introducing carboxyl or amino groups to modify the surface of biochar, the interaction between microorganisms and biochar is enhanced, which solves the problem of insufficient biofilm formation in anaerobic fermentation and improves the yield of medium-chain fatty acids and the conversion rate of lactic acid.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- CHONGQING UNIV
- Filing Date
- 2026-01-23
- Publication Date
- 2026-04-17
AI Technical Summary
In the current technology, there is a lack of research on the mechanism of biochar in regulating biofilm formation and medium-chain fatty acid synthesis, which limits its application in anaerobic fermentation.
By introducing carboxyl or amino groups onto the surface of biochar through acid-base modification, the interaction between microorganisms and biochar is enhanced, promoting the formation and stability of biofilms and increasing the yield of medium-chain fatty acids.
It significantly improves the yield of medium-chain fatty acids and the conversion rate of lactic acid, reduces the generation of by-products, enhances the functionality of biochar in anaerobic fermentation systems, and is suitable for both synthetic and practical organic wastewater treatment.
Smart Images
Figure CN121874012A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the fields of biomass resource utilization and microbial fermentation technology, specifically to a method for inducing biofilm formation and enhancing medium-chain fatty acid production using modified biochar. Background Technology
[0002] With the global energy structure transformation and the advancement of the "dual carbon" goal, the development of renewable chemical production technologies has become a research hotspot. Anaerobic fermentation can convert organic waste into short-chain fatty acids, but its low energy density and high separation cost limit its industrial application. Medium-chain fatty acids (MCFAs, C6~C12) have advantages such as strong hydrophobicity, high energy density, and ease of separation, and can be used as precursors for biofuels and other high-value-added chemicals, showing broad application prospects.
[0003] Currently, methods to increase MCFA yield mainly include adding conductive materials (such as nano-zero-valent iron and activated carbon), optimizing fermentation conditions (pH, temperature, electron donor-acceptor ratio), and using pure or mixed microbial communities. Biochar, due to its porous structure, high specific surface area, and abundant surface functional groups, is widely used as a microbial carrier to enhance microbial attachment, promote electron transfer, and reduce product inhibition. However, existing research focuses primarily on the physical properties of biochar (such as porosity and specific surface area), and systematic studies on how its surface functional groups regulate biofilm formation and the MCFA synthesis mechanism are still relatively lacking.
[0004] Therefore, developing a method to precisely regulate biofilm formation and enhance MCFAs production by modifying biochar with surface functional groups is of great scientific significance and application value. Summary of the Invention
[0005] To address the aforementioned shortcomings of existing technologies, this invention provides a method for inducing biofilm formation and enhancing medium-chain fatty acid production using modified biochar. This involves introducing carboxyl groups onto the surface of the biochar through acid-base modification. COOH) or amino ( NH2) enhances its interaction with microorganisms, promotes the formation and stability of biofilms, thereby improving lactic acid utilization and MCFA yield.
[0006] To achieve the above-mentioned objectives, the technical solution adopted by this invention is as follows: A method for inducing biofilm formation using modified biochar is provided, the specific steps of which are as follows: A1: Walnut shells were washed, dried, and then pyrolyzed under a nitrogen atmosphere to obtain walnut biochar. A2: Walnut biochar was soaked and stirred in 36% acetic acid or 28% ammonia water for 48 hours to obtain modified biochar; A3: Modified biochar was mixed with the target microorganism and co-cultured in LB liquid medium.
[0007] Furthermore, in step A1, the pyrolysis method is as follows: the temperature is increased to 600℃ at a rate of 10℃ / min, and the temperature is maintained for 2 hours for pyrolysis.
[0008] Furthermore, in step A2, the ratio of 36% acetic acid or 28% ammonia to walnut biochar is 45 mL: 3 g.
[0009] Furthermore, in step A3, the ratio of modified biochar to LB liquid culture medium is 10 g: 1 L.
[0010] This invention also provides a method for producing medium-chain fatty acids using modified biochar, the specific steps of which are as follows: B1: After washing and drying the walnut shells, pyrolyze them under a nitrogen atmosphere to obtain walnut biochar; soak the walnut biochar in acetic acid or ammonia water to obtain modified biochar. B2: The activated sludge is domesticated with lactic acid-acetic acid to obtain domesticated sludge inoculum enriched with target microorganisms that metabolize and synthesize medium-chain fatty acids; B3: Add lactic acid, modified biochar, and acclimatized sludge inoculum to a petri dish and anaerobic ferment at pH 5.5 and 37°C.
[0011] Furthermore, in step B1, the pyrolysis is specifically performed at 600℃ for 2 hours, and the ratio of acetic acid or ammonia to walnut biochar is 45mL:3g.
[0012] Furthermore, in step B3, the ratio of the culture medium solution containing 15 g / L lactic acid, the modified biochar, and the acclimatized sludge inoculum is 180 mL: 10 g: 20 mL.
[0013] Furthermore, in step B3, a methanogenic inhibitor is added to the petri dish, and the volume ratio of the methanogenic inhibitor to the anaerobic reaction system is 10 g: 1 L.
[0014] Furthermore, the methanogenic inhibitor is sodium 2-bromoethylsulfonate.
[0015] The beneficial effects of this invention are as follows: This invention significantly enhances the formation and stability of functional biofilms in anaerobic fermentation systems and efficiently promotes the synthesis of medium-chain fatty acids (MCFAs) by introducing modified biochar. The abundant functional groups on the biochar surface enhance microbial adhesion, forming a dense and highly active biofilm. In the system, it can alleviate product inhibition by undissociated MCFAs, improve microbial tolerance, thereby increasing MCFA yield by up to 60%, significantly improving lactic acid conversion, and reducing byproduct formation. This method is not only applicable to synthetic wastewater but also demonstrates good treatment effects and engineering application potential for actual organic wastewater. Related mechanistic studies provide a theoretical basis for the precise control of biochar in anaerobic fermentation. Attached Figure Description
[0016] Figure 1 This is a comparison diagram of the changes in biofilm amount on the surfaces of three types of biochar for the two strains in Example 2; Figure 2 This is a QCM-D real-time monitoring curve of two strains and three types of biochar in Example 2; Figure 3 This is a Logistic model fitting curve for the two strains and three types of biochar in Example 3; Figure 4 This is a graph showing the theoretical interaction energy curves between the two strains and the three types of biochar XDLVO in Example 3; Figure 5 This is a comparison chart of the changes in hexanoic acid production between the three types of biochar and the control group in Example 3. Detailed Implementation
[0017] The specific embodiments of the present invention are described below to enable those skilled in the art to understand the present invention. However, it should be understood that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, various changes are obvious as long as they are within the spirit and scope of the present invention as defined and determined by the appended claims. All inventions utilizing the concept of the present invention are protected.
[0018] Unless otherwise specified, all equipment used in the embodiments is commercially available and all raw materials are commercially available standard purity.
[0019] Example 1: Preparation of Modified Biochar Walnut shells were used as raw material. After being washed with deionized water and dried at 80℃, the shells were heated to 600℃ at 10℃ / min under a nitrogen atmosphere and pyrolyzed for 2 hours. The resulting material was then sieved to obtain walnut biochar (BC) with a particle size of 0.5~1mm.
[0020] Walnut biochar was soaked in acetic acid and stirred for 48 hours to obtain carboxyl-modified biochar (CBC); walnut biochar was soaked in ammonia water and stirred for 48 hours to obtain amino-modified biochar (NBC). The ratio of acetic acid or ammonia water to walnut biochar was 45 mL: 3 g.
[0021] Example 2: Induction of biofilm Biochars BC, CBC, and NBC prepared in Example 1 were co-cultured with *Escherichia coli* and *Staphylococcus aureus* in LB medium for 72 hours, with the medium being changed every 24 hours. During the culture process, the biofilm components of *E. coli* and *Staphylococcus aureus* in the medium were quantitatively detected every 6 hours. The detection results are as follows: Figure 1 As shown, where, Figure 1 In the figure, 'a' represents a comparison of changes in protein content in E. coli biofilms; Figure 1 In the figure, b is a comparison of the changes in polysaccharide content in Escherichia coli biofilm; Figure 1 In the figure, c represents a comparison of the changes in the dissolved chemical oxygen demand (SCOD) content of Escherichia coli biofilm; Figure 1 In the figure, d represents a comparison of changes in protein content in Staphylococcus aureus biofilm; Figure 1 In the figure, 'e' represents a comparison of changes in polysaccharide content in E. coli biofilms; Figure 1 In the figure, f represents a comparison of changes in SCOD content in E. coli biofilms; from Figure 1 It can be seen that the modified biochar CBC and NBC prepared by the present invention can effectively increase the content of each biofilm component of the two strains compared with the original biochar, proving that the two modified biochars can enhance the formation of biofilm.
[0022] The adhesion process was monitored in real time using a quartz microcrystalline balance (QCM-D), and the results are as follows: Figure 2 As shown, f3, f5, f7, and f9 represent the frequency variations of the quartz crystal under the 3rd, 5th, 7th, and 9th harmonics, respectively. A decrease in f indicates an increase in the surface mass of the crystal, including bacteria, biofilms, or adsorbed moisture. D3, D5, D7, and D9 represent the dissipation variations under the corresponding harmonics, reflecting the mechanical properties of the attached layer. Figure 2 In the figure, 'a' represents the real-time curve of E. coli attachment on the BC surface; Figure 2 In the graph, b represents the real-time curve of E. coli attachment on the NBC surface; Figure 2 c in the figure represents the real-time curve of E. coli attachment on the CBC surface; Figure 2 In the figure, d represents the real-time curve of Staphylococcus aureus attachment on the BC surface; Figure 2 In the figure, 'e' represents a real-time curve of Staphylococcus aureus attachment on the NBC surface; Figure 2 f in the figure represents the real-time curve of Staphylococcus aureus attachment on the CBC surface; Figure 2 The study demonstrated at the molecular level that the surface functional groups of CBC and NBC promote microbial attachment.
[0023] Example 3 Production of medium-chain fatty acids Anaerobic sludge from a wastewater treatment plant in Chongqing was subjected to 60 days of lactic acid-acetic acid acclimation. The acclimation process was as follows: Equal proportions of sludge and lactic acid culture medium were placed in an anaerobic reactor, and 10 g / L of sodium 2-bromoethylsulfonate was added, followed by nitrogen purging for deoxygenation. Fermentation was carried out at 35℃ and 150 rpm, with equal amounts of material replaced daily for 30 days in a semi-continuous culture. Biochar BC, CBC, and NBC prepared in Example 1 were added to 250 mL serum bottles, with 15 g / L of lactic acid as the carbon source, 10 g / L of acclimated sludge, and then 10 g / L of the biochar BC, CBC, and NBC prepared in Example 1, respectively. Additionally, 10 g / L of sodium 2-bromoethylsulfonate was added as a methanogenic inhibitor. The pH was controlled at 5.5, and anaerobic fermentation was carried out at 37℃ for 30 days.
[0024] The growth of pure bacterial biofilm was fitted using a Logistic model, and the results are as follows: Figure 3 As shown, where, Figure 3 In the figure, 'a' represents the fitting curve of the Logistic model for E. coli. Figure 3 In the figure, b represents the fitting curve of the Logistic model for Staphylococcus aureus, derived from... Figure 3 It can be seen that, compared with BC, the absorbance (Abs 595nm) saturation value of CBC and NBC groups is significantly improved, and the slope of the logarithmic growth period is larger. This indicates that the specific functional groups (-COOH or -NH2) introduced by acid-base modification enhance the surface affinity of biochar, shorten the attachment lag period of microorganisms, and induce the formation of a higher density biofilm structure.
[0025] The XDLVO theory was used to calculate and statistically analyze the biochar-microorganism interaction energy during the fermentation process. The results are as follows: Figure 4 As shown, where, Figure 4 In the figure, 'a' represents the energy interaction curve between E. coli and BC; Figure 4 In the figure, b represents the energy interaction curve between Escherichia coli and CBC; Figure 4 In the figure, c represents the energy interaction curve between E. coli and NBC; Figure 4 In the figure, d represents the energy interaction curve between Staphylococcus aureus and BC; Figure 4 In this figure, 'e' represents the energy interaction curve between Staphylococcus aureus and CBC. Figure 4 In the figure, f represents the energy interaction curve between *E. coli* and NBC. Figure 4It can be seen that, compared with BC, the total interaction energy (TOT) barrier between CBC and NBC is significantly reduced. This indicates that the carboxyl groups (-COOH) or amino groups (-NH2) introduced through acid-base modification effectively improve the physicochemical properties of the biochar surface, significantly reduce the repulsive resistance that microorganisms need to overcome when approaching the biochar surface, thereby enhancing biofilm formation.
[0026] The yield of hexanoic acid during fermentation was measured, and a blank group (i.e., no biochar added) was set up as a control group. The results are as follows: Figure 5 As shown, by Figure 5 It is known that the modified biochar CBC or NBC prepared by the present invention, and through the method of the present invention, effectively increases the yield of hexanoic acid compared with biochar BC prepared from walnut shells or other ordinary biochars. Moreover, CBC has the highest hexanoic acid yield, with a 60.12% increase in hexanoic acid yield and a lactic acid conversion rate of 68.5% compared with the control group.
[0027] In summary, the modified biochar prepared by the method of the present invention can form a dense and highly active biofilm, thereby enhancing the production of medium-chain fatty acids, significantly improving lactic acid conversion rate, and reducing by-product generation.
Claims
1. A method for inducing biofilm formation using modified biochar, characterized in that, The specific steps are as follows: A1: Walnut shells were washed, dried, and then pyrolyzed under a nitrogen atmosphere to obtain walnut biochar. A2: Walnut biochar was soaked and stirred in 36% acetic acid or 28% ammonia water for 48 hours to obtain modified biochar; A3: Modified biochar was mixed with the target microorganism and co-cultured in a liquid culture medium.
2. The method for inducing biofilm formation using modified biochar according to claim 1, characterized in that, In step A1, the pyrolysis method is as follows: the temperature is increased to 600℃ at a rate of 10℃ / min, and the temperature is maintained for 2 hours for pyrolysis.
3. The method for inducing biofilm formation using modified biochar according to claim 1, characterized in that, In step A2, the ratio of 36% acetic acid or 28% ammonia to walnut biochar is 45 mL: 3 g.
4. The method for inducing biofilm formation using modified biochar according to claim 1, characterized in that, In step A3, the ratio of modified biochar to LB liquid culture medium is 10 g: 1 L.
5. A method for producing medium-chain fatty acids using modified biochar, characterized in that, The specific steps are as follows: B1: After washing and drying the walnut shells, pyrolyze them under a nitrogen atmosphere to obtain walnut biochar; soak the walnut biochar in acetic acid or ammonia water to obtain modified biochar. B2: The activated sludge is domesticated with lactic acid-acetic acid to obtain domesticated sludge inoculum enriched with target microorganisms that metabolize and synthesize medium-chain fatty acids; B3: Add lactic acid, modified biochar and acclimatized sludge inoculum to a petri dish and anaerobic ferment at pH 5.5 and 37°C.
6. The method for producing medium-chain fatty acids using modified biochar according to claim 5, characterized in that, In step B1, the pyrolysis is specifically performed at 600℃ for 2 hours, and the ratio of acetic acid or ammonia to walnut biochar is 45 mL: 3 g.
7. The method for producing medium-chain fatty acids using modified biochar according to claim 5, characterized in that, In step B3, the ratio of the culture medium solution containing 15 g / L lactic acid, the modified biochar, and the acclimatized sludge inoculum is 180 mL: 10 g: 20 mL.
8. The method for producing medium-chain fatty acids using modified biochar according to claim 5, characterized in that, In step B3, a methanogenic inhibitor is added to the petri dish, and the volume ratio of the methanogenic inhibitor to the anaerobic reaction system is 10 g: 1 L.
9. The method for producing medium-chain fatty acids using modified biochar according to claim 8, characterized in that, The methanogenesis inhibitor is sodium 2-bromoethylsulfonate.