A lignin-grafted graphene oxide quantum dot material and its preparation and degradation method for polycyclic aromatic hydrocarbons.

Abstract

This invention relates to the field of organic pollution treatment technology, specifically to a lignin-grafted graphene oxide quantum dot material and its preparation and degradation method for polycyclic aromatic hydrocarbons (PAHs). The material is prepared by grafting alkaline lignin with graphene oxide quantum dots under alkaline conditions. When applied to PAH degradation, it forms a synergistic degradation system with the Microbacterium sp. W1-2 strain (CGMCC NO.35223). This material significantly promotes bacterial growth, enhances interfacial mass transfer and bioavailability of pollutants, and achieves efficient degradation in a dark environment without photocatalysis. Experiments show that, with an initial benzo[a]pyrene concentration of 5000 µg / L, adding this material increases the degradation rate to 651.4 µg / h within 6 hours, a 2.9-fold increase compared to a single strain; the removal rate reaches 86.4% within 72 hours. This invention has advantages such as high degradation efficiency, short cycle, strong environmental adaptability, and independence from light, making it suitable for the rapid remediation of PAHs in industrial wastewater.

Description

Technical Field

[0001] This invention relates to the field of organic pollution treatment technology, specifically to a lignin-grafted graphene oxide quantum dot material and a method for its preparation and degradation of polycyclic aromatic hydrocarbons. Background Technology

[0002] Polycyclic aromatic hydrocarbons (PAHs) are a class of persistent organic pollutants composed of two or more fused benzene rings. They have carcinogenic, teratogenic, and mutagenic effects and have been listed as priority pollutants for control. Benzo[a]pyrene, as a typical PAH, is widely found in the soil and water of industrial contaminated sites such as coking plants and oil refineries. Its recalcitrant nature and bioaccumulation pose a serious threat to the ecological environment and human health.

[0003] Microbial degradation has become a research hotspot for the remediation of polycyclic aromatic hydrocarbon (PAH) pollution due to its environmental friendliness and low cost. However, existing technologies using single degrading bacteria often suffer from low degradation efficiency and long degradation cycles. In recent years, the technology of using nanomaterials to assist microbial degradation of pollutants has attracted widespread attention. Chinese patent CN109748733A discloses a technology for promoting the degradation of organic pollutants by microorganisms using graphene quantum dots, demonstrating that nanomaterials can enhance microbial activity; however, this technology relies on photocatalysis, limiting its application scenarios. Chinese patent CN111875464A discloses a method for remediating heavy metal pollution using carbon quantum dots, but does not address the remediation of organic compound pollution. Furthermore, in existing bioremediation technologies, exogenous bacterial strains often become inactive due to competitive pressure in complex environments, leading to unstable remediation effects. Therefore, it is necessary to develop a new method that can enhance the environmental adaptability and degradation activity of degrading bacteria. Summary of the Invention

[0004] To address the above problems, this invention provides a lignin-grafted graphene oxide quantum dot material and a method for its preparation and degradation of polycyclic aromatic hydrocarbons (PAHs). This method significantly improves the degradation efficiency of benzo[a]pyrene by promoting the growth of degrading bacteria, enhancing substrate bioavailability, and improving interfacial contact, thus shortening the remediation cycle and achieving efficient and rapid remediation of benzo[a]pyrene pollution without relying on light conditions. The preparation method of the lignin-grafted graphene oxide quantum dot material specifically includes the following steps:

[0005] Step 1: Mix graphene oxide powder, 1-3M NaOH solution, and deionized water at a ratio of 30-80mg:5-15mL:30-80mL, and sonicate at 200-400W and 20-50kHz for 20-40min to form a uniform suspension. Perform hydrothermal reaction at 160-200℃ for 8-16h. After the reaction is complete, neutralize with 0.5-2M HCl solution to pH 6.5-7.5, centrifuge at 8000-12000rpm for 20-40min, and dialyze the supernatant at 0.5-3kDa for 16-48h (replace the deionized water every 8h) to obtain a graphene oxide quantum dot dispersion of 0.5-2.0mg / mL.

[0006] Step 2: Mix alkaline lignin with deionized water and stir at 300-600 rpm for 5-20 min. Then add ethylenediamine (EDA) and adjust the pH to 8-9 with analytical grade 25% ammonia water. Continue stirring for 5-20 min. Finally, add graphene oxide quantum dot dispersion and sonicate at 150-250 W and 20-50 kHz for 3-10 min. Stir in an oil bath at 110-130℃ and 300-600 rpm for 2-4 h. After the reaction, allow it to cool naturally to room temperature, pre-freeze at (-50)-(-20)℃ for 2-6 h, and freeze-dry at 5-50 Pa for 12-48 h to obtain lignin-grafted graphene oxide quantum dot material. The ratio of alkaline lignin, deionized water, EDA, and graphene oxide quantum dot dispersion is 30-60 mg: 20-40 mL: 30-60 μL: 10-20 mL.

[0007] Preferably, to reduce the interference of free small molecules and residual amines on subsequent biological experiments, the lignin-grafted graphene oxide quantum dot material is mixed with ultrapure water at a ratio of (1-10) mg:1 mL, dialyzed in a 1-3 kDa dialysis bag for 12-48 h (with deionized water replaced ≥4 times every 6 h) or purified by ultrafiltration centrifugation (using ultrafiltration centrifuge tubes with a molecular weight cutoff of 10-50 kDa, centrifugation speed of 4000-8000 rpm, centrifugation time of 10-40 min. Most preferably, centrifugation at 5000-6000 rpm for 15-30 min is performed, and repeated 1-3 times as needed to remove free small molecule impurities and residual amines).

[0008] This invention also provides a method for degrading polycyclic aromatic hydrocarbons using the lignin-grafted graphene oxide quantum dot material combined with microorganisms, specifically including the following steps:

[0009] Step 1: Inoculate the benzo[a]pyrene-degrading bacterium Microbacterium sp. W1-2 onto LB solid agar plates and incubate at 25-35°C for 24-48 h. Then, pick single colonies and inoculate them onto LB liquid agar plates, incubate at 25-35°C and 160-200 rpm until the late logarithmic phase. Collect the bacterial cells by centrifugation at 4000-6000 rpm for 5-15 min, wash twice with sterile water, and resuspend in sterile water. Adjust the OD of the bacterial suspension. 600 =0.8.

[0010] The benzo[a]pyrene degrading bacterium is named Microbacterium sp. W1-2, and is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.35223, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences.

[0011] Step 2: Mix the bacterial suspension, benzo[a]pyrene wastewater, and lignin-grafted graphene oxide quantum dot material at a ratio of 0.5-2L: 5-15L: 500-1500mg, and culture under shaking conditions at 25-35℃, 150-220rpm, and in the dark for 48-96 hours to degrade the benzo[a]pyrene pollutant.

[0012] Preferably, the concentration of benzo[a]pyrene in the benzo[a]pyrene wastewater is 2000-80000 µg / L.

[0013] The present invention has the following advantages:

[0014] (1) Significantly promotes the growth of degrading bacteria: Lignin-grafted graphene oxide quantum dots promote the growth of Microbacterium sp. W1-2 by increasing its OD. 600 The value increased from 0.05 to 0.149, which enhanced the growth ability of the degrading bacteria in the benzo[a]pyrene contaminated environment and solved the limitation of low biomass of single degrading bacteria.

[0015] (2) Significantly improves degradation efficiency and shortens remediation cycle: Under the condition of 5000µg / L benzo[a]pyrene pollution, after adding 100mg / L lignin-grafted graphene oxide quantum dots, the degradation rate of benzo[a]pyrene reached 651.4µg / h within 6h, which is 2.9 times higher than that of using degradation bacteria alone (221.92µg / h); the removal rate of benzo[a]pyrene reached 86.4% within 72h, which is 1.6 times higher than that of using degradation bacteria alone, overcoming the problem of long cycle of traditional bioremediation. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0017] Figure 1 Transmission electron microscopy image of lignin-grafted graphene oxide quantum dot material.

[0018] Figure 2 This is a crystal structure diagram of lignin-grafted graphene oxide quantum dot material.

[0019] Figure 3 The growth curve of Microbacterium sp. W1-2 under lignin-grafted graphene oxide quantum dot material treatment.

[0020] Figure 4 The figure shows the degradation of benzo[a]pyrene by the synergistic system.

[0021] Figure 5 This is a comparison of the degradation efficiency of the synergistic system and the Microbacterium sp. W1-2 strain.

[0022] Figure 6 This is a co-polymer laser microscope image of the synergistic system. Detailed Implementation

[0023] 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.

[0024] Example 1

[0025] Step 1: Take 50 mg of graphene oxide powder (purity ≥95%), add it to 50 mL of deionized water, and sonicate for 30 min (300 W, 40 kHz) to form a uniform brownish-yellow suspension. Add 10 mL of 2 M NaOH solution to the suspension, stir well, and transfer to a 100 mL PTFE-lined stainless steel high-pressure reactor. Place the reactor in an oven and perform a hydrothermal reaction at 180 °C for 12 h. After the reaction, allow it to cool naturally to room temperature. Open the reactor; the solution will exhibit bright fluorescence (observed under UV light). Adjust the pH to 7.0 by adding 1 M HCl solution dropwise, centrifuge at 10,000 rpm for 30 min to remove unreacted large graphene oxide particles. Take the supernatant and place it in a dialysis bag with a molecular weight cutoff of 1 kDa. Dialyze in 2 L of deionized water for 24 h to remove small molecule impurities and salt ions. After dialysis, a purified graphene oxide quantum dot suspension is obtained and stored at 4 °C for later use.

[0026] Step 2: Dissolve 45 mg of alkaline lignin in 30 mL of deionized water and stir magnetically for 10 min until fully dissolved. Add 45 µL of ethylenediamine, adjust the pH to 8.5 with ammonia solution, and continue stirring for 10 min. Add 15 mL of the 1 mg / mL graphene oxide quantum dot dispersion prepared above, and sonicate for 5 min (200 W, 40 kHz). Transfer the mixture to a PTFE-lined stainless steel high-pressure reactor and stir in a 120 °C oil bath for 3 h. After the reaction, allow it to cool naturally to room temperature, pre-freeze at -40 °C for 4 h, and freeze-dry at 10 Pa for 24 h. This yields lignin-grafted graphene oxide quantum dot material. To reduce interference from free small molecules and residual amines in subsequent biological experiments, redisperse the crude product in ultrapure water to a concentration of 1 mg / mL. Dialyze using a dialysis bag with a molecular weight cutoff of 2 kDa for 24 h (changing the water every 6 h), or purify by ultrafiltration and centrifugation. The purified lignin-grafted graphene oxide quantum dot material was stored at 4°C in the dark for later use.

[0027] Step 3: Collect surface soil samples from polycyclic aromatic hydrocarbon (PAH) contaminated soil in a coking plant in Jiangsu Province. Weigh 10g of soil and add it to 90mL of sterile water. Shake at 180rpm for 30 minutes. Take 1mL of the supernatant and inoculate it into a mineral salt medium (1.0g / L (NH4)2SO4, 1.5g / L K2HPO4, 0.5g / L KH2PO4, 0.2g / L MgSO4·7H2O, 0.02g / L CaCl2 and 0.5g / L NaCl) containing 500mg / L benzo[a]pyrene as the sole carbon source. Incubate at 30℃ and 180rpm in the dark for 7 days. Transfer to fresh medium for further enrichment. Repeat 5 times, gradually increasing the benzo[a]pyrene concentration to 1000mg / L. The enrichment solution was diluted and spread onto mineral salt solid plates containing 500 mg / L benzo[a]pyrene. Incubation was carried out at 30°C for 4 days. Single colonies forming clear degradation zones were picked and purified by streaking five times. 49 mL of pre-prepared inorganic salt medium was aseptically mixed with 1 mL of 25 mg / mL benzo[a]pyrene stock solution to prepare 50 mL of benzo[a]pyrene mineral salt liquid medium with a final concentration of 500 mg / L. The purified strain was inoculated into the liquid medium to determine the degradation rate, and strain W1-2 with the highest degradation rate was selected.

[0028] Genomic DNA was extracted from strain W1-2, and the 16S rRNA gene was amplified using universal primers 27F and 1492R. The PCR products were bidirectionally sequenced, and the sequencing results were compared with the NCBI database using BLAST. The similarity to bacteria of the genus *Microbacterium* was ≥99%. The strain was named *Microbacterium sp.* W1-2 and deposited at the China General Microbiological Culture Collection Center (CGMCC) on July 14, 2025, with accession number CGMCC No. 35223. Morphological observation showed that this strain is a Gram-positive short rod-shaped bacterium belonging to the phylum Actinobacteria. It grows stably within the pH range of 5-9, with an optimal pH of 7 and an optimal growth temperature of 30℃.

[0029] Microbacterium sp. W1-2 was streaked onto LB agar plates and incubated at 30°C for 48 hours. Single colonies were picked and inoculated into 50 mL of LB liquid medium, and incubated at 30°C and 180 rpm for 18 hours until the late logarithmic phase. The cells were collected by centrifugation at 5000 rpm for 10 minutes, washed twice with sterile water, and resuspended in sterile water. The OD of the bacterial suspension was adjusted. 600 Reserved for 0.8.

[0030] Step four: Under aseptic conditions, add 5 mL of benzo[a]pyrene stock solution (1 g / L) to 995 mL of prepared mineral salt medium and mix well to obtain a mineral salt medium with a final benzo[a]pyrene concentration of 5000 μg / L. Degradation experiments were conducted using this medium, with three systems established in total:

[0031] A control system was constructed with a total volume of 20 mL, consisting of a mineral salt culture medium containing 5000 μg / L benzo[a]pyrene.

[0032] A single-strain system was constructed by adding activated Microbacterium sp. W1-2 bacterial suspension and mineral salt medium with a benzo[a]pyrene concentration of 5000 μg / L to the system at a volume ratio of 1:9, with a total system volume of 20 mL.

[0033] A degradation system was constructed in which the volume ratio of activated Microbacterium sp. W1-2 bacterial suspension, mineral salt culture medium with a benzo[a]pyrene concentration of 5000 μg / L, and lignin-grafted graphene oxide quantum dot dispersion was 1:8.5:0.5, with a total system volume of 20 mL. The concentration of the lignin-grafted graphene oxide quantum dot dispersion was 100 mg / L.

[0034] The above system was dispensed into conical flasks sealed with vented sealing film and cultured under shaking conditions at 30℃, 180 rpm, and in the dark for a total culture time of 72 h.

[0035] Experimental Example 1

[0036] The morphology of the lignin-grafted graphene oxide quantum dot material prepared in Example 1 was observed using transmission electron microscopy (TEM). The results showed that the lignin-grafted graphene oxide quantum dots were uniform, near-spherical particles with an average particle size of 5.95 nm. Figure 1 The crystal structure was observed using high-resolution transmission electron microscopy (HRTEM). The lattice spacing was 0.25 nm, corresponding to the (002) crystal plane of graphene. Figure 2 ).

[0037] Experimental Example 2

[0038] Samples were taken at 6, 12, 24, 48, and 72 hours during the fourth step of the culture process in Example 1 for bacterial growth determination and benzo[a]pyrene concentration detection.

[0039] The results of bacterial cell proliferation are as follows Figure 3 The results show that the synergistic system can effectively improve the growth of degrading bacteria (OD). 600 This indicates that the synergistic effect between quantum dots and bacterial cells is beneficial to bacterial growth and functional maintenance. Under the above-mentioned dark environment and process parameters, the synergistic system achieved a benzo[a]pyrene degradation rate of 78.2% within 6 hours. Figure 4 The corresponding degradation rate was 651.4 µg / h; the degradation rate reached 86.4% after 72 hours. Figure 5 ).

[0040] To support the synergistic mechanism, this invention further employs laser confocal microscopy to observe the complexes formed during the culture process. Figure 6 The lignin-grafted graphene oxide quantum dots shown can cross the cell membrane and enter the cell interior, forming a stable composite structure with the cell, thereby enhancing interfacial compatibility and material transfer, and promoting the biotransformation of benzo[a]pyrene.

[0041] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for preparing lignin-grafted graphene oxide quantum dot materials, characterized in that, Alkaline lignin, deionized water, EDA, and graphene oxide quantum dot dispersion were mixed at a ratio of 30-60 mg: 20-40 mL: 30-60 μL: 10-20 mL, and the pH was adjusted to 8.0-9.0 with ammonia. The mixture was then sonicated and stirred in an oil bath at 110-130 °C for 2-4 h. After the reaction was completed, the mixture was allowed to cool naturally to room temperature and then freeze-dried under vacuum to obtain lignin-grafted graphene oxide quantum dot material.

2. The method for preparing a lignin-grafted graphene oxide quantum dot material according to claim 1, characterized in that, The concentration of the graphene oxide quantum dot dispersion is 0.5-2.0 mg / mL.

3. The method for preparing a lignin-grafted graphene oxide quantum dot material according to claim 2, characterized in that, The preparation method of the graphene oxide quantum dot dispersion is as follows: graphene oxide powder, NaOH solution and deionized water are mixed at a ratio of 30-80mg:5-15mL:30-80mL and ultrasonicated. The mixture is then subjected to hydrothermal reaction at 160-200℃ for 8-16h. After the reaction is completed, the mixture is neutralized with HCl solution to pH 6.5-7.5, centrifuged, and the supernatant is dialyzed to obtain a graphene oxide quantum dot dispersion of 0.5-2mg / mL.

4. The method for preparing a lignin-grafted graphene oxide quantum dot material according to claim 3, characterized in that, The concentration of the NaOH solution is 2M, and the concentration of the HCl solution is 1M.

5. The method for preparing a lignin-grafted graphene oxide quantum dot material according to claim 1, characterized in that, The lignin-grafted graphene oxide quantum dot material was redispersed in ultrapure water and purified by dialysis or ultrafiltration centrifugation.

6. The lignin-grafted graphene oxide quantum dot material prepared by the method according to any one of claims 1-5.

7. A method for degrading polycyclic aromatic hydrocarbons using the lignin-grafted graphene oxide quantum dot material as described in claim 6, characterized in that, Includes the following steps: Step 1: The benzo[a]pyrene degrading bacteria with accession number CGMCC NO.35223 were used... Microbacterium strain W1-2 was inoculated onto LB agar plates for purification and culture. Single colonies were then picked and inoculated onto LB liquid medium for further culture. The cells were collected by centrifugation, washed with sterile water, and resuspended in sterile water. The OD of the bacterial suspension was adjusted. 600 =0.8; Step 2: Mix the bacterial suspension, benzo[a]pyrene wastewater, and lignin-grafted graphene oxide quantum dot material at a ratio of 0.5-2L: 5-15L: 500-1500mg, and culture under shaking conditions in the dark to degrade the benzo[a]pyrene pollutant.

8. The method according to claim 7, characterized in that, The concentration of benzo[a]pyrene in the wastewater was 2000-8000 µg / L.

Images