A method to enhance the degradation of adsorbed phenanthrene on biochar by sphingosine bacteria

Abstract

This invention discloses a method for enhancing the degradation of adsorbed phenanthrene on biochar by sphingosine-containing bacteria. The method includes the following steps: 1) mixing sphingosine-containing bacteria solution with phenanthrene-adsorbed biochar in a culture medium solution; 2) adding an exogenous iron chelating agent and a ferric salt solution to the mixture, and then incubating under natural conditions to degrade and remove the phenanthrene adsorbed on the biochar. This invention uses an exogenous iron chelating agent and ferric salt to enhance the degradation efficiency of microorganisms on adsorbed phenanthrene on biochar, strengthens the regeneration of biochar adsorption performance through microbial degradation, improves the long-term effectiveness of biochar in soil pollution fixation and control, reduces the risk of secondary pollution, and provides technical support for improving soil environmental quality. It has good economic and environmental benefits in practical applications.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a method for enhancing the degradation of adsorbed phenanthrene on biochar by sphingosine bacteria. Background Technology

[0002] Polycyclic aromatic hydrocarbons (PAHs) are a class of persistent organic pollutants that are widely present in polluted soils. They are highly carcinogenic, mutagenic, and teratogenic, posing a great threat to the ecological environment and human health (Sci.Total.Environ.2013,461,341-347;Environ.Earth Sci.2015,74(3),2743-2748).

[0003] Using biochar to adsorb and fix polycyclic aromatic hydrocarbons (PAHs) in soil is a rapid and effective method for controlling PAH pollution in soil, and it has been widely used in soil organic pollution prevention and remediation projects (Appl. Sci.-Basel 2019, 9(7), 1365; Ecotoxicol. Environ. Saf. 2016, 130, 248-255). However, the PAHs adsorbed by biochar are not completely removed, which not only reduces the adsorption performance of biochar, but also may pose a risk of secondary pollution. Existing research indicates that microbial degradation can be used to remove polycyclic aromatic hydrocarbons (PAHs) adsorbed on biochar in soil in situ (Environ. Sci. ECHNOL. 2012, 46(22), 12445-12451; Geoderma. 2018, 328, 100-108; Environ. Sci.-Nano 2020, 7(9), 2486-2508; Environ. Sci. Technol. 2019, 53(22), 13201-13208). Although PAHs on biochar are difficult to desorb, microorganisms can degrade PAHs adsorbed on biochar by generating hydroxyl radicals through extracellular Fenton-like reactions (Environ. Pollut. 2024, 346, 123583; J. Hazard. Mater. 2022, 439, 129625). However, the microbial degradation method for removing adsorbed polycyclic aromatic hydrocarbons (PAHs) from biochar still suffers from low degradation efficiency, especially for biochar with high ash content, which severely restricts PAH degradation and biochar regeneration. Therefore, it is urgent to develop enhanced methods for microbial degradation of PAHs on biochar to improve the long-term effectiveness of biochar adsorption and reduce the risk of secondary pollution. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide a method for enhancing the degradation of adsorbed phenanthrene on biochar by sphingosine bacteria.

[0005] The technical solution of the present invention is as follows:

[0006] This invention provides a method for enhancing the degradation of adsorbed phenanthrene on biochar by sphingosine bacteria, comprising the following steps:

[0007] 1) Mix the sphingosine bacillus suspension with phenanthrene-adsorbed biochar;

[0008] 2) Add exogenous iron chelating agent and ferric salt solution to the mixture, and culture it under natural conditions to degrade and remove phenanthrene adsorbed on biochar.

[0009] According to an embodiment of the present invention, the biochar is prepared by oxygen-limited pyrolysis of raw materials such as rice straw, wheat straw, corn straw, pine needles, and sawdust at 700°C.

[0010] The sphingosine bacterium of this invention is *Sphingobium yanoikuyae* B1, a heterotrophic, aerobic, Gram-negative bacterium. Its colonies are yellow, round, with regular edges, a smooth and glossy surface, a moist and viscous texture, and a raised center. It can degrade biphenyls, substituted aromatic compounds, and polycyclic aromatic hydrocarbons. Aerobic microbial systems typically contain abundant H2O2. In the *Sphingobium yanoikuyae* B1 system, H2O2 mainly originates from the following two pathways: 1. The microorganism possesses extracellular amino acid oxidases that produce H2O2. The reaction principle is: L-amino acid + O2 + H2O → α-keto acid + NH4+. + +H₂O₂. Sphingobium yanoikuyae B1 is a heterotrophic aerobic bacterium that produces superoxide radicals extracellularly. These superoxide radicals undergo a disproportionation reaction to produce H₂O₂. The reaction principle is: O₂ + e⁻ - (microbial cells)→O2 ·– ;2O2 ·– +2H + →H₂O₂ + O₂. This is caused by the conventional superoxide dismutation reaction.

[0011] The sphingobium yanoikuyae B1 bacterial suspension was obtained as follows: Sphingobium yanoikuyae B1 was cultured in liquid medium for 18–24 h until the mid-to-late logarithmic growth phase. After centrifugation to remove the medium components, the suspension was resuspended in inorganic salt medium (MSM). The bacterial concentration was adjusted using a UV spectrophotometer at a wavelength of 600 nm to achieve the desired OD value. 600 =1.0.

[0012] Preferably, the amount of biochar containing phenanthrene adsorbed added per liter of sphingosine bacillus suspension is 10-100g.

[0013] Preferably, the liquid culture medium has the following composition: 1L of distilled water containing: 10g tryptone, 5g yeast extract, 10g NaCl, pH 6.5-7.5; the culture conditions in the liquid culture medium are: culture at 30℃ and 150rpm for 18-24h.

[0014] According to a preferred embodiment of the present invention, the exogenous iron chelating agent is deferoxamine B (DFOB), with a final concentration of 1.0 mmol / L in the mixed solution. The ferric salt solution is FeCl3 solution, with a final concentration of 1.0 mmol / L in the mixed solution. The culture conditions are natural aerobic conditions at 30°C for 7 days.

[0015] This invention adds an exogenous iron chelating agent to the microbial degradation system of adsorbed phenanthrene on biochar to mimic the function of microbial siderophore secretion, while simultaneously increasing the Fe(III) concentration. This enhances the extracellular Fenton-like reaction of the microorganisms, generating more hydroxyl radicals. The amount of hydroxyl radicals generated determines the degradation efficiency of adsorbed phenanthrene on biochar by the microorganisms. This invention not only enhances the degradation efficiency of adsorbed phenanthrene on biochar by microorganisms but also strengthens the regeneration of biochar adsorption performance through microbial degradation. It can improve the long-term effectiveness of biochar in soil pollution fixation and control, reduce the risk of secondary pollution, and provide technical support for improving soil environmental quality. It has good economic and environmental benefits in practical applications. Detailed Implementation

[0016] The present invention will be further described and illustrated below with reference to specific embodiments. The technical features of each embodiment of the present invention can be combined accordingly, provided that there is no mutual conflict.

[0017] Sphingobium bacteria are excellent bioremediation agents for aromatic hydrocarbon-contaminated environments. The sphingobium bacteria described in this invention is *Sphingobium yanoikuyae* B1. .Literature Yabuuchi,E.;Kosako,Y.;Fujiwara,N.;Naka,T.;Matsunaga,I.;Ogura,H.;Kobayashi,K.,Emendation of the genus SphingomonasYabuuchi et al.1990and junior objective synonymy of the species of threegenera,Sphingobium,Novosphingobium and Sphingopyxis,in conjunction with Blastomas ursincola. Int. Sphingopyxis,on the basis of phylogenetic and The chemotaxonomic analyses.Int.J.Syst.Evol.Microbiol.2001,51,1405-1417. have reported and studied it, with accession numbers: DSM 6900,JCM 30198.

[0018] The *Sphingobium yanoikuyae* B1 bacterial suspensions in each embodiment were obtained as follows: *Sphingobium yanoikuyae* B1 was cultured in liquid medium for 18–24 h until the mid-to-late logarithmic growth phase. After centrifugation to remove the medium components, the suspension was resuspended in inorganic salt medium (MSM). The bacterial concentration was adjusted using a UV spectrophotometer at a wavelength of 600 nm to achieve the desired OD value. 600 =1.0.

[0019] The liquid culture medium consists of the following components: 1L of distilled water containing 10g tryptone, 5g yeast extract, 10g NaCl, and pH 6.5–7.5; the shaking culture conditions are as follows: culture at 30℃ and 150rpm for 18–24h.

[0020] The composition of the inorganic salt culture medium MSM is as follows: 1L of distilled water contains: 2.8g NaH2PO4, 1g KH2PO4, 0.5g (NH4)2SO4, 0.053g MgCl2, 0.05g Ca(NO3)2·4(H2O), 0.0005g EDTA-2Na, 0.00003g H3BO3, 0.00002g CoCl2·6H2O, 0.0002g FeSO4·7H2O, 0.00001g ZnSO4·7H2O, 0.000003g MnCl2·4H2O, 0.000003g Na2MoO4·2H2O, 0.000002g NiCl2·6H2O, and 0.000001g CuCl2·2H2O, with a pH of 6.5–7.5.

[0021] The biochar in each embodiment is prepared by oxygen-limited pyrolysis of the corresponding raw materials (rice straw, wheat straw, corn straw, pine needles, or sawdust) at 700°C. The method for adsorbing phenanthrene is as follows:

[0022] First, phenanthrene was dissolved in methanol to prepare a high-concentration methanol mother liquor. An appropriate amount of the methanol mother liquor was injected into biochar using a microsyringe, and then ultrapure water was added to the system for dilution. After sealing the system, it was placed on a shaker for adsorption experiments.

[0023] Example 1

[0024] 1.98 mL OD 600 A suspension of *Sphingobium yanoikuyae* B1 bacteria with a concentration of 1.0 μL was mixed with 100 mg of rice straw biochar adsorbed with 20 mg / g phenanthrene. 20 μL of a 0.1 mol / L exogenous iron chelating agent DFOB solution and 20 μL of a 0.1 mol / L FeCl3 solution were added. The mixture was cultured under natural aerobic conditions at 30 °C and pH 7 to degrade the phenanthrene adsorbed on the biochar. After 7 days, solid-liquid separation was performed by centrifugation (3500 rpm, 15 min). The concentration of phenanthrene in the supernatant was determined by HPLC. The phenanthrene adsorbed on the biochar was extracted with toluene, and the concentration of phenanthrene in the extract was determined by HPLC. Finally, the degradation rate of adsorbed phenanthrene on the biochar was calculated using mass conservation.

[0025] Blank group: 2.02 mL OD 600 A suspension of Sphingobium yanoikuyae B1 bacteria with a concentration of 1.0 was mixed with 100 mg of rice straw biochar that had adsorbed 20 mg / g phenanthrene.

[0026] Fe(III) addition group: 1.98 mL OD 600A suspension of *Sphingobium yanoikuyae* B1 bacteria with a concentration of 1.0 μL was mixed with 100 mg of rice straw biochar adsorbed with 20 mg / g phenanthrene, and 40 μL of 0.1 mol / L FeCl3 solution was added.

[0027] Add DFOB group: Add 1.98 mL OD 600 A suspension of *Sphingobium yanoikuyae* B1 bacteria with a concentration of 1.0 μL was mixed with 100 mg of rice straw biochar adsorbed with 20 mg / g phenanthrene, and 40 μL of a 0.1 mol / L exogenous iron chelating agent DFOB solution was added.

[0028] The above groups were cultured together under natural aerobic conditions at 30℃ and pH=7 to degrade phenanthrene adsorbed on biochar (the culture medium and conditions were exactly the same as those in the experimental group DFOB+Fe(III)). After 7 days, solid-liquid separation was performed by centrifugation (3500 rpm, 15 min), and the concentration of phenanthrene in the supernatant was determined by HPLC. The phenanthrene adsorbed on the biochar was extracted with toluene, and the concentration of phenanthrene in the extract was determined by HPLC. Finally, the degradation rate of adsorbed phenanthrene on the biochar was calculated by mass conservation.

[0029] Studies have shown that the addition of Fe(III), DFOB, and DFOB+Fe(III) increased the degradation rate of adsorbed phenanthrene on rice straw biochar by microorganisms from 30.3% in the blank to 33.2% (Fe(III)), 40.7% (DFOB), and 72.4% (DFOB+Fe(III)), respectively.

[0030] Example 2

[0031] 1.98 mL OD 600A suspension of *Sphingobium yanoikuyae* B1 bacteria with a concentration of 1.0 μL was mixed with 100 mg of corn straw biochar adsorbed with 20 mg / g phenanthrene. 20 μL of a 0.1 mol / L exogenous iron chelating agent DFOB solution and 20 μL of a 0.1 mol / L FeCl3 solution were added. The mixture was cultured under natural aerobic conditions at 30 °C and pH 7 to degrade the phenanthrene adsorbed on the biochar. After 7 days, solid-liquid separation was performed by centrifugation (3500 rpm, 15 min). The concentration of phenanthrene in the supernatant was determined by HPLC. The phenanthrene adsorbed on the biochar was extracted with toluene, and the concentration of phenanthrene in the extract was determined by HPLC. Finally, the degradation rate of adsorbed phenanthrene on the biochar was calculated using mass conservation. Studies have shown that the addition of DFOB+Fe(III) significantly improved the degradation rate of adsorbed phenanthrene on corn straw biochar compared with the control group, the Fe(III) group, and the DFOB group, with the degradation rate increasing from 43.3% in the control group to 71.2% (DFOB+Fe(III)).

[0032] Example 3

[0033] 1.98 mL OD 600 A suspension of *Sphingobium yanoikuyae* B1 bacteria with a concentration of 1.0 μL was mixed with 100 mg of wheat straw biochar adsorbed with 20 mg / g phenanthrene. 20 μL of a 0.1 mol / L exogenous iron chelating agent DFOB solution and 20 μL of a 0.1 mol / L FeCl3 solution were added. The mixture was cultured under natural aerobic conditions at 30 °C and pH 7 to degrade the phenanthrene adsorbed on the biochar. After 7 days, solid-liquid separation was performed by centrifugation (3500 rpm, 15 min). The concentration of phenanthrene in the supernatant was determined by HPLC. The phenanthrene adsorbed on the biochar was extracted with toluene, and the concentration of phenanthrene in the extract was determined by HPLC. Finally, the degradation rate of adsorbed phenanthrene on the biochar was calculated using mass conservation. Studies have shown that the addition of DFOB+Fe(III) significantly improved the degradation rate of adsorbed phenanthrene on wheat straw biochar compared with the control group, the Fe(III) group, and the DFOB group, with the degradation rate increasing from 42.7% in the control group to 73.5% (DFOB+Fe(III)).

[0034] Example 4

[0035] 1.98 mL OD 600A suspension of *Sphingobium yanoikuyae* B1 with a concentration of 1.0 μL was mixed with 100 mg of sawdust biochar adsorbed with 20 mg / g phenanthrene. 20 μL of a 0.1 mol / L exogenous iron chelating agent DFOB solution and 20 μL of a 0.1 mol / L FeCl3 solution were added. The mixture was cultured under natural aerobic conditions at 30 °C and pH 7 to degrade the phenanthrene adsorbed on the biochar. After 7 days, solid-liquid separation was performed by centrifugation (3500 rpm, 15 min). The concentration of phenanthrene in the supernatant was determined by HPLC. The phenanthrene adsorbed on the biochar was extracted with toluene, and the concentration of phenanthrene in the extract was determined by HPLC. Finally, the degradation rate of adsorbed phenanthrene on the biochar was calculated using mass conservation. Studies have shown that the addition of DFOB+Fe(III) significantly improved the degradation rate of adsorbed phenanthrene on wood biochar compared with the control group, the Fe(III) group, and the DFOB group, with the degradation rate increasing from 55.7% in the control group to 68.5% (DFOB+Fe(III)).

[0036] Example 5

[0037] 1.98 mL OD 600 A suspension of *Sphingobium yanoikuyae* B1 bacteria with a concentration of 1.0 μL was mixed with 100 mg of pine needle biochar adsorbed with 20 mg / g phenanthrene. 20 μL of a 0.1 mol / L exogenous iron chelating agent DFOB solution and 20 μL of a 0.1 mol / L FeCl3 solution were added. The mixture was cultured under natural aerobic conditions at 30 °C and pH 7 to degrade the phenanthrene adsorbed on the biochar. After 7 days, solid-liquid separation was performed by centrifugation (3500 rpm, 15 min). The concentration of phenanthrene in the supernatant was determined by HPLC. The phenanthrene adsorbed on the biochar was extracted with toluene, and the concentration of phenanthrene in the extract was determined by HPLC. Finally, the degradation rate of adsorbed phenanthrene on the biochar was calculated using mass conservation. Studies have shown that the addition of DFOB+Fe(III) significantly improved the degradation rate of adsorbed phenanthrene on pine needle biochar compared with the blank group, the Fe(III) group, and the DFOB group, with the degradation rate increasing from 53.7% in the blank group to 66.3% (DFOB+Fe(III)).

[0038] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the appended claims.

Claims

1. A method for enhancing the degradation of adsorbed phenanthrene on biochar by sphingosine bacteria, characterized in that, Includes the following steps: 1) Mix the sphingosine bacteria suspension with phenanthrene-adsorbed biochar; the biochar is prepared by oxygen-limited pyrolysis of rice straw, wheat straw, corn straw, pine needles, or sawdust at 700℃; the sphingosine bacteria suspension is obtained by the following method: sphingosine bacteria Sphingobium yanoikuyae B1 was cultured at 30°C in liquid medium for 18-24 hours until the logarithmic growth phase. After centrifugation to remove the medium components, the culture was resuspended in MSM (mineral-free medium) and the bacterial concentration was adjusted to achieve the desired OD value. 600 =1.0; 2) Add an exogenous iron chelating agent and a ferric salt solution to the mixture, and culture it under natural conditions at 30°C for 7 days to degrade and remove phenanthrene adsorbed on the biochar; the exogenous iron chelating agent is deferoxamine B (DFOB), and its final concentration in the mixture is 1.0 mmol / L; the ferric salt solution is FeCl3 solution, and the final concentration of FeCl3 in the mixture is 1.0 mmol / L.

2. The method for enhancing the degradation of adsorbed phenanthrene on biochar by sphingosine bacteria according to claim 1, characterized in that, The liquid culture medium is composed of the following: 1 L of distilled water contains: 10 g tryptone, 5 g yeast extract, 10 g NaCl, and pH 6.5–7.

5.

3. The method for enhancing the degradation of adsorbed phenanthrene on biochar by sphingosine bacteria according to claim 1, characterized in that, The inorganic salt culture medium MSM has the following composition: 1 L of distilled water contains: 2.8 g NaH2PO4, 1 g KH2PO4, 0.5 g (NH4)2SO4, 0.053 g MgCl2, 0.05 g Ca(NO3)2·4(H2O), 0.0005 g EDTA-2Na, 0.00003 g H3BO3, 0.00002 g CoCl2·6H2O, 0.0002 g FeSO4·7H2O, 0.00001 g ZnSO4·7H2O, 0.000003 g MnCl2·4H2O, 0.000003 g Na2MoO4·2H2O, 0.000002 g NiCl2·6H2O, and 0.000001 g CuCl2·2H2O, with a pH of 6.5–7.5.