An immobilized bacterial agent for polycyclic aromatic hydrocarbons degradation and a preparation method thereof

Abstract

The present invention discloses an immobilized bacterial agent for polycyclic aromatic hydrocarbon-degrading strains and a preparation method thereof. The average pore size of the brewer's grains biochar prepared by the present invention presents obvious mesoporous characteristics (2 to 50 nm), which is very conducive to the attachment and growth of microorganisms therein and the continuous performance of pollutant degradation efficiency, and also has a good protective effect on the loaded microorganisms. In addition, the brewer's grains biochar prepared by the present invention has a promoting effect on the growth number of functional bacteria and the intracellular dioxygenase activity, further characterizing the synergistic protective effect of the brewer's grains biochar immobilization technology on the functional bacteria, and improving the resistance to adverse external environments.

Description

Technical Field

[0001] The invention relates to an immobilized bacterial agent for polycyclic aromatic hydrocarbons degradation and a preparation method thereof, belonging to the field of environmental technology. Background Art

[0002] Microbial immobilization technology typically uses physical or chemical methods to fix dispersed, free-living functional microbial cells within a defined spatial area. This approach, while reducing the competitive and toxic effects of indigenous microorganisms and other bacterial species, maintains the biomass, cell density, and metabolic activity of functional microorganisms for a certain period of time. Microbial immobilization methods primarily include adsorption, embedding, and cross-linking. Adsorption, however, has minimal impact on cell viability and is simple to operate, making it widely used for enhanced removal of recalcitrant organic matter.

[0003] According to existing literature and patent reports, various biochar-based and modified porous materials have different degrees of microbial adsorption properties, and different immobilization carriers have been developed. Currently, microbial biochar immobilization carriers are mainly corn straw, rice husks, fir, sugarcane bagasse, peanut shells, and bamboo. Existing patents (such as Chinese patent CN109628353B and Chinese patent CN104946620A) mainly focus on the supply of nutrients in the inoculum, the cross-linking adsorption effect of the bacteria to enhance the maintenance of strain activity and biomass attachment, or the combined effect of plant root secretions to promote the adsorption of degrading bacteria on biochar-based materials (Chinese patent CN112387778A). Chinese patent (CN111088187B) obtained the immobilization method and application conditions of cotton stalk and rice husk biochar for specific PAHs-degrading bacteria by optimizing solid culture medium and immobilization conditions. The Chinese patent (CN113293157A) is mainly based on the Fe3O4 modification of cotton stalk biochar to increase the specific surface area and Zeta potential, and enhance the removal efficiency of phenanthrene by increasing the bacterial load of biochar.

[0004] The pore structure and surface properties of biochar materials, as well as their matching with the characteristics of functional bacteria, are key factors influencing the biomass of live cells immobilized on the carrier and its pollution remediation efficiency. Brewer's grains are a byproduct of the beer brewing process, primarily containing cellulose, lignin, starch, and other substances. my country produces approximately 3 million tons of brewer's grains annually, with a moisture content of approximately 80%. Currently, they are primarily used as livestock feed and for cultivating edible fungi. Due to their high moisture content and acidic components, brewer's grains pose a significant risk of environmental pollution if not promptly processed or utilized. Optimizing the preparation of microbial immobilization carriers using brewer's grain biochar can provide new insights into the resource utilization of brewer's grains, achieving the goals of waste utilization and a circular economy.

[0005] Polycyclic aromatic hydrocarbons (PAHs) are a typical class of persistent organic pollutants with teratogenic, carcinogenic, and mutagenic effects, posing a significant potential hazard to ecological and environmental health. They have been listed as priority persistent pollutants by countries such as China and the United States, and are widely present in environmental media such as soil, sediments, and water bodies. Chinese Patent No. CN111088246A discloses an application technology for the enhanced removal of naphthalene from soil by using microcrystalline cellulose to prepare biochar-immobilized microorganisms. The main steps include the preparation of a mixed solution of biochar and degrading bacteria, and the preparation of an immobilized naphthalene-degrading bacterial agent by a cross-linking reaction of sodium alginate and calcium chloride. Existing reported results mostly focus on the removal effect of different biochar-adsorbed immobilized microorganisms on the target pollutant cycle. There is still limited research on the increase in the biomass load of biochar-based materials, the continuous and efficient degradation of pollutants, the coupling of structure-activity characteristics, and the synergistic protection of immobilized microorganisms. Summary of the Invention

[0006] To solve the above technical problems, the present invention provides an immobilized bacterial agent for polycyclic aromatic hydrocarbons-degrading strains and a preparation method thereof, and utilizes brewer's grains waste as a resource. Based on the geometric size of the functional bacteria and the physical and chemical characteristics of the strains, a high-efficiency adsorption immobilized biochar material is proposed through the optimization of preparation conditions, as well as the effect of its immobilization on the physiological characteristics of the functional bacteria. The biochar material is then applied to the pollution remediation of three low- and high-ring PAHs compounds: phenanthrene, fluoranthene, and pyrene.

[0007] The first object of the present invention is to provide an immobilized bacterial agent for polycyclic aromatic hydrocarbons-degrading strains, wherein the immobilized bacterial agent uses brewer's grains biochar as a carrier to load the polycyclic aromatic hydrocarbons-degrading strains.

[0008] Furthermore, the polycyclic aromatic hydrocarbons-degrading strain is Arthrobacter nicotinovoransr, which was deposited in the China Center for Type Culture Collection on March 7, 2022, with the deposit address being Wuhan University, Wuhan, China, and the deposit number being CCTCC NO: M2022207.

[0009] Furthermore, the brewer's grains biochar is prepared by the following method: drying the brewer's grains and then crushing them to obtain brewer's grains powder, pyrolyzing the brewer's grains powder at 450-550° C. for 1-3 hours, cooling and then crushing them to obtain the brewer's grains biochar.

[0010] The second object of the present invention is to provide a method for preparing the immobilized bacterial agent of the polycyclic aromatic hydrocarbon-degrading strain, comprising the following steps:

[0011] S1. Drying and crushing brewer's grains to obtain brewer's grain powder, pyrolyzing the brewer's grain powder at 450-550° C. for 1-3 hours, cooling, and then crushing to obtain the brewer's grain biochar;

[0012] S2. Inoculate the polycyclic aromatic hydrocarbons-degrading bacterial strain suspension into a mixture of brewer's grains biochar and liquid culture medium, cultivate for 12 to 24 hours, and obtain the immobilized bacterial agent after filtering and washing.

[0013] Furthermore, in step S1, the drying is performed at 80-120° C. for 10-30 hours.

[0014] Furthermore, in step S1, the brewer's grains powder is heated to 450-550° C. at a rate of 4-6° C. / min.

[0015] Furthermore, in step S1, the brewer's grain powder is passed through a 50-70 mesh sieve, and the brewer's grain biochar is passed through a 20-40 mesh sieve.

[0016] Furthermore, in step S2, the bacterial suspension of the polycyclic aromatic hydrocarbon-degrading strain is prepared by the following method: a single colony of the polycyclic aromatic hydrocarbon-degrading strain is expanded and cultured in liquid culture medium for 12 to 24 hours, the supernatant is removed by centrifugation, and the suspension is washed and resuspended in physiological saline to obtain the bacterial suspension.

[0017] Furthermore, the polycyclic aromatic hydrocarbons degradation strain suspension OD 600 The value is 1 to 1.5.

[0018] Furthermore, the liquid culture medium is LB culture medium, comprising the following components: yeast extract 4-6 g / L, peptone 8-12 g / L, and NaCl 8-12 g / L.

[0019] The third object of the present invention is to provide the use of the immobilized bacterial agent of the polycyclic aromatic hydrocarbons-degrading strain in the degradation of polycyclic aromatic hydrocarbons.

[0020] Furthermore, the polycyclic aromatic hydrocarbons are one or more of phenanthrene, fluoranthene, pyrene or their derivatives.

[0021] The beneficial effects of the present invention are:

[0022] The average pore size of the brewer's grains biochar prepared by the present invention presents obvious mesoporous characteristics (2 to 50 nm), which is very conducive to the attachment and growth of microorganisms inside it and the continuous performance of pollutant degradation efficiency, and also has a good protective effect on the loaded microorganisms; and the brewer's grains biochar prepared by the present invention has a promoting effect on the growth number of functional bacteria and the activity of intracellular dioxygenases, further characterizing the synergistic protective effect of the brewer's grains biochar immobilization technology on the functional bacteria, and improving the resistance to adverse external environments.

[0023] Biodeposit Information:

[0024] Arthrobacter nicotinovoransr was deposited in the China Center for Type Culture Collection on March 7, 2022, address: Wuhan University, Wuhan, China; Postal Code 430072, with the accession number: CCTCC NO: M2022207. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Adsorption changes of biochar on Arthrobacter nicotinamiphilus CCTCC NO:M2022207 at different pyrolysis temperatures;

[0026] Figure 2 Microstructure of biochar and immobilized functional bacteria at different pyrolysis temperatures (×10000);

[0027] Figure 3 The removal performance of PAHs in water by free bacteria, biochar and immobilized functional bacteria;

[0028] Figure 4 Changes in the number of bacteria in the free bacteria and immobilized functional bacteria reaction systems;

[0029] Figure 5 Changes in dioxygenase activity in free bacteria and immobilized functional bacteria reaction systems;

[0030] Figure 6 The removal performance of PAHs in sediments by free bacteria, biochar and immobilized functional bacteria;

[0031] Figure 7 Removal performance of PAHs in water by different biochar-immobilized functional bacteria. DETAILED DESCRIPTION

[0032] The present invention will be further described below with reference to specific examples so that those skilled in the art can better understand the present invention and implement it, but the examples are not intended to limit the present invention.

[0033] The contents in the examples without specific conditions were carried out under conventional conditions; the reagents or instruments used without indicating the manufacturer were all common commercial products.

[0034] Example 1: Preparation of brewer's grains biochar

[0035] Brewer's grains were dried at 100°C for 24 hours, ground through a 60-mesh sieve, and stored dry. 20g of brewer's grain powder was placed in a quartz boat and heated to 400°C, 500°C, 600°C, and 700°C in a tube furnace at a rate of 5°C / min. Pyrolysis was performed under nitrogen for 2 hours, cooled to room temperature, ground through a 30-mesh sieve, and stored dry. These were BL400 biochar, BL500 biochar, BL600 biochar, and BL700 biochar, respectively.

[0036] Example 2: Adsorption and immobilization of Arthrobacter nicotinicola CCTCC NO: M2022207 by brewer's grains biochar

[0037] 0.5 g of brewer's grain biochar with different pyrolysis temperatures was weighed into a 100 mL conical flask, and 20 mL (OD 600 =0.5) bacterial suspension. Batch adsorption experiments were carried out in a constant temperature oscillator at 30°C and 160r / min, and the number of functional bacteria adsorbed and fixed on the brewer's grains biochar carrier was determined at 2, 6, 12, 24h and 48h. The biochar was separated from the bacterial suspension and transferred to a 100mL conical flask. 38mL of extraction solution (chloroform: methanol: distilled water = 1:2:0.8) was added, and the mixture was shaken for 10min and allowed to stand for 12h. 10mL of chloroform and distilled water were added, and after standing for 12h, 10mL of the lower chloroform phase was taken and transferred to a 25mL colorimetric tube, evaporated to dryness in a water bath, and the lipid phosphorus of the biofilm was digested by potassium sulfate and the phosphorus content was determined. The amount of adsorbed bacteria was expressed as nmol P·g -1 The result is as follows. Figure 1 The results showed that the adsorption capacity of Arthrobacter nicotinamiphilus CCTCC NO:M2022207 by brewer's grains biochar at different pyrolysis temperatures was as follows: BL500>BL600>BL700>BL400, among which BL500 had the highest adsorption capacity of 825.71 nmol P·g -1 (approximately 3.78×10 10 cfu / g), and the adsorption capacity reached saturation after 24 h.

[0038] Figure 2 The SEM images of brewer's grain biochar and immobilized functional bacteria show that during the pyrolysis process of brewer's grain, pore structures of different specifications are formed on the surface. 2 / g, pore size 23.928nm, specific capacitance 10.313F·g -1 ) The surface of the lower channel is clean and free of impurities, making it more suitable as a fixed carrier. 400℃ (specific surface area 2.585m 2 / g, pore size 3.27nm, specific capacitance 8.402F·g -1 ) The cellulose and lignin in the biochar formed at 600℃ (specific surface area 1.002m 2 / g, pore size 20.754nm, specific capacitance 5.699F·g -1 ) and 700℃ (specific surface area 0.408m 2 / g, pore size 15.859nm, specific capacitance 7.441F·g -1) caused some pores to collapse, hindering the attachment and growth of microorganisms within the biochar. SEM images of BL500 immobilization revealed that some Arthrobacter nicotinicola CCTCCNO:M2022207 bacteria were firmly attached to the pores, demonstrating that brewer's grains biochar has a protective effect on functional bacteria.

[0039] Example 3: Immobilized functional bacteria in brewer's grains and their removal properties of PAHs in water

[0040] Weigh 1 g of BL500 biochar in Example 1, add it to 100 mL of LB liquid culture medium, sterilize it at 121°C for 20 min, and cool it to room temperature for later use. Inoculate the bacterial suspension (OD 600 =1.0), and incubated at 30°C and 160 rpm for 24 h. Filtered with an 80-mesh sieve and rinsed 2-3 times with physiological saline to obtain BL500 immobilized functional bacteria. Its microstructure is as follows Figure 2 shown.

[0041] A 30 mL inorganic salt culture medium containing 30 mg / L each of phenanthrene, fluoranthene, and pyrene was prepared. 0.05 g of BL500 biochar, 0.05 g of immobilized functional bacteria, and an equal amount of free bacteria were added, with three replicates set up. After degradation for 40 days in a shaking incubator at 30°C and 160 rpm, an equal volume of anhydrous ethanol (chromatographic grade) was added for solubilization and extraction. After filtration, the residual PAH content was determined by high-performance liquid chromatography. The inorganic salt culture medium consisted of: 5 g / L NaCl, 0.25 g / L MgSO₄·7H₂O, 1 g / L (NH₄)₂SO₄, 2 g / L NaNO₃, 10 g / L K₂HPO₄·3H₂O, and 4 g / L KH₂PO₄. The pH was adjusted to 7 and sterilized at 121°C for 20 min.

[0042] The results are as follows Figure 3 The results showed that the removal rates of phenanthrene, fluoranthene and pyrene by immobilized functional bacteria can reach 71%, 38% and 35%, respectively, which are 13% to 34% higher than the removal rates of PAHs compounds in other groups, showing significant differences (P<0.05). At 15 days, the adsorption of PAHs by the biochar group gradually reached saturation, while the PAHs removal rate of the immobilized group showed a steady upward trend from 15 to 30 days. At this time, the activity of functional bacteria was enhanced, and the degradation of microorganisms played a dominant role. Among the three PAHs, phenanthrene is a low-ring PAHs, while fluoranthene and pyrene are high-ring PAHs. Figure 3 It can be seen that immobilization has a stronger removal effect on low-ring PAHs, which is more than 20% higher than the removal rate of high-ring PAHs, indicating that immobilization has a more significant effect on the adsorption-degradation of low-ring PAHs.

[0043] Example 4: Effects of immobilization of brewer's grains biochar on the physiological characteristics of functional bacteria

[0044] In the system of Example 3, samples were taken on the 5th, 10th, 15th, 20th, 30th and 40th days of the reaction cycle, and the changes in the number of active microorganisms in the system were analyzed by lipid phosphorus determination; the trend of dioxygenase activity in the system was determined by catechol colorimetry. The results of the changes in the number of functional bacteria are shown in Figure 3. Figure 4 As shown in Figure 2, after 15 days, the number of bacteria in the immobilized functional bacteria system gradually exceeded that in the free bacteria system. Until the end of the reaction cycle, the number of bacteria in the immobilized functional bacteria system was 1238.81 nmol P·g -1 The number of bacteria in the free bacterial reaction system was 1004.28 nmol P·g -1 The experiment showed that using brewer's grains biochar as a fixed carrier can effectively weaken the adverse effects of environmental factors on functional bacteria and increase the number of active functional bacteria. Figure 5 As shown, the dioxygenase activity in both reaction systems showed an initial increase followed by a decrease over time. Between days 10 and 15, the overall enzyme activity of the immobilized functional bacteria gradually surpassed that of the free bacteria reaction group. The maximum increase in 2,3-dioxygenase activity due to immobilization was 261.8 U / mL, 1.7 times that of the free bacteria; the maximum increase in 1,2-dioxygenase activity was 117.23 U / mL. This indicates that brewer's grains biochar immobilization technology can protect functional bacteria by providing loading sites, thereby enhancing their enzyme activity and promoting the degradation of polycyclic aromatic hydrocarbons. The trends in the number of functional bacteria and dioxygenase activity were consistent with the PAH removal trends over the reaction period. By day 15, both the number and dioxygenase activity of the immobilized functional bacteria exceeded those of the free bacteria, indicating a gradual increase in microbial degradation. Simultaneously, the PAH removal efficiency of the immobilized group also gradually increased, exceeding the adsorption efficiency of the biochar group.

[0045] Example 5: Removal characteristics of PAHs in sediments by functional bacteria immobilized on brewer's grains biochar

[0046] 1g of the BL500 immobilized functional bacteria prepared in Example 3 was weighed and evenly mixed with 100g of a PAH-contaminated sediment sample (1% w / w). The mixture was placed in a 100ml beaker. The concentrations of phenanthrene, fluoranthene, and pyrene in the sediment sample were 14.1, 15.34, and 14.97 mg / kg, respectively. A blank control group, a BL500 biochar experimental group, and a free bacteria experimental group were also set up. Aerated tap water was added, and after settling for 15 minutes, the mixture was placed in a 50L water tank filled with tap water. Water was replenished every two weeks, with three control groups set up for each experimental group. The reaction was run for 50 days, with samples measured at 10, 20, 30, 40, and 50 days. The immobilized functional bacteria experimental group achieved 64%, 43%, and 38% removal rates of phenanthrene, fluoranthene, and pyrene in the sediment, respectively. Compared with the free bacteria group, it increased by 14-28%, indicating that in the sediment water-solid interface environment, brewer's grains biochar immobilization still has a high promoting effect on the degradation of PAHs by functional bacteria.

[0047] Comparative Example 1: Removal characteristics of PAHs in water by different biochar-immobilized functional bacteria

[0048] Rice husk biochar and straw biochar were prepared according to the method in Example 1 (heated to 500°C). Rice husk-immobilized functional bacteria and straw-immobilized functional bacteria were then prepared according to the method in Example 3. A 30 mL inorganic salt culture medium containing 30 mg / L each of phenanthrene, fluoranthene, and pyrene was prepared, and 0.05 g of rice husk biochar-immobilized functional bacteria, 0.05 g of straw biochar-immobilized functional bacteria, and 0.05 g of brewer's grains biochar-immobilized functional bacteria were added, respectively. Three replicates were set up. After degradation in an oscillating incubator at 30°C and 160 rpm for 40 days, an equal volume of anhydrous ethanol (chromatographic grade) was added for solubilization and extraction. After filtration, the residual PAH content was determined by high-performance liquid chromatography. Inorganic salt culture medium components include: NaCl 5g / L, MgSO4·7H2O 0.25g / L, (NH4)2SO4 1g / L, NaNO3 2g / L, K2HPO4·3H2O 10g / L, KH2PO4 4g / L, pH adjustment to 7, sterilization at 121℃ for 20min.

[0049] The results are as follows Figure 7 As shown, the removal efficiencies of phenanthrene, fluoranthene, and pyrene in the rice husk biochar immobilization group were 55%, 22%, and 19%, respectively, while those in the straw biochar immobilization group were 61%, 13%, and 8%, respectively, indicating similar removal rates. The PAHs removal efficiency of the brewer's grains biochar immobilization group increased by 16-25% compared to the rice husk and straw biochars, respectively, with the increases for fluoranthene and pyrene being more significant. This suggests that brewer's grains biochar is a more suitable immobilization and remediation material for Arthrobacter nicotinicus CCTCC NO: M2022207.

[0050] The above embodiments are merely preferred embodiments for the purpose of fully illustrating the present invention, and the scope of protection of the present invention is not limited thereto. Equivalent substitutions or modifications made by those skilled in the art based on the present invention are within the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

Claims

1. An immobilized bacterial agent for polycyclic aromatic hydrocarbons-degrading strains, characterized in that: The immobilized bacterial agent uses brewer's grains biochar as a carrier and carries a polycyclic aromatic hydrocarbons-degrading strain, wherein the polycyclic aromatic hydrocarbons-degrading strain is Arthrobacter nicotinamiphilus ( Arthrobacter nicotinovorans r ), the deposit number is CCTCC NO: M2022207.

2. The immobilized bacterial agent according to claim 1, characterized in that The brewer's grains biochar is prepared by the following method: drying the brewer's grains and then crushing them to obtain brewer's grains powder; pyrolyzing the brewer's grains powder at 450-550° C. for 1-3 hours; cooling and then crushing them to obtain the brewer's grains biochar.

3. A method for preparing an immobilized bacterial agent for polycyclic aromatic hydrocarbon-degrading strains according to claim 1 or 2, characterized in that: The steps include: S1. Drying and crushing brewer's grains to obtain brewer's grain powder, pyrolyzing the brewer's grain powder at 450-550° C. for 1-3 hours, cooling, and then crushing to obtain the brewer's grain biochar; S2. Inoculate the polycyclic aromatic hydrocarbons-degrading bacterial strain suspension into a mixture of brewer's grains biochar and liquid culture medium, cultivate for 12 to 24 hours, and obtain the immobilized bacterial agent after filtering and washing.

4. The preparation method according to claim 3, characterized in that In step S1, the brewer's grains powder is heated to 450-550°C at a rate of 4-6°C / min.

5. The preparation method according to claim 3, characterized in that In step S1, the brewer's grain powder is passed through a 50-70 mesh sieve, and the brewer's grain biochar is passed through a 20-40 mesh sieve.

6. The preparation method according to claim 3, characterized in that In step S2, the PAH-degrading strain suspension is prepared by the following method: a single colony of the PAH-degrading strain is cultured in a liquid medium for 12 to 24 hours, the supernatant is removed by centrifugation, and the suspension is washed and resuspended in physiological saline to obtain the bacterial suspension. The OD value of the bacterial suspension is 0.

05. 600 The value is 1~1.

5.

7. The preparation method according to claim 3, characterized in that The liquid culture medium is LB culture medium, which includes the following components: yeast extract 4-6 g / L, peptone 8-12 g / L, and NaCl 8-12 g / L.

8. Use of the immobilized bacterial agent of the polycyclic aromatic hydrocarbon-degrading strain according to claim 1 or 2 in the degradation of polycyclic aromatic hydrocarbons.

9. The use according to claim 8, characterized in that The polycyclic aromatic hydrocarbons are one or more of phenanthrene, fluoranthene and pyrene.

Images