Microbacterium sp. and application thereof

Abstract

The application discloses a microbacterium and application thereof, and the microbacterium is Microbacterium sp. NJS-5, which is preserved in the China General Microbiological Culture Collection Center on September 28, 2025, and has a preservation number of CGMCC No. 36103, and a gene sequence of 16S rRNA of the microbacterium is shown as SEQ ID No. 1. Microbacterium sp. The microbacterium provided by the application has high efficient nitrobenzene degradation capacity, can be used for bioremediation of nitrobenzene contaminated soil and underground water, and has the advantages of high efficiency, greenness and no pollution.

Description

Technical Field

[0001] This invention belongs to the field of environmental microbial engineering and bioremediation technology, and in particular relates to a strain of Microbacterium sp. NJS-5 and its application in the remediation of nitrobenzene-contaminated soil and groundwater. Background Technology

[0002] Nitrobenzene (NB) is a highly representative recalcitrant organic pollutant, widely used in industries such as explosives, pharmaceuticals, pesticides, dyes, and rubber synthesis. Due to its high water solubility, chemical stability, and persistence in the environment, NB can cause severe pollution to soil and water bodies if it enters the environment through transportation leaks, industrial emissions, or improper disposal. NB has been listed as a priority pollutant by the U.S. Environmental Protection Agency (USEPA) and China's environmental authorities, and is classified as a Group 2B carcinogen by the International Agency for Research on Cancer (IARC). Its toxicity to ecosystems is mainly manifested in its teratogenic and mutagenic effects on microorganisms, plants, and aquatic organisms, and it readily accumulates through the food chain, threatening human health.

[0003] Traditional nitrobenzene treatment methods mainly include physical adsorption (such as activated carbon adsorption) and chemical oxidation (such as Fenton oxidation and ozone oxidation). However, physical methods only achieve pollutant transfer rather than mineralization, while chemical methods have drawbacks such as high operating costs, large reagent consumption, and the potential for secondary pollution (e.g., intermediate products with higher toxicity). In contrast, bioremediation technology utilizes the metabolic capabilities of microorganisms to convert nitrobenzene into harmless substances (such as carbon dioxide, water, and ammonia), offering significant advantages such as low cost, environmental friendliness, and no secondary pollution.

[0004] Although existing literature reports the ability of bacteria such as Streptomyces, Bacillus, and Pseudomonas to degrade nitrobenzene, there are few reports on the application of Microbacterium sp. in nitrobenzene degradation. Furthermore, existing strains often suffer from low concentration tolerance and poor environmental adaptability, making them unsuitable for the actual remediation needs of sites with high concentrations of contamination. Summary of the Invention

[0005] Therefore, embodiments of the present invention provide a strain of microbacterium and its application.

[0006] In a first aspect, embodiments of the present invention provide a strain of microbacterium, namely Microbacterium sp. NJS-5, which was registered and deposited at the China General Microbiological Culture Collection Center on September 28, 2025, with accession number CGMCC No. 36103. The gene sequence of the 16S rRNA of the microbacterium is shown in SEQ ID No. 1.

[0007] Secondly, embodiments of the present invention provide an application of the aforementioned microbacteria in the degradation of nitrobenzene.

[0008] Furthermore, the application process includes:

[0009] Microbacterium NJS-5 was activated in sterilized seed culture medium and cultured in a constant temperature shaking incubator at 30℃-40℃.

[0010] Inoculate the seed culture solution into the liquid culture medium at an inoculation rate of 0.5%-3%, and place it in a constant temperature shaking incubator at 30℃-40℃ for anaerobic culture for more than 3 days;

[0011] Take soil or groundwater contaminated with nitrobenzene and add it to the liquid culture medium. Place the mixed mud-water solution in a constant temperature shaking incubator at 30℃-40℃ and incubate for more than 5 days.

[0012] Furthermore, the seed culture medium consists of: 10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, and pH 7.0.

[0013] Furthermore, the liquid culture medium has the following composition: NaNO3 2-8 g / L, KH2PO4 2-8 g / L, MgSO4 0.1-1 g / L, NaCl 0.5-3 g / L, KCl 0.5-3 g / L, CaCl2 0.1-1 g / L, H3PO4 2-10 mL / L, pH=6.0-8.0.

[0014] Preferably, the concentrations are: NaNO3 3-5 g / L, KH2PO4 3-5 g / L, MgSO4 0.1-0.5 g / L, NaCl 0.5-1.5 g / L, KCl 0.5-1.5 g / L, CaCl2 0.2-0.5 g / L, H3PO4 3-8 mL / L, and pH = 6.5-7.5.

[0015] Preferably, the concentrations are: NaNO3 4 g / L, KH2PO4 4 g / L, MgSO4 0.2 g / L, NaCl 1 g / L, KCl 1 g / L, CaCl2 0.4 g / L, H3PO4 5 mL / L, and pH=7.0.

[0016] Furthermore, the mass-to-volume ratio of nitrobenzene-contaminated soil to liquid culture medium was 1:1 to 1:4, and the content of benzene series compounds in the contaminated soil was 10 to 500 mg / kg;

[0017] Furthermore, the volume ratio of groundwater contaminated with nitrobenzene to liquid culture medium was 1:1 to 1:4, and the content of benzene series compounds in the contaminated groundwater was 10 to 500 mg / L.

[0018] Thirdly, embodiments of the present invention provide a microbial agent containing the aforementioned microbacteria.

[0019] Fourthly, embodiments of the present invention provide a method for preparing a bacterial agent, the method comprising: fermenting and culturing the above-mentioned bacilli.

[0020] Fifthly, embodiments of the present invention provide a method for remediating nitrobenzene-contaminated soil and / or groundwater, the method comprising adding the aforementioned microbacteria or the aforementioned bacterial agent to the nitrobenzene-contaminated soil and / or groundwater to be remediated.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0022] (1) This invention has for the first time screened and identified a microbacterium sp. NJS-5 that can efficiently degrade nitrobenzene. Currently, most reports on the biodegradation of nitrobenzene focus on Pseudomonas or Bacillus. The discovery of this invention broadens the microbial germplasm resource bank for nitrobenzene degradation and provides a new choice of microbial materials for the remediation of nitrobenzene pollution.

[0023] (2) The Microbacterium sp. NJS-5 strain obtained by screening in this invention has extremely strong environmental tolerance and can survive and maintain degradation activity in environments with nitrobenzene concentrations as high as 500 mg / L. Experimental data show that Microbacterium NJS-5 can effectively function within a broad concentration range of 10-500 mg / L, overcoming the shortcomings of traditional functional strains that are easily inactivated under high concentrations of toxic pollutants, and is particularly suitable for the remediation of heavily contaminated sites.

[0024] (3) The Microbacterium sp. NJS-5 strain provided by this invention has high degradation efficiency and significant remediation effect: This strain has a strong mineralization ability for nitrobenzene, with a degradation rate as high as 93.7% at low concentrations (10 mg / L). In simulated real-world application scenarios, whether targeting highly adsorbed soil slurry systems (degradation rate >80%) or highly fluid groundwater systems (degradation rate >84%), this strain exhibits excellent removal effects, demonstrating its good environmental adaptability and potential for practical engineering applications.

[0025] (4) This invention utilizes microbial metabolism to convert nitrobenzene into harmless substances, and can achieve efficient remediation through optimized culture conditions (pH 7.0, conventional temperature 30-40℃). Compared with traditional methods such as physical adsorption or chemical oxidation, this method has the advantages of low cost, simple operation, no secondary pollution, and good environmental compatibility, and is an environmentally friendly green bioremediation technology. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0027] Figure 1 Microscopic images of the microbacterium NJS-5 provided in the embodiments of the present invention. Detailed Implementation

[0028] 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.

[0029] It should be noted that, unless otherwise specified, the features in the following embodiments and implementation methods can be combined with each other.

[0030] Example 1: Isolation, screening and identification of microbacterium NJS-5

[0031] 1. Enrichment of bacterial strains

[0032] 2 g of soil sample taken from soil heavily contaminated with polycyclic aromatic hydrocarbons was placed in a shake flask containing 60 mL of sterile distilled water and kept at 37°C. oAfter culturing on a constant temperature shaker for 15 minutes, 2 mL of the supernatant was placed in 60 mL of LB liquid medium for enrichment culture for 12 hours to obtain strains that can survive in soil with high concentrations of polycyclic aromatic hydrocarbons.

[0033] 2. Screening of strains using nitrobenzene as the sole carbon source.

[0034] Take 2 mL of the bacterial enrichment solution and add it to 60 mL of liquid culture medium containing 200 mg / L nitrobenzene, then incubate at 37°C. o The culture was carried out in a constant temperature shaking incubator at C. After more than 3 days of culture, the strain NJS-5, which can degrade nitrobenzene, was isolated by dilution plating method.

[0035] After re-inoculating Microbes NJS-5 into a liquid culture medium with nitrobenzene as the sole carbon source, and culturing for 72 hours, the pH and bacterial concentration of the liquid were measured. A significant change in pH was observed, and the bacterial concentration decreased from OD... 600 =0.35 increased to OD 600 =0.67, which proves that the strain can take up and utilize nitrobenzene, and therefore has the ability to degrade nitrobenzene.

[0036] The liquid culture medium consisted of: NaNO3 5 g / L, KH2PO4 4 g / L, MgSO4 0.1 g / L, NaCl 1 g / L, KCl 1 g / L, CaCl2 0.4 g / L, H3PO4 5 mL / L, and pH=7.0.

[0037] 3. Taxonomic identification of strains

[0038] Microscopic images of microbacterium NJS-5 are shown below. Figure 1 As shown, the bacteria appear deep purple or bluish-purple, indicating that they are Gram-positive. The bacteria are mainly rod-shaped or microrod-shaped, small in size, and have regular edges. They are mostly scattered in the field of view, but some are also observed to aggregate into irregular small clusters or clumps. No obvious long chains are observed, and there are no flagella or spore structures, consistent with the typical morphological characteristics of the genus *Microbacterium*.

[0039] The screened degrading strain was identified using molecular biology methods. Its 16S rRNA sequence, shown in SEQ ID No. 1, was compared with the GenBank nucleic acid database. Sequence alignment confirmed it as *Microbacterium*, named *Microbacterium* NJS-5, and it was deposited at the China General Microbiological Culture Collection Center (CGMCC) on September 28, 2025, with accession number CGMCC No. 36103. The deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0040] Example 2: Selection of the optimal liquid culture medium for the growth of Microbacterium NJS-5

[0041] The isolated microbes NJS-5 were inoculated into a liquid culture medium with glucose as the carbon source (5 g / L) and a pH of 7.0. The inorganic salt content of the liquid culture medium is shown in Table 1. The medium was incubated at 37°C with shaking for 2 days, and then the growth of the strain was measured. The culture medium with good strain growth was considered the optimal medium. A blank control without the strain was also included to reduce the impact of volatilization.

[0042] Table 1: Growth of NJS-5 under different culture medium conditions

[0043]

[0044] Specifically, as shown in Table 1 above, from the first group to the fifth group, the growth rate (OD) of the strains increased with the gradual increase of the concentration of inorganic salt components. 600 The values ​​(values) showed a typical trend of first increasing and then decreasing. The first to third groups were the rising period, and the growth of the strain accelerated significantly with the increase of nutrient concentration, indicating that inorganic salts were the limiting factor for growth in the low concentration range; the third group reached the highest growth point; the fourth to fifth groups were the inhibition period, and the growth decreased instead of increasing with the concentration, indicating that excessively high inorganic salt concentrations inhibited the growth of the strain.

[0045] The first group has the lowest inorganic salt content (e.g., NaNO3 is only 2 g / L, KH2PO4 is only 2 g / L). OD 600 The low value (2.0±0.7) indicates that the essential nutrients such as nitrogen, phosphorus and metal ions in the culture medium are insufficient, which become the limiting factor for the growth of the strain, resulting in the inability to accumulate biomass effectively.

[0046] Compared to the first group, the inorganic salt concentration in the second group was moderately increased, and the nutrient limitation was broken. The growth rate of the strain was improved (OD). 600The value reached 2.7±0.1, indicating that the nutrient supply within this concentration range began to better meet the needs of bacterial division, but it was still not saturated.

[0047] The third group represents the optimal growth state. Under this formulation (NaNO3 4g / L, KH2PO4 4g / L, etc.), the strain achieved the highest biomass (OD). 600 The concentration reached 4.5 ± 0.5. This indicates that the nitrogen-to-phosphorus ratio and metal ion concentration of this component are optimal for the metabolic needs of Microbeobacterium NJS-5. At this point, the nutrient supply is sufficient and has not yet reached the threshold for toxicity or high osmotic pressure, representing the optimal growth window for the strain.

[0048] The fourth group showed slight inhibition; despite being richer in nutrients (NaNO3 5 g / L), its growth rate decreased to 3.3 ± 0.2, lower than the third group. This suggests that the inorganic salt concentration had exceeded the optimal range, which may have increased the osmotic pressure of the culture medium, forcing the strain to consume energy to maintain the osmotic balance inside and outside the cell, thereby reducing the energy allocation for growth and reproduction.

[0049] The fifth group showed significant inhibition; high concentrations of salt (such as NaNO3 and KH2PO4, both up to 8 g / L) led to OD... 600 The value dropped sharply to 1.3±0.4, the lowest among all groups. This indicates that excessively high salt concentrations may have caused significant salt stress or ion toxicity to the cells, severely inhibiting enzyme activity or disrupting cell membrane permeability, resulting in poor bacterial growth and a biomass significantly lower than the optimal group.

[0050] In summary, as shown in Table 1 above, the inorganic salt liquid culture medium composition (g / L) of the third group of cultures is more suitable for the growth of the strains, namely: NaNO3 4 g / L, KH2PO4 4 g / L, MgSO4 0.2 g / L, NaCl 1 g / L, KCl 1 g / L, CaCl2 0.4 g / L, H3PO4 5 mL / L, achieving the optimal combination of nutrient supply and osmotic pressure balance. This formula not only yields the highest biomass (OD200)... 600 =4.5), which was confirmed as the optimal inorganic salt liquid culture medium composition for Microbes NJS-5.

[0051] Example 3: pH selection for the optimal growth of microbacterium NJS-5

[0052] The isolated microbes NJS-5 were inoculated into a liquid culture medium with glucose as the carbon source at a concentration of 5 g / L. The inorganic salt composition of the liquid culture medium was: NaNO3 4 g / L, KH2PO4 4 g / L, MgSO4 0.2 g / L, NaCl 1 g / L, KCl 1 g / L, CaCl2 0.4 g / L, and H3PO4 5 mL / L. The medium was incubated at 37°C with shaking for 2 days, and then the growth of the strain was measured. The optimal pH of the medium was determined based on the growth of the strain. A blank control without the strain was included to reduce the impact of volatilization.

[0053] Table 2: Growth of NJS-5 under different pH conditions

[0054]

[0055] As shown in Table 2 above, as the pH of the liquid culture medium gradually increased from 6.0 to 8.0, the growth (OD600 value) of strain NJS-5 exhibited a typical single-peak curve characteristic of first increasing and then decreasing. The experimental data clearly divided the growth into three stages: pH 6.0 to 6.5 was the adaptive growth period, during which biomass accumulated rapidly as acidity decreased; pH 7.0 was the optimal metabolic period, reaching the peak growth; and pH 7.5 to 8.0 was the inhibitory decline period, during which growth was significantly inhibited as alkalinity increased.

[0056] In a slightly acidic environment of pH 6.0, the higher hydrogen ion concentration may affect the proton kinetic potential of the strain's cell membrane, forcing the cell to consume more ATP to maintain intracellular pH homeostasis via the proton pump, rather than for anabolism. Furthermore, the acidic environment may inhibit the catalytic activity of some key enzymes.

[0057] In group 2 (pH 6.5), growth increased by approximately 52% compared to group 1. This indicates that *Microbensis NJS-5* has a good adaptability to a slightly acidic environment. As the acidity of the environment decreases, the energy consumption of cells to maintain homeostasis decreases, and the metabolic focus shifts back to division and proliferation.

[0058] Group 3, pH=7.0, represents the optimal growth window for the strain. A neutral environment best suits the physiological characteristics of this microbacterium. At this point, the extracellular and intracellular pH are closest, resulting in the highest transmembrane transport efficiency. Furthermore, the enzymes responsible for glycolysis and macromolecule synthesis are within their optimal pH range, maximizing catalytic efficiency.

[0059] At pH 7.5, although there was a slight decrease, the biomass remained at a high level (OD). 600 =4.2), indicating that NJS-5 is more tolerant of slightly alkaline environments than slightly acidic environments (compared to OD at pH=6.5). 600=3.5). However, when the pH is further increased to 8.0, the high concentration of OH⁻ ions may damage the cell wall structure or denature enzyme proteins, resulting in significant alkaline stress, causing a precipitous drop in growth, falling back to a low level similar to that at pH 6.0.

[0060] In summary, *Microbacterium NJS-5* is quite sensitive to environmental pH, and its metabolic activity exhibits a clear neutral preference. pH 7.0 achieves the optimal balance between enzyme activity and energy distribution, resulting in the highest biomass (OD). 600 Based on the best growth stability (pH = 4.7), pH 7.0 was determined to be the optimal growth pH for this strain.

[0061] Example 4: Determination of the ability of microbacterium NJS-5 to degrade nitrobenzene

[0062] The isolated microbacterium NJS-5 was inoculated into liquid culture media with different nitrobenzene contents (10, 50, 100, 200, 300, 400, 500 mg / L) and cultured in a constant temperature shaking incubator at 37℃ for 5 days. The residual nitrobenzene content in the culture medium was then analyzed, and the degradation rate of nitrobenzene was calculated. A blank control without the addition of the strain was set up to reduce the impact of volatilization.

[0063] The liquid culture medium consisted of: NaNO3 4 g / L, KH2PO4 4 g / L, MgSO4 0.2 g / L, NaCl 1 g / L, KCl 1 g / L, CaCl2 0.4 g / L, H3PO4 5 mL / L, and pH=7.0.

[0064] Table 3: Degradation Rate of NJS-5 Nitrobenzene

[0065]

[0066] The degradation rates calculated and subtracted from the blank control are shown in Table 3 above. It can be seen that Microbacterium NJS-5 can efficiently degrade nitrobenzene. As the concentration of nitrobenzene increases, its degradation rate decreases. Even at a concentration of 500 mg / L, there is a degradation rate of 49.3%, indicating that this strain can tolerate high concentrations of nitrobenzene.

[0067] Specifically, as shown in Table 3, as the initial nitrobenzene concentration increased in stages from 10 mg / L to 500 mg / L, the degradation rate of NJS-5 exhibited a significant negative trend (decreasing from 93.7% to 49.3%). This trend is consistent with the typical kinetic characteristics of microbial degradation of toxic organic matter: at low concentrations, the substrate mainly serves as a carbon and energy source to promote growth; as the concentration increases, the cytotoxicity of nitrobenzene itself (such as damaging cell membranes and inhibiting enzyme activity) begins to emerge, producing a substrate inhibition effect, leading to a decrease in degradation efficiency.

[0068] Example 5: Determination of inoculum size for nitrobenzene degradation by Microbacterium NJS-5

[0069] The isolated microbacterium NJS-5 was inoculated into a liquid culture medium with nitrobenzene as the sole carbon source at a concentration of 100 mg / L. The medium was then incubated in a constant temperature shaking incubator at 37°C for 2 days. The residual nitrobenzene content in the culture medium was then analyzed, and the degradation rate of nitrobenzene was calculated. A blank control without the addition of the strain was set up to reduce the impact of volatilization.

[0070] The liquid culture medium consisted of: NaNO3 4 g / L, KH2PO4 4 g / L, MgSO4 0.2 g / L, NaCl 1 g / L, KCl 1 g / L, CaCl2 0.4 g / L, H3PO4 5 mL / L, and pH=7.0.

[0071] Table 4: Degradation capacity of NJS-5 at different inoculum sizes

[0072]

[0073] As shown in Table 4 above, under the condition of nitrobenzene concentration of 100 mg / L, the degradation rate showed a continuous upward trend after 48 hours (from 51.1% to 60.3%) as the initial inoculum amount increased from 0.5% to 3%. This indicates that the initial bacterial density is one of the key process parameters affecting degradation efficiency. A higher initial bacterial count means stronger biocatalytic ability and a faster initiation of the metabolic process of pollutants. Within the experimental range, the degradation effect was optimal at an inoculum amount of 3%.

[0074] Example 6: Treatment of nitrobenzene-contaminated soil with Microbacterium NJS-5

[0075] Microbacterium NJS-5 was inoculated into sterilized LB medium and cultured in a constant temperature shaking incubator at 37°C (260 rpm) to form a seed culture solution. The seed culture solution was then inoculated into the liquid medium at a volume ratio of 3%.

[0076] The liquid culture medium consisted of: NaNO3 4 g / L, KH2PO4 4 g / L, MgSO4 0.2 g / L, NaCl 1 g / L, KCl 1 g / L, CaCl2 0.4 g / L, H3PO4 5 mL / L, and pH=7.0.

[0077] 10g of soil contaminated with nitrobenzene (hereinafter referred to as "contaminated soil") was taken and added to 10mL, 20mL, 30mL, and 40mL of liquid culture medium, respectively. The nitrobenzene content in the contaminated soil was 133 mg / kg. The mixed mud-water solution was placed in a 37℃ constant temperature shaking incubator (260 rpm) for 5 days, and the residual nitrobenzene content in the contaminated soil was analyzed. A blank control group without bacteria was also set up. The calculated result is the result after subtracting the influence of the blank control.

[0078] Table 5: Degradation of nitrobenzene by NJS-5 under different soil and culture medium ratios

[0079]

[0080] The results in Table 5 above show that in contaminated soil with an initial nitrobenzene content of 133 mg / kg, the degradation rate of all experimental groups exceeded 80% after 5 days of treatment. With the increase of the liquid culture medium ratio (from 1:1 to 1:4), the residual amount of nitrobenzene generally showed a decreasing trend, while the degradation rate showed an increasing trend. Even in the 1:1 group (with the thickest slurry), the solid-liquid ratio was the highest, with a degradation rate as high as 80.6%, indicating that this strain can maintain extremely high metabolic activity in complex matrices with low water content and high solid content, demonstrating excellent potential for practical engineering applications.

[0081] When the soil-to-medium medium ratio is 1:1, meaning the mass-to-volume ratio of soil and water is equal, the mixture is a viscous slurry. Under these conditions, oxygen transfer is typically hindered (mass transfer limitation), and desorption of pollutants from soil particles is difficult. However, NJS-5 still achieved a removal rate exceeding 80%. In practical soil remediation projects, a low liquid-to-medium ratio means less wastewater production and lower subsequent dewatering costs. Although the degradation rate was slightly lower than the high liquid-to-medium ratio group, an 80% removal rate is significant for reducing treatment costs.

[0082] When the soil-to-culture medium ratio is 1:4, the large amount of liquid culture medium acts as a solvent, promoting the diffusion (desorption) of nitrobenzene adsorbed in the micropores of soil particles into the liquid phase, thus improving the bioavailability of pollutants. At this point, strain NJS-5 is mainly present in the liquid phase. The high liquid-to-soil ratio allows for more thorough contact between the strain and suspended soil particles, thereby achieving the highest degradation rate and reducing the residue to below 20 mg / kg.

[0083] The data in Table 5 above strongly demonstrate the coupling mechanism of "desorption-biodegradation". The liquid culture medium not only provides nutrients but also serves as the medium for pollutants to transfer from the solid phase to the liquid phase and be captured by bacteria. The more liquid there is, the lower the mass transfer resistance and the more thorough the degradation. In practical soil remediation projects, if the remediation targets are extremely stringent, a soil:culture medium ratio of 1:4 can be used. If the remediation targets are relatively lenient, a soil:culture medium ratio of 1:1 or 1:2 is a more cost-effective choice, because a 1:1 ratio requires only one-quarter the water consumption of a 1:4 ratio and can significantly reduce the energy consumption of subsequent mud-water separation while still ensuring a removal efficiency of over 80%.

[0084] Example 7: Treatment of nitrobenzene-contaminated groundwater with Microbacterium NJS-5

[0085] Microbacterium NJS-5 was inoculated into sterilized LB medium and cultured in a constant temperature shaking incubator at 37°C (260 rpm) to form a seed culture solution. The seed culture solution was then inoculated into the liquid medium at a volume ratio of 3%.

[0086] The liquid culture medium consisted of: NaNO3 4 g / L, KH2PO4 4 g / L, MgSO4 0.2 g / L, NaCl 1 g / L, KCl 1 g / L, CaCl2 0.4 g / L, H3PO4 5 mL / L, and pH=7.0.

[0087] Take 20 mL of water contaminated with nitrobenzene and add 20 mL, 40 mL, 60 mL, and 80 mL of culture medium, respectively. The nitrobenzene concentration in the contaminated water is 149 mg / L. Incubate the mixtures in a 37℃ constant temperature shaking incubator (260 rpm) for 5 days. Analyze the residual nitrobenzene concentration in the water. A blank control group without added bacteria is also included. The calculated results are the result after subtracting the influence of the blank control.

[0088] Table 6: Degradation of nitrobenzene by NJS-5 under different groundwater and culture medium ratios

[0089]

[0090] The results in Table 6 above show that in contaminated groundwater with an initial nitrobenzene concentration of 149 mg / L, the degradation rate of all experimental groups remained stable between 84% and 86% after 5 days of treatment. Unlike the soil remediation experiment, which was affected by mass transfer limitations, in the homogeneous aqueous solution system, changing the ratio of groundwater to culture medium (from 1:1 to 1:4) had a very small effect on the final degradation rate (the range was only 2.15%). The degradation rate of the 1:4 group (maximum dilution) (85.33%) was almost the same as that of the 1:1 group (85.20%). This indicates that in water remediation, simply increasing the volume of the culture medium does not significantly improve the removal rate. The degradation capacity of the strain is mainly limited by its own metabolic kinetics, rather than the bioavailability of the substrate (this is quite different from soil remediation). This shows that Microbacterium NJS-5 has a strong adaptability to the aquatic environment and can maintain high metabolic activity in both high-load (1:1) and low-load (1:4) systems.

[0091] Microbacterium NJS-5 exhibits excellent dilution insensitivity in groundwater remediation. Because groundwater is a liquid environment, the contaminants are in full contact with the strain, and there is no mass transfer resistance, resulting in minimal differences in degradation rates among different groups. Considering economic cost and treatment efficiency, a 1:1 dosage ratio is recommended for practical engineering applications. This ratio achieves degradation effects (>85%) comparable to higher dilutions while minimizing culture medium usage, offering the highest cost-effectiveness. Even with significant fluctuations in influent concentration, the strain's stable performance across a wide range of dosage ratios demonstrates its strong resistance to shock loads and provides process flexibility.

[0092] In summary, the Microbacterium sp. NJS-5 provided by this invention has the ability to degrade nitrobenzene and can be used for the bioremediation of nitrobenzene-contaminated soil and groundwater. The Microbacterium provided by this invention exhibits tolerance to high concentrations of nitrobenzene, high degradation efficiency for nitrobenzene, strong tolerance to nitrobenzene, and advantages such as high efficiency, greenness, and no pollution. It is a beneficial supplement to current bioremediation technologies for nitrobenzene-contaminated soil and provides a new solution for the treatment and control of nitrobenzene pollution problems.

[0093] The above description is merely a preferred example of the present invention and is not intended to limit the scope of protection of the present invention. In addition to the above embodiments, the present invention may have other implementations. All technical solutions formed by equivalent substitutions or equivalent changes fall within the scope of protection claimed by the present invention.

Claims

1. A strain of microbacterium (Microbacterium sp.), Microbacterium sp. ), characterized in that, The microbacterium is microbacterium NJS-5, which was registered and deposited at the China General Microbiological Culture Collection Center on September 28, 2025, with accession number CGMCC No. 36103. The gene sequence of the 16S rRNA of the microbacterium is shown in SEQ ID No.

1. The microbacterium has the ability to degrade nitrobenzene in the concentration range of 10 to 500 mg / L nitrobenzene.

2. The application of the microbacterium as described in claim 1 in the degradation of nitrobenzene.

3. Use according to claim 2, characterized in that, The application process includes: Microbacterium NJS-5 was activated in sterilized seed culture medium and cultured in a constant temperature shaking incubator at 30℃-40℃. Inoculate the seed culture solution into the liquid culture medium at an inoculation rate of 0.5%-3%, and place it in a constant temperature shaking incubator at 30℃-40℃ for anaerobic culture for more than 3 days; Take soil or groundwater contaminated with nitrobenzene and add it to the liquid culture medium. Place the mixed mud-water solution in a constant temperature shaking incubator at 30℃-40℃ and incubate for more than 5 days.

4. Use according to claim 3, characterized in that, The seed culture medium consists of: 10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, and pH 7.

0.

5. Use according to claim 3, characterized in that, The liquid culture medium has the following composition: NaNO3 2-8 g / L, KH2PO4 2-8 g / L, MgSO4 0.1-1 g / L, NaCl 0.5-3 g / L, KCl 0.5-3 g / L, CaCl2 0.1-1 g / L, H3PO4 2-10 mL / L, pH=6.0-8.

0.

6. Use according to claim 3, characterized in that, The mass-to-volume ratio of soil contaminated with nitrobenzene to liquid culture medium was 1:1 to 1:4, and the content of benzene series compounds in the contaminated soil was 10 to 500 mg / kg.

7. The application according to claim 3, characterized in that, The volume ratio of groundwater contaminated with nitrobenzene to liquid culture medium is 1:1 to 1:4, and the content of benzene series compounds in the contaminated groundwater is 10 to 500 mg / L.

8. A microbial agent, characterized in that, The bacterial agent contains the microbacteria described in claim 1.

9. A method for preparing a microbial agent, characterized in that, The method includes: fermenting and culturing the microbacteria described in claim 1.

10. A method for remediating nitrobenzene-contaminated soil and / or groundwater, characterized in that, The method includes adding the microbacterium of claim 1 or the microbial agent of claim 8 to the soil and / or groundwater contaminated with nitrobenzene to be remediated.

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