Clostridium and application thereof in groundwater pollution treatment

Abstract

The application provides a Clostridium sp. LXY-7, the taxonomic name of the Clostridium is as follows: Clostridium sp. LXY-7, the preservation unit is as follows: China General Microbiological Culture Collection Center, the address is as follows: No. 1, Yihuangyuan, Beichen West Road, Chaoyang District, Beijing, the preservation date is as follows: October 22, 2024, and the preservation number is as follows: CGMCC No. 1.18144. The application also provides application of the Clostridium sp. in microbial sulfate reduction and application of the Clostridium sp. in underground water pollution treatment.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of microorganisms and relates to application in groundwater pollution treatment, in particular to a Clostridium sp. and application thereof. BACKGROUND

[0002] As an important water resource, groundwater pollution is increasingly serious. Industrial wastewater discharge, agricultural non-point source pollution, landfill leakage and the like result in accumulation of harmful substances (such as organic matter and heavy metals) in groundwater, threatening drinking water safety and ecosystem health. Although traditional remediation technologies such as extraction treatment and chemical oxidation are effective, they have bottlenecks such as high cost and secondary pollution. Biological remediation technology has become a research hotspot due to its environmental friendliness, and microbial degradation of pollutants has the advantages of low cost and no secondary pollution, but existing strains are often limited by the environment (such as temperature and pH value) and have insufficient degradation efficiency.

[0003] Clostridium sp. is widely used in bioremediation due to its strong stress resistance and ability to form spores to survive in harsh environments. However, the degradation efficiency of existing Clostridium strains in complex pollution systems (such as coexistence of multi-component organic matter and heavy metals) still needs to be improved. In addition, in situ bioremediation of groundwater often results in long remediation period or failure due to poor environmental adaptability of strains and insufficient nutrients.

[0004] Therefore, screening of new remediation strains with better degradation capacity or wider environmental adaptability has become a problem to be solved. SUMMARY

[0005] To solve the above problems, the purpose of the present application is to provide a Clostridium sp. with good groundwater remediation effect.

[0006] Another purpose of the present application is to provide application of the above-mentioned Clostridium sp.

[0007] To achieve the above purpose, the present application provides a Clostridium sp. with the classification name of Clostridium sp. LXY-7, the preservation unit of China General Microbiological Culture Collection Center, the address of No. 3, Institute of Microbiology, Chinese Academy of Sciences, Beijing City, Chaoyang District, Beichen West Road No. 1, the preservation date of October 22, 2024, and the preservation number of CGMCC No. 1.18144.

[0008] The present application also provides application of the above-mentioned Clostridium sp. in sulfate reduction.

[0009] The present application also provides application of the above-mentioned Clostridium sp. in groundwater pollution treatment.

[0010] The present application has the following advantages:

[0011] This invention provides a Clostridium strain that can efficiently reduce sulfates in a weakly acidic environment and can be used to treat soil and groundwater pollution. Attached Figure Description

[0012] Figure 1 This is a diagram showing the growth status of Clostridium LXY-7 on a culture medium, as provided by the present invention.

[0013] Figure 2 The phylogenetic tree of Clostridium LXY-7 based on 16S rDNA provided by the present invention.

[0014] Figure 3 The Clostridium LXY-7 strain provided by this invention has different sulfate-reducing abilities under different initial pH values.

[0015] Figure 4 The Clostridium LXY-7 provided by this invention is used in different initial SO4 2- It has a highly efficient sulfate reducing ability.

[0016] Figure 5 A schematic diagram showing the layout of monitoring points at the engineering site. Detailed Implementation

[0017] The embodiments of the present invention will now be described in detail and comprehensively so that the advantages and features of the present invention can be more easily understood by those skilled in the art, thereby providing a clearer and more explicit definition of the scope of protection of the present invention.

[0018] Material

[0019] The DNA extraction kit was purchased from Beijing Jinsha Biotechnology Co., Ltd., product number: DE703-50.

[0020] Preparation of culture medium

[0021] Microbial liquid culture medium (1L): 2g anhydrous magnesium sulfate, 5g sodium citrate dihydrate, 1g calcium sulfate dihydrate, 1g ammonium chloride, 0.5g dipotassium hydrogen phosphate, 3.5g sodium lactate, 1g yeast extract, pH=7.2. Adjust the pH with 1mol / L dilute sulfuric acid as needed. After evacuating the air in an anaerobic workbench, seal the container and sterilize in a high-pressure steam autoclave at 121℃ for 20min.

[0022] Microbial solid culture medium (1L): 2g anhydrous magnesium sulfate, 5g sodium citrate dihydrate, 1g calcium sulfate dihydrate, 1g ammonium chloride, 0.5g dipotassium hydrogen phosphate, 3.5g sodium lactate, 1g yeast extract, 15g agar, pH=7.2. Adjust the pH as needed with 1mol / L dilute sulfuric acid. After evacuation in an anaerobic workbench, seal the container and autoclave at 121℃ for 20min. After sterilization, pour the hot mixture into sterile petri dishes in the anaerobic workbench and allow it to cool to obtain culture medium plates.

[0023] Example 1: Isolation and Identification of Strains

[0024] Soil samples were collected from a lead-zinc smelter in Guangxi Province. Sterilized shovels or samplers were used to collect samples from depths of 10-40 cm. The samples were placed in sterile mean sampling bags and labeled with sample information. The collected samples were then sent to the laboratory at low temperature for microbial isolation and screening.

[0025] Take 5g of soil sample and add it to 45mL of sterile water. Shake thoroughly for 10 minutes to fully suspend the soil particles. Let the soil mixture stand for 10 minutes to allow the solid particles to settle. The supernatant is the bacterial suspension used for screening. In an anaerobic operating table, add 100μL of the bacterial suspension to 900μL of sterile physiological saline and mix thoroughly to form a 10... -1 Diluent. Take 100 μL of 10 -1 The diluent was added to 900 μL of sterile physiological saline to form a 10... -2 Diluent, and so on, up to 10 -6 Dilution. Take 200 μL of 10... -4 10 -5 and 10 -6 The serially diluted solution was added to the culture medium plates and spread evenly using a sterile spreader. The plates were then inverted and incubated at 30°C for 48 hours, during which time the colony growth was observed.

[0026] After clear single colonies have grown on the culture medium plate, select representative and well-grown colonies and inoculate them using a sterile inoculation loop. Streak the colonies on a fresh culture medium plate to ensure the isolation of single colonies. Invert the plate and incubate at 30°C for 48 hours. Observe the growth of the colonies and repeat the streaking process until a purified strain is obtained. Figure 1 The image shown is a photograph of the growth on a solid culture medium.

[0027] The strain was expanded using microbial enrichment medium. Genomic DNA was extracted from single colonies using a DNA extraction kit and amplified by PCR. The PCR products were then subjected to agarose gel electrophoresis to confirm the amplification effect. The 16S rRNA of this bacterium was amplified by PCR and sequenced, as shown in Seq ID No. 1, with a sequence length of 1320 bp. Comparison with the Ezbiocloud database (https: / / www.ezbiocloud.net / ) showed that the maximum full-length similarity of the 16S rRNA gene sequence of this strain with all standard strains of the Clostridium genus was 98.26%, which is below the 98.5% threshold for new species classification. Figure 2As shown, based on physiological and biochemical characteristics, this bacterium was identified as a new species of the genus *Clostridium*, named *Clostridium* sp. LXY-7, or simply *Clostridium* LXY-7, and deposited for preservation. The preservation number is CGMCC No. 1.18144; the preservation date is October 22, 2024; the depositary institution is the China General Microbiological Culture Collection Center (CGMCC), located at Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0028] Example 2: Detection of the sulfate reducing ability of the strain

[0029] Microbial liquid culture medium was prepared, and the pH was adjusted to 4.5, 5, 5.5, 6, 6.5, and 7 respectively using 1 mol / L dilute sulfuric acid as needed. After evacuation and sealing in an anaerobic operating table, the medium was sterilized in a high-pressure steam autoclave at 121°C for 20 minutes. The Clostridium LXY-7 strain obtained in Example 1 was inoculated at a 1% inoculum, with an initial OD... 600 =1 was inoculated into the cooled liquid culture medium described above and incubated in a 30°C incubator. Samples were taken at 25, 50, 75, 100, 125, 150, 175, and 300 hours, and the relationship between sulfate reduction rate and time was plotted as follows: Figure 3 As shown.

[0030] from Figure 3 It can be seen that when the pH is 6-7, the sulfate reduction rate is stable at around 80%; when the pH is 5.5, the sulfate reduction rate is around 20%; and when the pH is 4.5-5.0, the sulfate reduction rate is less than 5%. This indicates that strain LXY-7 has a high sulfate reduction capacity under weakly acidic conditions, and the capacity is relatively persistent.

[0031] Example 3: Strain LXY-7 under different initial SO4 2- It has a high sulfate reducing capacity.

[0032] To prepare a microbial liquid culture medium, the SO4 content in the medium is reduced by increasing or decreasing the mass of anhydrous magnesium sulfate while keeping other components constant. 2- The concentrations were adjusted to 0.5, 1, 2, 3, 4, 5, and 10 g / L, respectively. After evacuation and sealing in the anaerobic operating table, the mixture was sterilized in a high-pressure steam autoclave at 121℃ for 20 min and cooled before being inoculated with 1% initial OD. 600 =1. The bacterial culture of Clostridium LXY-7 obtained in Example 1 was observed from day 0 to day 7. The relationship between sulfate reduction rate and time was plotted as follows: Figure 4 As shown.

[0033] from Figure 4 It can be seen that the Clostridium LXY-7 of the present invention, when the SO4 in the solution... 2- At a concentration of 1 g / L, the highest sulfate removal rate of 79% is achieved, demonstrating a high sulfate reducing capacity; when the SO4 in the solution... 2- Within a concentration range of 2 g / L to 5 g / L, the sulfate reducing ability of the strain tends to stabilize, with a reduction rate between 49% and 60%, demonstrating stable sulfate reducing capacity; when SO42-... 2- At a concentration of 10 g / L, the sulfate reduction energy is 26%, indicating that when the sulfate concentration in the solution is too high, the growth and reducing ability of the strain are inhibited, but a reducing effect still exists. In reality, most heavy metal contaminated soils contain SO42-... 2- The concentration is less than 10 g / L, around 5 g / L. Compared with existing strains, Clostridium LXY-7 has a lower concentration in SO4. 2- Sites with a concentration of less than 10 g / L all have sulfate reduction capabilities, making them extremely valuable for application.

[0034] Example 4: Engineering application of strain LXY-7 in groundwater pollution remediation

[0035] The sulfate-reducing strain LXY-7 was applied to a production enterprise where groundwater levels exceeded standards for heavy metals. Based on the "Groundwater Quality Standard" (GB / T14848), the groundwater in this area was evaluated using Class IV as a reference. Testing revealed that, except for arsenic (not detected) and a few other indicators meeting the standards, while volatile phenols, hexavalent chromium, and lead were within acceptable limits, ammonia nitrogen, nickel, mercury, and thallium levels were severely exceeded, especially mercury and thallium, with the exceedances concentrated primarily in the sintering workshop area. The groundwater pH was acidic, with most sampling points exceeding the standard range.

[0036] Since the overall flow direction of groundwater in the area is roughly from northeast to southwest, monitoring points are mainly set up in the relatively downstream southern part of the region to monitor the spread of pollution. For example... Figure 5 As shown, five new monitoring wells (numbered D1 to D5, with a construction depth of 40m) and eight bacterial agent injection wells (numbered T1 to T8) were built, with sampling depths below 0.5m of the stable water level.

[0037] A microbial reactor was selected based on the project scale and expected treatment effect. Then, the bacterial solution and culture medium were injected into the reactor according to a specific ratio, maintaining a suitable pH value. The concentration and activity of the microorganisms in the reactor were monitored regularly using a biomass analyzer and enzyme activity analysis technology. When the microbial concentration in the reactor reached a preset threshold, i.e., the biomass required for efficient treatment of the target pollutant, eight pre-designed and installed bacterial agent injection wells were injected around the D1 monitoring well to maximize the diffusion effect of the bacterial solution in the underground aquifer. The statistical results are shown in Table 1.

[0038] Table 1

[0039]

[0040] Table 1 shows that the September data is before the injection of microbial agents, while the October and November data are after the injection. From the implementation process, the injection around well D1 effectively impacted wells D2 and D3, achieving a good remediation effect. The monitoring results show that the content of heavy metals nickel, thallium, and mercury in monitoring wells D1, D2, and D3, located relatively upstream in the groundwater flow field, generally showed a decreasing trend, and the exceedance data in October and November were less than those in September. Comparing the groundwater monitoring results before and after the treatment, the in-situ microbial mineralization technology has begun to show results.

[0041] As can be seen from the above embodiments, the Clostridium LXY-7 provided by the present invention has a good groundwater remediation effect, providing an innovative solution for future groundwater pollution control.

[0042] 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 this patent should be determined by the appended claims.

Claims

1. A Clostridium species, characterized in that, The taxonomic name of this clostridium is: Clostridium sp. LXY-7, deposited at: China General Microbiological Culture Collection Center (CGMCC); address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing; deposit date: October 22, 2024; accession number: CGMCC No. 1.18144.

2. The application of Clostridium as described in claim 1 in sulfate reduction.

3. The application of Clostridium in groundwater pollution remediation as described in claim 1, characterized in that, The groundwater pollution is caused by nickel, thallium, and mercury.

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