Clostridium and application thereof in groundwater pollution treatment

Abstract

The invention provides a clostridium, the classification name of the clostridium is Clostridium sp.LXY-7, and the clostridium is preserved in China General Microbiological Culture Collection Center (CGMCC). The address is Institute of Microbiology, Chinese Academy of Sciences, No.3, Yard 1, Beichen West Road, Chaoyang District, Beijing; the preservation date is October 22, 2024, and the preservation number is CGMCC (China General Microbiological Culture Collection Center) No. 1.18144. The invention also provides application of the clostridium in microbial sulfate reduction and application of the clostridium in groundwater pollution treatment.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, relates to applications in groundwater pollution control, and particularly relates to a clostridium strain and applications thereof. Background Art

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

[0003] Clostridium species are widely used in bioremediation due to their strong stress resistance and ability to form spores and survive in harsh environments. However, the degradation efficiency of existing Clostridium strains in complex pollution systems (such as those with multi-component organic matter and heavy metals) needs to be improved. Furthermore, in situ groundwater bioremediation often results in long remediation cycles or even failure due to poor strain adaptability and insufficient nutrients.

[0004] Therefore, screening new remediation bacteria with better degradation ability or wider environmental adaptability has become an urgent problem to be solved. Summary of the Invention

[0005] In order to solve the above problems, the object of the present invention is to provide a Clostridium strain having good groundwater remediation effect.

[0006] Another object of the present invention is to provide a use of the above-mentioned Clostridium.

[0007] In order to achieve the above-mentioned object, the present invention provides a strain of Clostridium, the classification name of which is: Clostridium sp. LXY-7, the preservation unit is: General Microbiology Center of China Culture Collection Administration Committee of Microorganisms; the address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; the preservation date is: October 22, 2024, and the preservation number is: CGMCC No.1.18144.

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

[0009] The present invention also provides application of the above-mentioned clostridium in groundwater pollution control.

[0010] The beneficial effects of the present invention are:

[0011] The present invention provides a clostridium strain, which has the effect of efficiently reducing sulfate in a weakly acidic environment and can be used to treat soil and groundwater pollution. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

[0014] Figure 3 The Clostridium LXY-7 provided by the present invention has different sulfate-reducing abilities at different initial pH values.

[0015] Figure 4 The Clostridium LXY-7 provided by the present invention is subjected to different initial SO4 2- It has high sulfate reducing ability.

[0016] Figure 5 This is a schematic diagram of the layout of monitoring points on the project site. DETAILED DESCRIPTION

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

[0018] Material

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

[0020] Prepare culture medium

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

[0022] Microbial solid culture medium (1 L): 2 g anhydrous magnesium sulfate, 5 g sodium citrate dihydrate, 1 g calcium sulfate dihydrate, 1 g ammonium chloride, 0.5 g dipotassium hydrogen phosphate, 3.5 g sodium lactate, 1 g yeast extract, 15 g agar, pH = 7.2. Adjust the pH as needed with 1 mol / L dilute sulfuric acid. Ventilate the medium in an anaerobic operating chamber, seal the container, and sterilize at 121°C and autoclave for 20 min. After sterilization, pour the medium into a sterile culture dish in an anaerobic operating chamber while still hot. Cool the plate to obtain the culture medium.

[0023] Example 1 Isolation and identification of strains

[0024] Soil samples were collected from a lead-zinc smelter in Guangxi. Using a sterilized shovel or sampler, soil samples were collected at a depth of 10-40 cm. The samples were placed in sterile, average sampling bags and labeled. The collected samples were then shipped to a laboratory at low temperatures 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 precipitate the solid particles. The supernatant is the bacterial suspension used for screening. In the anaerobic operating table, take 100μL of bacterial suspension and add it to 900μL of sterile saline, mix thoroughly, and form 10 -1 diluent. Take 100μL 10 -1 The dilution was added to 900 μL of sterile saline to form 10 -2 dilution, and so on, up to 10 -6 Dilution. Take 200 μL of 10 -4 , 10 -5 and 10 -6 Add the gradient dilutions to the culture medium plate and evenly spread it on the plate with a sterile applicator. Place the plate upside down in a 30°C incubator for 48 hours and observe the growth of the colonies.

[0026] After a single colony has grown on the culture medium plate, select a representative and well-growing colony and pick it up with a sterile inoculation loop. Perform streak culture on a new culture medium plate to ensure that a single colony is isolated. Place it upside down in a 30°C incubator and culture for 48 hours. Observe the growth of the colony and repeat the streak process until a purified strain is obtained. Figure 1 Shown are photographs of growth on solid medium.

[0027] The strain was expanded using microbial enrichment medium, and genomic DNA was extracted from a single colony using a DNA extraction kit for PCR amplification. The PCR product was subjected to agarose gel electrophoresis to confirm the amplification effect. The 16SrRNA of the bacterium was PCR amplified and sequenced, as shown in Seq ID No.1, with a sequence length of 1320bp. By comparing with the Ezbiocloud database (https: / / www.ezbiocloud.net / ), the maximum similarity of the full-length 16SrRNA gene sequence of this strain with all standard strains of the Clostridium genus was 98.26%, which is lower than the new species classification threshold of 98.5%. Figure 2Based on the physiological and biochemical characteristics, the bacterium was identified as a new species of the genus Clostridium and named Clostridium sp. LXY-7, abbreviated as Clostridium LXY-7. It was deposited with the China General Microbiology Center, China Culture Collection Administration, on October 22, 2024. The deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0028] Example 2 Detection of sulfate reducing ability of strains

[0029] Prepare a microbial liquid culture medium, adjust the pH to 4.5, 5, 5.5, 6, 6.5 and 7 respectively with 1 mol / L dilute sulfuric acid as needed, ventilate in an anaerobic operating table, seal the medium, and sterilize in a high-pressure steam sterilizer at 121°C for 20 minutes. 600 = 1 was inoculated into the cooled liquid culture medium and cultured 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 shown in Figure 2. Figure 3 shown.

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

[0031] Example 3 strain LXY-7 at different initial SO4 2- Highly efficient sulfate reduction capability

[0032] Prepare microbial liquid culture medium by increasing or decreasing the mass of anhydrous magnesium sulfate in the culture medium while keeping other components unchanged. 2- The concentrations were adjusted to 0.5, 1, 2, 3, 4, 5, and 10 g / L, respectively. The tubes were sealed after ventilation in an anaerobic operating table, sterilized in a high-pressure steam sterilizer at 121°C for 20 min, and then cooled and inoculated with 1% and initial OD 600 = 1, the bacterial solution of Clostridium LXY-7 obtained in Example 1 was observed from day 0 to day 7, and the relationship between sulfate reduction rate and time was plotted as follows: Figure 4 shown.

[0033] from Figure 4 It can be seen that the Clostridium LXY-7 of the present invention is SO4 2- When the concentration is 1g / L, the maximum sulfate removal rate is 79%, which has a high sulfate reduction capacity; when the SO4 2- When the concentration is in the range of 2g / L to 5g / L, the sulfate reducing ability of the strain tends to be stable, and the reduction rate is between 49% and 60%, which means that the strain has stable sulfate reducing ability. 2- When the concentration is 10g / L, the sulfate reduction energy is 26%, which means that when the concentration of sulfate in the solution is too high, the growth and reduction ability of the strain will be inhibited, but there is still a reduction effect. In reality, SO4 in the soil of most heavy metal contaminated sites is 2- The concentration is less than 10g / L, around 5g / L. Compared with the existing strains, Clostridium LXY-7 has a higher concentration in SO4 2- Sites with concentrations less than 10g / L have sulfate reduction capabilities and have extremely high application value.

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

[0035] The sulfate-reducing strain LXY-7 was applied to a production enterprise where groundwater levels exceeded standards for heavy metals. According to the "Groundwater Quality Standard" (GB / T14848), the groundwater in this area was evaluated against Class IV. Testing revealed that, while arsenic was not detected and a few indicators met standards, volatile phenols, hexavalent chromium, and lead were within acceptable limits. However, levels of ammonia nitrogen, nickel, mercury, and thallium were significantly above standards. Mercury and thallium were particularly widespread, with the excess concentrations primarily concentrated in the sintering workshop area. The groundwater pH was slightly acidic, with most points exceeding the standard.

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

[0037] A microbial reactor was selected based on the project scale and expected treatment effect. A bacterial solution and culture medium were then injected into the reactor in a specific ratio to maintain an appropriate pH. The concentration and activity of the microorganisms within the reactor were regularly monitored using a biomass meter and enzyme activity analysis techniques. When the microbial concentration within the reactor reached a preset threshold, representing the biomass required for efficient treatment of the target pollutant, the solution was injected into eight pre-designed and installed microbial injection wells surrounding the D1 detection well to maximize the diffusion of the bacterial solution into the underground aquifer. The statistical results are shown in Table 1.

[0038] Table 1

[0039]

[0040] As shown in Table 1, the September data are before microbial inoculant injection, while the October and November data are after. In terms of implementation, injection around Well D1 significantly impacted Wells D2 and D3, achieving excellent remediation results. Test results indicate that the heavy metal nickel, thallium, and mercury levels in Wells D1, D2, and D3, located relatively upstream of the groundwater flow field, have generally shown a downward trend, with fewer exceeding standards in October and November compared to September. Comparing groundwater monitoring results before and after remediation, the microbial in situ mineralization technology has shown initial success.

[0041] It can be seen from the above examples that the Clostridium LXY-7 provided by the present invention has a good groundwater remediation effect and provides an innovative solution for future groundwater pollution control.

[0042] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be determined by the appended claims.

Claims

1. A Clostridium strain, characterized in that The classification name of this clostridium is: Clostridium sp.LXY-7, and the preservation unit is: General Microbiology Center of China Culture Collection Administration; the address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Building 1, Beichen West Road, Chaoyang District, Beijing; the preservation date is: October 22, 2024, and the preservation number is: CGMCCNo.1.18144.

2. Use of the Clostridium as claimed in claim 1 in sulfate reduction.

3. Use of the Clostridium as claimed in claim 1 in the treatment of groundwater pollution.

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