A method for repairing Cr(VI) contaminated water bodies by using escherichia coli and leersia hexandra

Abstract

The present application belongs to the field of environmental microorganism-plant technology, and particularly relates to a method for repairing Cr(VI) polluted water bodies by using Escherichia coli and Leersia hexandra Swallen. A strain of Escherichia coli 2-2b is isolated from the rhizosphere soil of a chromium hyperaccumulating plant Leersia hexandra Swallen, and has a growth promoting effect. The present application simulates the composition of domestic sewage, establishes Cr(VI) polluted water bodies, and uses Leersia hexandra Swallen and the Escherichia coli 2-2b to jointly treat the Cr(VI) polluted water bodies. The results show that the Escherichia coli 2-2b has a growth promoting effect, can promote the contents of chlorophyll, carotenoid, POD and CAT of Leersia hexandra Swallen, and improve the repair effect of Leersia hexandra Swallen on the Cr(VI) polluted water bodies.

Description

Technical Field

[0001] This invention belongs to the field of environmental microbiology-plant technology, specifically relating to a method for remediating Cr(VI) polluted water using Escherichia coli and Listeria monocytogenes. Background Technology

[0002] Chromium (Cr) is a metallic element widely used in various chemical industries such as leather, paint, dyes, and metal processing, thus easily causing chromium pollution in the environment. Chromium typically exists in two oxidation states in the aquatic environment: trivalent chromium (Cr(III)) and hexavalent chromium (Cr(VI)). The most persistent form of chromium in the aquatic environment is soluble, mobile, and the most toxic Cr(VI). Therefore, removing Cr(VI) is of great significance for the remediation of chromium pollution.

[0003] Leymus chinensis is a Cr hyperaccumulator plant that can absorb Cr(III) and Cr(VI) from the surrounding environment through its roots, accumulating over 1000 mg / kg of Cr within its own body, thereby remediating Cr pollution in water bodies. Moreover, Leymus chinensis grows rapidly and has a short harvest cycle. Compared to traditional physicochemical remediation methods that are costly and prone to secondary pollution, utilizing the chromium hyperaccumulation properties of Leymus chinensis for chromium-polluted water remediation is a green, low-cost, and highly promising heavy metal pollution remediation technology. However, when the concentration of heavy metals around the plant reaches a certain threshold, the heavy metals can affect the plant's metabolism and physiological functions, thus affecting the plant's absorption rate of heavy metals. To increase phytoremediation yield, existing technologies typically employ chemically assisted phytoremediation techniques, using synthetic or organic chelating agents to improve the bioavailability of metals, such as ethylenediaminetetraacetic acid (EDTA) and diethylenetriaminepentaacetic acid (DTPA). However, these chelating agents also have significant drawbacks during application; for example, EDTA itself is often toxic and not easily biodegradable. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art, improve the ability of Listeria monocytogenes to remediate Cr(VI) and domestic sewage in water bodies, and efficiently remove Cr(VI), chemical oxygen demand (COD), nitrate nitrogen and total phosphorus from Cr(VI) polluted water bodies.

[0005] To achieve the above objectives, the present invention provides the application of Escherichia coli 2-2b in improving the remediation effect of Listeria monocytogenes on Cr(VI) polluted water; the Escherichia coli 2-2b is deposited at the Institute of Microbiology, Guangdong Academy of Sciences, China, with accession number GDMCC 64075.

[0006] Preferably, the concentration of Cr(VI) in the Cr(VI) polluted water is 18-22 mg / L, the chemical oxygen demand is 394.9-434.0 mg / L, the nitrate nitrogen concentration is 168.5-204.1 mg / L, and the total phosphorus concentration is 35.0-44.3 mg / L.

[0007] This invention also provides a method for remediating Cr(VI) contaminated water using a combination of Escherichia coli and Listeria monocytogenes, comprising the following steps:

[0008] Pre-cultured Rheum palmatum seedlings were placed in Cr(VI)-contaminated water, and Escherichia coli 2-2b in its logarithmic growth phase was introduced to form a co-treatment system for the co-treatment of Cr(VI)-contaminated water.

[0009] The conditions for the combined treatment are: daytime temperature 28-32℃, nighttime temperature 22-27℃, and daily light exposure time 10-14h.

[0010] The preservation number of the Escherichia coli 2-2b is GDMCC 64075.

[0011] Preferably, the concentration of Cr(VI) in the Cr(VI) polluted water is 18-22 mg / L, the chemical oxygen demand is 394.9-434.0 mg / L, the nitrate nitrogen concentration is 168.5-204.1 mg / L, and the total phosphorus concentration is 35.0-44.3 mg / L.

[0012] Preferably, the combined treatment method includes replacing the Cr(VI) contaminated water and logarithmic growth phase Escherichia coli 2-2b every 3 days of combined treatment.

[0013] Preferably, the number of seedlings of Leymus chinensis in the pre-cultured combined treatment system is 25-40; the OD600 value of the combined treatment system is 1.3-1.6.

[0014] The pre-cultured seedlings of Leymus chinensis reached a height of 15-20 cm.

[0015] Preferably, the preparation of the pre-cultured Leymus chinensis seedlings includes: pre-culturing the Leymus chinensis seedlings using Hoagland nutrient solution to obtain the pre-cultured Leymus chinensis seedlings.

[0016] Preferably, the Hoagland nutrient solution comprises the following components at the following concentrations: Ca(NO3)2·4H2O 1.18-1.19 g / L, KNO3 0.50-0.51 g / L, KH2PO4 0.13-0.14 g / L, MgSO4·7H2O 0.49-0.50 g / L, FeSO4·7H2O 5.55-5.60 mg / L, CuSO4·5H2O 0.07-0.08 mg / L, Na2-EDTA·2H2O 6.70-6.80 mg / L, H3BO3 3.60-3.65 mg / L, MnCl2·4H2O 1.90-2.00 mg / L, ZnSO4·7H2O 0.02-0.03 mg / L, and NaMoO4·2H2O 0.02-0.03 mg / L;

[0017] The pH of the Hoagland nutrient solution is 5.8-6.2;

[0018] The pre-culture time is 3-7 days.

[0019] Preferably, the preparation of the logarithmic growth phase Escherichia coli 2-2b includes: culturing the Escherichia coli 2-2b to the logarithmic growth phase, freezing and centrifuging, collecting the bacterial cells, and obtaining the logarithmic growth phase Escherichia coli 2-2b.

[0020] Preferably, the culture temperature is 28-32℃, the rotation speed is 150-200rpm, and the time is 6-18h;

[0021] The refrigerated centrifuge is performed at a temperature of 0-4℃, a rotation speed of 6000-10000 rpm, and a time of 5-15 min.

[0022] Beneficial effects

[0023] This invention isolated a strain of *Escherichia coli* 2-2b from the rhizosphere soil of the chromium hyperaccumulator *Listeria oryzae*. Reintroducing this strain into *Listeria oryzae* promoted the production of chlorophyll, carotenoids, POD, and CAT, thereby enhancing the remediation effect of *Listeria oryzae* on Cr(VI)-contaminated water and efficiently removing Cr(VI), chemical oxygen demand (COD), nitrate nitrogen, and total phosphorus from Cr(VI)-contaminated water. The results of the examples show that the removal rate of Cr(VI) from Cr(VI)-contaminated water can reach up to 43.35%, the removal rate of COD can reach up to 89%, the removal rate of nitrate nitrogen can reach up to 97%, and the removal rate of total phosphorus can reach up to 70%.

[0024] Biological Preservation Information

[0025] Escherichia coli 2-2b was deposited on November 27, 2023, at the Institute of Microbiology, Guangdong Academy of Sciences, China, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province, 510075, China, with accession number GDMCC 64075. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0027] Figure 1 This is a colony morphology diagram of strain 2-2b;

[0028] Figure 2 Phylogenetic tree of the 16S rDNA sequence of strain 2-2b;

[0029] Figure 3 The graph shows the changes in chlorophyll content of Rheum palmatum in the combined experimental and control systems.

[0030] Figure 4 The graph shows the changes in carotenoid content of Rheum palmatum in the combined experimental and control systems.

[0031] Figure 5 The graph shows the changes in POD activity of Rhesperidium argyi in the experimental and control groups combined system;

[0032] Figure 6 The graph shows the changes in CAT activity of Rhesperidin in the experimental and control groups combined system.

[0033] Figure 7 The graph shows the changes in the removal rate of Cr(VI) in Cr(VI)-contaminated water by the combined experimental and control systems.

[0034] Figure 8 The graph shows the changes in COD removal rate of Cr(VI) polluted water by the combined experimental and control systems.

[0035] Figure 9 The graph shows the changes in nitrate nitrogen removal rates in Cr(VI)-contaminated water bodies by the combined experimental and control systems.

[0036] Figure 10 The graph shows the changes in the removal rate of total phosphorus in Cr(VI) polluted water by the combined experimental and control systems. Detailed Implementation

[0037] This invention provides the application of Escherichia coli 2-2b in improving the remediation effect of Listeria monocytogenes on Cr(VI) polluted water; the Escherichia coli 2-2b is deposited at the Institute of Microbiology, Guangdong Academy of Sciences, China, with accession number GDMCC 64075.

[0038] In this invention, the concentration of Cr(VI) in the Cr(VI)-polluted water is preferably 18-22 mg / L, more preferably 20 mg / L; the chemical oxygen demand (COD) in the Cr(VI)-polluted water is preferably 394.9-434.0 mg / L, more preferably 417.6 mg / L; the nitrate nitrogen concentration in the Cr(VI)-polluted water is preferably 168.5-204.1 mg / L, more preferably 182.4 mg / L; and the total phosphorus concentration in the Cr(VI)-polluted water is preferably 35.0-44.3 mg / L, more preferably 40.4 mg / L. The Cr(VI) polluted water body described in this invention preferably comprises the following components at the following concentrations: 18-22 mg / L Cr(VI), 50% v / v Hoagland nutrient solution, CH3COONa 550-560 mg / L, NH4Cl 150-160 mg / L, K2HPO4·3H2O 50-60 mg / L, CaCl2 110-120 mg / L, MgCl2·6H2O 200-210 mg / L, NaHCO3 115-125 mg / L, NaCl 795-805 mg / L, EDTA 0.8-1.2 mg / L, and trace elements 10-30 μL / L; the trace elements preferably comprise the following components at the following concentrations: ZnSO4 2.1-2.3 g / L, MnCl2·4H2O 5.00-5.10 g / L, FeSO4·7H2O 4.90-5.10 g / L, (NH4)6Mo7O2·4H2O 1.05-1.15 g / L, CuSO4·5H2O 150-160 g / L, CoCl2·6H2O 1.60-1.62 g / L; further preferred components include the following concentrations: 18-22 mg / L Cr(VI) 50% v / v Hoagland nutrient solution, CH3COONa 556 mg / L, NH4Cl 1153 mg / L, K2HPO4·3H2O 52 mg / L, CaCl2 115 mg / L, MgCl2·6H2O 203 mg / L, NaHCO3 120 mg / L, NaCl 800 mg / L, EDTA 1 mg / L, trace elements 20 μL / L; the trace elements preferably include the following concentrations: ZnSO4 2.2 g / L, MnCl2·4H2O 5.06 g / L, FeSO4·7H2O 5.0 g / L, (NH4)6Mo7O2·4H2O 1.1 g / L, CuSO4·5H2O 157 g / L, CoCl2·6H2O 1.61 g / L.

[0039] This invention isolates a strain of *Escherichia coli* 2-2b with growth-promoting effects from the rhizosphere soil of the chromium hyperaccumulating plant *Listeria oryzae*. This invention simulates the composition of domestic sewage, establishes a Cr(VI)-contaminated water body, and uses *Listeria oryzae* and the aforementioned *E. coli* 2-2b for combined treatment of the Cr(VI)-contaminated water. Results show that *E. coli* 2-2b has a growth-promoting effect, increasing the chlorophyll, carotenoid, POD, and CAT content of *Listeria oryzae*, thus improving the remediation effect of *Listeria oryzae* on Cr(VI)-contaminated water.

[0040] This invention provides a method for remediating Cr(VI) contaminated water using a combination of Escherichia coli and Listeria monocytogenes, comprising the following steps:

[0041] Pre-cultured Rhes oryzae seedlings were placed in Cr(VI) polluted water and Escherichia coli 2-2b in logarithmic growth phase was introduced to form a co-treatment system for the co-treatment of Cr(VI) polluted water.

[0042] The conditions for the combined treatment are: daytime temperature 28-32℃, nighttime temperature 22-27℃, and daily light exposure time 10-14h.

[0043] The preservation number of the Escherichia coli 2-2b is GDMCC 64075.

[0044] In this invention, the pre-cultured *Leymus chinensis* seedlings preferably have a height of 15-20 cm, more preferably 17-19 cm. The preparation of the pre-cultured *Leymus chinensis* seedlings preferably includes: pre-culturing the *Leymus chinensis* seedlings using Hoagland nutrient solution to obtain the pre-cultured *Leymus chinensis* seedlings. The pre-culture time is preferably 3-7 days, more preferably 5 days. The Hoagland nutrient solution is preferably replaced every 3 days. The pre-culture conditions are preferably: 30°C during the day, 25°C at night, and 12 hours of light per day. The Hoagland nutrient solution of this invention preferably comprises the following components at the following concentrations: Ca(NO3)2·4H2O 1.18-1.19 g / L, KNO3 0.50-0.51 g / L, KH2PO4 0.13-0.14 g / L, MgSO4·7H2O 0.49-0.50 g / L, FeSO4·7H2O 5.55-5.60 mg / L, CuSO4·5H2O 0.07-0.08 mg / L, Na2-EDTA·2H2O 6.70-6.80 mg / L, H3BO3 3.60-3.65 mg / L, MnCl2·4H2O 1.90-2.00 mg / L, ZnSO4·7H2O 0.02-0.03 mg / L, NaMoO4·2H2O 0.02-0.03 mg / L; further preferred components include the following concentrations: Ca(NO3)2·4H2O 1.1808 g / L, KNO3 0.5055 g / L, KH2PO4 0.1361 g / L, MgSO4·7H2O 0.4929 g / L, FeSO4·7H2O 5.5600 mg / L, CuSO4·5H2O 0.0749 mg / L, Na2-EDTA·2H2O 6.7242 mg / L, H3BO3 3.6414 mg / L, MnCl2·4H2O 1.9791 mg / L, ZnSO4·7H2O 0.0230 mg / L, NaMoO4·2H2O 0.0242 mg / L. The pH of the Hoagland nutrient solution described in this invention is preferably 5.8-6.2, and more preferably 6.0. This invention utilizes the Hoagland nutrient solution to pretreat Leymus chinensis seedlings, enabling seedlings collected from the wild to adapt to the hydroponic environment and obtain sufficient nutrients for growth. The Leymus chinensis seedlings described in this invention are preferably healthy seedlings with intact rhizomes, unaffected by Cr(VI) damage; the seedlings are preferably collected from paddy fields in Yanshan District, Guilin City, Guangxi Zhuang Autonomous Region.

[0045] In this invention, the preparation of *E. coli* 2-2b in the logarithmic growth phase preferably includes: culturing *E. coli* 2-2b to the logarithmic growth phase, centrifuging at room temperature, collecting the bacterial cells, and obtaining *E. coli* 2-2b in the logarithmic growth phase. The culture temperature is preferably 28-32℃, more preferably 30℃; the culture speed is preferably 150-200 rpm, more preferably 180 rpm; the culture time is preferably 6-18 h, more preferably 12 h. The OD600 of the logarithmic growth phase is preferably 1.3-1.6, more preferably 1.4-1.5. The centrifugation temperature is preferably 0-4℃, more preferably 4℃; the centrifugation speed is preferably 6000-10000 rpm, more preferably 8000 rpm; the centrifugation time is preferably 5-15 min, more preferably 10 min. Culturing *E. coli* 2-2b to the logarithmic growth phase in this invention enables the cells to be in an active state, enhancing cell proliferation and metabolic capacity.

[0046] After obtaining pre-cultured Leymus chinensis seedlings and culturing Escherichia coli 2-2b to the logarithmic growth phase, the present invention places the pre-cultured Leymus chinensis seedlings in Cr(VI) polluted water and inoculates them with Escherichia coli 2-2b in the logarithmic growth phase to form a combined treatment system for the combined treatment of Cr(VI) polluted water.

[0047] In this invention, the number of seedlings of Leymus chinensis after pre-culture in the combined treatment system is preferably 25-40, more preferably 30-35; the OD600 value of the combined treatment system is preferably 1.3-1.6, more preferably 1.4-1.5.

[0048] In this invention, the combined treatment temperature is 28-32℃ during the day and 22-27℃ at night; the preferred daytime temperature is 30℃; the preferred nighttime temperature is 25℃; and the combined treatment light duration is 10-14 h / d, preferably 12 h. This invention specifies the combined treatment conditions to ensure that *Rhizophora stearacea* grows normally at a suitable temperature and receives sufficient light. The preferred method of combined treatment in this invention includes replacing the Cr(VI)-contaminated water and logarithmic-phase *Escherichia coli* 2-2b every 3 days of combined treatment. The combined treatment method provided by this invention can achieve the cyclical remediation of Cr(VI)-contaminated water. Example results show that the removal rate of Cr(VI) can reach up to 43.35%, the removal rate of COD can reach up to 89%, the removal rate of nitrate nitrogen can reach up to 97%, and the removal rate of total phosphorus can reach up to 70%.

[0049] To further illustrate the present invention, the following detailed description, in conjunction with the accompanying drawings and embodiments, describes a method for remediating Cr(VI) contaminated water using a combination of Escherichia coli and Listeria monocytogenes, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0050] Unless otherwise specified, all culture media used in the specific implementation of this invention are conventional culture media in the art. Specific experimental methods not mentioned in the following examples are generally performed according to conventional experimental methods.

[0051] Example 1

[0052] Strains Isolation and Identification

[0053] (1) In a paddy field in Yanshan District, Guilin City, well-grown Rhizophora oryzae were uprooted, and loose, sticky soil attached to the roots was removed by shaking the plants. The soil firmly attached to the roots (i.e., rhizosphere soil) was collected into sterilized centrifuge tubes. Take 10g of soil sample and add it to an Erlenmeyer flask containing 90ml of PBS (phosphate buffered solution) or 0.85% physiological saline. Sonicate in an ultrasonic cleaner for 15min. After standing, remove the supernatant and keep the soil sample. Wash and remove impurities. Place the soil sample in LB liquid medium containing 50mg / L Cr(VI) (10g / L tryptone, 10g / L sodium chloride, 5g / L yeast extract and the remainder water; sterilize at 121℃ for 20min before use). Incubate at 30℃ and 200rpm for 24h with shaking. Centrifuge at 8000rpm for 10min, remove the supernatant, and then continue to place the soil sample in LB liquid medium containing 50mg / L Cr(VI) and incubate at 30℃ and 200rpm for another 24h with shaking (bacterial enrichment).

[0054] (2) Take 0.5-1 mL of the soil sample suspension after shaking in step (1), and dilute it 10-fold in a serial manner. Spread the solution onto LB solid medium containing 50 mg / L Cr(VI). Invert the medium and incubate at 30°C for 72 h. Select colonies with good growth, record the characteristics of different colony morphologies, and perform multiple separation and purification using the streak plating method to obtain a single colony of Cr(VI)-resistant Rhizobium ritrium, which is named 2-2b. The obtained colony is then cultured and enriched on fresh LB solid medium to obtain a pure culture medium. The bacteria are round, white, and opaque. Figure 1 ).

[0055] (3) The strain 2-2b obtained in step (2) was sent to Shanghai Sangon Biotech Co., Ltd. for 16S rDNA bidirectional sequencing. The obtained sequences were compared with the NCBI database using BLAST to obtain the information of the strain with the highest sequence similarity to the test strain. The sequences were then uploaded to the GenBank database to obtain sequence numbers. Then, the obtained strain sequences and the highly similar 16S gene sequences were arranged using MEGA11 software, and a phylogenetic tree was constructed using the Neighbor-joining method. The results are as follows: Figure 2 .according to Figure 2

[0056] Example 2

[0057] 1. In a paddy field in Yanshan District, Guilin City, healthy seedlings of *Leymus chinensis* were uprooted and preserved intact before being sent to the laboratory. The collected seedlings were repeatedly rinsed with clean water to remove mud and impurities from the roots. They were then placed in small plastic containers containing 50% Hoagland nutrient solution for hydroponic pre-culture for 5 days, with the nutrient solution being changed every 3 days. The culture temperature was set at 30℃ during the day and 25℃ at night; the daily light duration was 12 hours. The resulting pre-cultured *Leymus chinensis* seedlings were obtained.

[0058] 2. The Escherichia coli 2-2b obtained in Example 1 was cultured in LB liquid medium at 30°C and 180 rpm for 12 h with shaking. The strain was cultured to the logarithmic growth phase with an OD600 value of 1.4-1.5. Then, it was centrifuged at 4°C and 8000 rpm for 10 min to collect the bacterial cells and obtain Escherichia coli 2-2b in the logarithmic growth phase.

[0059] 3. Simulate the composition of domestic sewage and prepare a Cr(VI) polluted water body, specifically composed of the following components at the following concentrations: 20 mg / L Cr(VI), 50% v / v Hoagland nutrient solution, CH3COONa 556 mg / L, NH4Cl 1153 mg / L, K2HPO4·3H2O 52 mg / L, CaCl2 115 mg / L, MgCl2·6H2O 203 mg / L, NaHCO3 120 mg / L, NaCl 800 mg / L, EDTA 1 mg / L, trace elements 20 μL / L, and the balance being pure water;

[0060] The trace element composition is as follows: ZnSO4 2.2 g / L, MnCl2·4H2O 5.06 g / L, FeSO4·7H2O 5.0 g / L, (NH4)6Mo7O2·4H2O 1.1 g / L, CuSO4·5H2O 157 g / L, CoCl2·6H2O 1.61 g / L, and the balance being pure water;

[0061] The Hoagland nutrient solution composition is as follows: Ca(NO3)2·4H2O 1.1808 g / L, KNO3 0.5055 g / L, KH2PO4 0.1361 g / L, MgSO4·7H2O 0.4929 g / L, FeSO4·7H2O 5.5600 mg / L, CuSO4·5H2O 0.0749 mg / L, Na2-EDTA·2H2O 6.7242 mg / L, H3BO3 3.6414 mg / L, MnCl2·4H2O 1.9791 mg / L, ZnSO4·7H2O 0.0230 mg / L, NaMoO4·2H2O 0.0242 mg / L, and the remainder being pure water;

[0062] 4. After the pre-culture in step 1, select seedlings of *Leymus chinensis* with similar growth conditions, i.e., plants with a height of 17-19 cm. Wash the roots with pure water, then place them in 250 ml Erlenmeyer flasks and randomly divide them into experimental and control groups, with 3 replicates in each group. Each replicate contains 30-35 *Leymus chinensis* plants, and the following treatments are performed:

[0063] Experimental group (Rhizoctonia solani + 20 mg / L Cr(VI) + strain): Escherichia coli 2-2b obtained in step 2 in logarithmic growth phase and 250 mL of Cr(VI) contaminated water from step 3 were inoculated to form a co-treatment system; the initial OD600 value of the co-treatment system was 1.4-1.5;

[0064] Control group (Rhizophora stylosa + 20 mg / L Cr(VI)): 250 mL of water contaminated with Cr(VI) from step 3 was added, without adding any bacterial strains, forming a control treatment system; the initial OD600 value of the control treatment system was 1.4-1.5;

[0065] Both the experimental and control groups were cultured under conditions of 30°C during the day and 25°C at night, with a light exposure of 12h / d. Every 3 days, the experimental group was replaced with new Escherichia coli 2-2b in the logarithmic growth phase and Cr(VI)-contaminated water, while the control group was replaced with new Cr(VI)-contaminated water every 3 days. Each 3-day remediation of Cr(VI)-contaminated water was recorded as one water intake.

[0066] (1) The chlorophyll and carotenoid contents, as well as the POD and CAT activities, were measured in the experimental and control groups at days 0, 12, and 30 of cultivation. Chlorophyll and carotenoid contents were determined using the ethanol extraction-spectrophotometric method (Gao Junfeng. Experimental Guide to Plant Physiology [M]. Beijing: Higher Education Press, 2006). POD activity was determined using the guaiacol method, and CAT activity was determined using the ultraviolet absorption method. The results are shown in Table 1 and... Figures 3-6 As shown.

[0067] Table 1. Measurement results of chlorophyll, carotenoids, POD, and CAT

[0068]

[0069] Chlorophyll is an essential substance for photosynthesis, which is necessary for plant growth. Changes in chlorophyll levels can reflect the damage caused by heavy metals to plants. Carotenoids can scavenge reactive oxygen species in leaves, protecting chlorophyll and chloroplasts from photo-oxidative loss or damage. POD is an important respiratory enzyme in plants, catalyzing H2O2 oxidase reactions and oxidizing guaiacol; CAT is a hemolytic protease that catalyzes the decomposition of H2O2 into water and oxygen. Chlorophyll, carotenoids, and the activities of both enzymes are all related to plant physiological metabolism and resistance.

[0070] According to Table 1 and Figures 3-6 It can be seen that the chlorophyll content in both the experimental and control groups showed a decreasing trend, but the chlorophyll content in the experimental group remained higher than that in the control group. Similarly, the carotenoid content in both treatment groups also showed a decreasing trend, but the carotenoid content in the experimental group remained higher than that in the control group. The continuous decrease in chlorophyll in both the experimental and control groups is presumably due to the initial Cr(VI) stress, which prevented chlorophyll from having a defense mechanism, thus causing a decrease. However, under Cr(VI) stress, the experimental group could still promote the production of carotenoids (or antioxidant enzymes) to help eliminate reactive oxygen species in the leaves, hence the consistently higher chlorophyll and carotenoid content in the experimental group compared to the control group. However, Cr(VI) also damages chloroplasts, inhibiting chlorophyll synthesis, thus causing a continuous decrease in chlorophyll and carotenoid content in both treatment groups. The POD activity in the experimental group reached its highest value of 24.55 U / gFW on day 12, showing an increase in POD activity after treatment compared to the control group. The highest CAT activity in the experimental group was 8.60 U / gFW on day 12, showing an increase in CAT activity compared to the control group. Both enzyme activities reflect plant resistance to Cr(VI). Generally, higher enzyme activity indicates stronger plant resistance to Cr(VI); the higher enzyme activity in the experimental group compared to the control group over time indicates that the experimental group improved plant resistance to Cr(VI).

[0071] (2) During the culture process, 5 mL samples were taken every 24 hours to measure OD600. The samples were then centrifuged at 8000 rpm for 5 min at 4℃. The supernatant was collected, and Cr(VI) was determined using the diphenylcarbazide spectrophotometric method. The removal rate of Cr(VI) was calculated according to the following formula. The results are shown in Table 2 and... Figure 7 .

[0072] Cr(VI) removal rate (%) = (Cr(VI) influent value - Cr(VI) measured value) / Cr(VI) influent value × 100;

[0073] Table 2. Cr(VI) Measurement Results

[0074]

[0075]

[0076]

[0077] According to Table 2 and Figure 7 It can be seen that the removal rate of Cr(VI) in the experimental group gradually increased from day 3, with the highest removal rate of 43.35% at day 27-30. In contrast, the removal rate of Cr(VI) in the control group generally did not exceed 15%. The longitudinal comparison over time shows that the removal rate of Cr(VI) in the experimental group was higher than that in the control group, indicating that the Escherichia coli 2-2b-Listeria system significantly enhanced the removal effect of Cr(VI) in water.

[0078] (3) During the cultivation process, at the beginning and end of each water influent intake, Cr(VI) polluted water was determined according to the "Determination of Nitrate Nitrogen in Water Quality - Ultraviolet Spectrophotometry (Trial)" (HJ / T 346-2007) and the "Determination of Total Phosphorus in Water Quality - Ammonium Molybdate Spectrophotometry" (GB 11893-89), and COD was determined using a COD meter. After each measurement, the removal rates of COD, nitrate nitrogen, and total phosphorus were calculated according to the following formulas. The results are shown in Tables 3-5. Figures 8-10 ;

[0079] COD removal rate (%) = (Initial COD value in influent - Ending COD value in influent) / Initial COD value in influent × 100;

[0080] Nitrate nitrogen removal rate (%) = (initial value of nitrate nitrogen in the influent - final value of nitrate nitrogen in the influent) / initial value of nitrate nitrogen in the influent × 100;

[0081] Total phosphorus removal rate (%) = (total phosphorus influent starting value - total phosphorus influent ending value) / total phosphorus influent starting value × 100.

[0082] Table 3 COD Measurement Results

[0083]

[0084]

[0085] Table 4. Nitrate Nitrogen Measurement Results

[0086]

[0087]

[0088] Table 5 Total Phosphorus Measurement Results

[0089]

[0090] According to Table 3 and Figure 8 It can be seen that the highest influent COD was 434.0 mg / L, the lowest was 394.9 mg / L, and the average influent COD was 417.6 mg / L. Both treatment groups were able to remove COD, and the COD removal efficiency of each group tended to stabilize after 6 days. For the control group, more than 97% of the COD could be removed every 3 days after 9 days; for the experimental group, the highest COD removal rate reached 89%. The results demonstrate that the Escherichia coli 2-2b-Listeria system can remove COD from water.

[0091] According to Table 4 and Figure 9 It can be seen that the highest influent nitrate nitrogen concentration was 204.1 mg / L, the lowest was 168.5 mg / L, and the average influent nitrate nitrogen concentration was 182.4 mg / L. Both treatment groups were able to remove nitrate nitrogen, and their removal rates tended to stabilize after 9 days. Among them, the control group had the highest nitrate nitrogen removal rate of 60%, while the experimental group had the highest removal rate of 97%. A longitudinal comparison over time shows that the experimental group consistently achieved higher nitrate nitrogen removal rates than the control group, and the removal effect of the experimental group became increasingly significant over time, indicating that the *E. coli* 2-2b-Listeria system has a promoting effect on the removal of nitrate nitrogen in water.

[0092] According to Table 5 and Figure 10 It can be seen that the highest influent total phosphorus concentration was 44.3 mg / L, the lowest was 35.0 mg / L, and the average influent total phosphorus concentration was 40.4 mg / L. Both treatment groups were able to remove total phosphorus, and the removal rate tended to stabilize after 9 days. The total phosphorus removal rate development curves of the two treatment groups were similar both horizontally and vertically, with the highest removal rates of total phosphorus in the control group and the experimental group being 73% and 70%, respectively. The results demonstrate that the *Escherichia coli* 2-2b-Listeria system can remove total phosphorus from water bodies.

[0093] As can be seen from the above, the technical solution provided by the present invention can effectively remove Cr(VI), chemical oxygen demand, nitrate nitrogen and total phosphorus from Cr(VI) polluted water, and Escherichia coli 2-2b can enhance the remediation effect of Listeria monocytogenes on Cr(VI) polluted water.

[0094] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Application of Escherichia coli 2-2b in improving the remediation effect of Listeria monocytogenes on Cr(VI) polluted water; the Escherichia coli 2-2b is deposited at the Institute of Microbiology, Guangdong Academy of Sciences, China, with accession number GDMCC64075.

2. The application according to claim 1, characterized in that, The concentrations of Cr(VI) in the Cr(VI)-polluted water bodies were 18-22 mg / L, chemical oxygen demand (COD) was 394.9-434.0 mg / L, nitrate nitrogen was 168.5-204.1 mg / L, and total phosphorus was 35.0-44.3 mg / L.

3. A method for remediating Cr(VI)-contaminated water using a combination of Escherichia coli and Listeria monocytogenes, characterized in that, Includes the following steps: Pre-cultured Rhes oryzae seedlings were placed in Cr(VI) polluted water and Escherichia coli 2-2b in logarithmic growth phase was introduced to form a co-treatment system for the co-treatment of Cr(VI) polluted water. The conditions for the combined treatment are: daytime temperature 28-32℃, nighttime temperature 22-27℃, and daily light exposure time 10-14h. The preservation number of the Escherichia coli 2-2b is GDMCC 64075.

4. The method according to claim 3, characterized in that, The concentrations of Cr(VI) in the Cr(VI)-polluted water bodies were 18-22 mg / L, chemical oxygen demand (COD) was 394.9-434.0 mg / L, nitrate nitrogen was 168.5-204.1 mg / L, and total phosphorus was 35.0-44.3 mg / L.

5. The method according to claim 3, characterized in that, The combined treatment method includes replacing the Cr(VI) contaminated water and logarithmic growth phase Escherichia coli 2-2b every 3 days of combined treatment.

6. The method according to claim 3, characterized in that, The number of seedlings of Leymus chinensis in the pre-cultured combined treatment system was 25-40; the OD600 value of the combined treatment system was 1.3-1.

6. The pre-cultured seedlings of Leymus chinensis reached a height of 15-20 cm.

7. The method according to claim 3, characterized in that, The preparation of the pre-cultured Leymus chinensis seedlings includes: pre-culturing the Leymus chinensis seedlings using Hoagland nutrient solution to obtain the pre-cultured Leymus chinensis seedlings.

8. The method according to claim 7, characterized in that, The Hoagland nutrient solution comprises the following components at the following concentrations: Ca(NO3)2·4H2O 1.18-1.19 g / L, KNO3 0.50-0.51 g / L, KH2PO4 0.13-0.14 g / L, MgSO4·7H2O 0.49-0.50 g / L, FeSO4·7H2O 5.55-5.60 mg / L, CuSO4·5H2O 0.07-0.08 mg / L, Na2-EDTA·2H2O 6.70-6.80 mg / L, H3BO3 3.60-3.65 mg / L, MnCl2·4H2O 1.90-2.00 mg / L, ZnSO4·7H2O 0.02-0.03 mg / L, and NaMoO4·2H2O 0.02-0.03 mg / L. The pH of the Hoagland nutrient solution is 5.8-6.2; The pre-culture time is 3-7 days.

9. The method according to claim 3, characterized in that, The preparation of the logarithmic growth phase Escherichia coli 2-2b includes: culturing the Escherichia coli 2-2b to the logarithmic growth phase, freezing and centrifuging, collecting the bacterial cells, and obtaining the logarithmic growth phase Escherichia coli 2-2b.

10. The method according to claim 9, characterized in that, The culture temperature is 28-32℃, the rotation speed is 150-200rpm, and the time is 6-18h; The refrigerated centrifuge is performed at a temperature of 0-4℃, a rotation speed of 6000-10000 rpm, and a time of 5-15 min.

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