Enterobacter ludwigii HHXJ1T1-1, a bacterial agent and application thereof

Abstract

The application belongs to the technical field of microorganisms, and specifically discloses Enterobacter ludwigii HHXJ1T1-1 as well as a bacterial agent and application thereof. The Enterobacter ludwigii HHXJ1T1-1 of the application has a preservation number of GDMCC No.62758. The Enterobacter ludwigii can effectively degrade pesticides and / or adsorb heavy metals, and can be used to resist the stress of heavy metals, thus being beneficial to the green development of agriculture.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to Enterobacter leuciscus HHXJ1T1-1, its inoculum and applications. Background Technology

[0002] Pollution from organic compounds (pesticides, herbicides, plastics, tannins, polyphenols, etc.) and inorganic compounds (cadmium, copper, lead, chromium, mercury, etc.) is a pervasive global problem. The long-term accumulation and non-degradable nature of these pesticides, herbicides, and heavy metals in the environment pose potential hazards to ecosystems and human health.

[0003] There are several ways to reduce pesticide or heavy metal residues, including reducing usage, chemical treatment, and microbial treatment. Some pesticides or inorganic chemicals remain widely used because suitable alternatives have not yet been found. Chemical methods, such as adding adsorbents and stabilizers, can sometimes be ineffective and may even cause new pollution. Microbial treatment, on the other hand, is a greener and more efficient method.

[0004] Therefore, screening a strain with efficient heavy metal adsorption and pesticide degradation capabilities is of great significance and can provide strong support for alleviating environmental heavy metal and pesticide pollution. Summary of the Invention

[0005] In view of the above, it is necessary to provide a method that can effectively degrade pesticides and / or adsorb heavy metals, thereby enabling the degradation of pesticides and resistance to heavy metal stress, which is conducive to the green development of agriculture.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] This invention provides a strain of Enterobacter ludwigii HHXJ1T1-1, whose taxonomic name is Enterobacter ludwigii. It is deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC) at the address of Building 59, No. 100 Xianlie Middle Road, Guangzhou, on September 2, 2022, with accession number GDMCC No. 62758.

[0008] The present invention also provides a microbial agent comprising Enterobacter leucisgenus HHXJ1T1-1 as described above and / or its fermentation products.

[0009] The present invention also provides the application of the above-mentioned Enterobacter leucisgenin HHXJ1T1-1 or the above-mentioned bacterial agent in the degradation of pesticides and / or adsorption of heavy metals.

[0010] Furthermore, the pesticides mentioned include pyrethroid pesticides.

[0011] Furthermore, the heavy metals mentioned include chromium and / or cadmium.

[0012] The present invention has the following beneficial effects:

[0013] The Enterobacter ludwigii HHXJ1T1-1 of this invention was isolated from the rhizosphere soil of plants in Xinjiang. It can effectively degrade pesticides and / or adsorb heavy metals. The degradation rate of pyrethroid pesticides can reach up to 87.43% after 8 days, and the adsorption rates of chromium and cadmium can reach up to 91.54% and 99.33%, respectively. It can provide strong support for alleviating environmental heavy metal pollution and pesticide pollution. Attached Figure Description

[0014] Figure 1 This is a colony diagram of the Enterobacter luteolyticus strain HHXJ1T1-1 of this invention on a plate.

[0015] Figure 2 This is a microscopic image of the Enterobacter lucifera strain HHXJ1T1-1 of this invention.

[0016] Information on the preservation of biological materials

[0017] The strain information deposited in this application is: Enterobacter ludwigii HHXJ1T1-1, with the taxonomic name Enterobacter ludwigii, deposited at Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, on September 2, 2022, with accession number GDMCC No. 62758. Detailed Implementation

[0018] To make the technical problems, technical solutions and advantages of the present invention clearer, a detailed description will be given below in conjunction with specific embodiments.

[0019] All features disclosed in this specification, or all steps in all disclosed methods or processes, may be combined in any way, except for mutually exclusive features and / or steps.

[0020] Unless otherwise stated, each feature disclosed in this specification (including any appended claims and abstract) is merely one example of a series of equivalent or similar features.

[0021] Example 1

[0022] This embodiment provides an *Enterobacter ludwigii* HHXJ1T1-1 strain, which was isolated from rhizosphere soil of plants in Xinjiang. The specific isolation and screening method is as follows:

[0023] (1) Isolation and purification of strains

[0024] Soil samples were collected from the rhizosphere soil of plants in Xinjiang, placed in sterile bags, and brought back to the laboratory for storage at 4℃. 10g of fresh soil sample was weighed and added to 90mL of sterile physiological saline, shaken at 180 rpm for 30 min, and then incubated at 28℃ for 24 h. After enrichment, the supernatant was diluted to 10⁻⁶. -3 10 -4 10 -5 Three gradients were used, and 100 μL of liquid from each dilution was spread onto LB solid medium. After incubation at 35°C for 1 day, single colonies grew. After colony growth, the strain was purified three times on LB medium plates using the streak method to obtain a single bacterial strain, numbered HHXJ1T1-1.

[0025] (2) Physiological and biochemical identification of colonies

[0026] Refer to Bergey's Manual of Bacteriological Identification (9th Edition) to observe the morphology, color, etc., of the colonies grown by the strain on the surface of LB medium. Figure 1 Colony morphology diagram and Figure 2 The microscopic image is shown.

[0027] (3) Sequence analysis of the strain's 16S rRNA

[0028] The strain HHXJ1T1-1 was sent to the sequencing and identification department of Shanghai Sangon Biotech for sequencing identification. The sequencing results are shown below:

[0029]

[0030] The sequencing results were compared and analyzed in a ribosome database. The 16S rDNA sequence analysis results showed that the strain of the present invention had 100% homology with Enterobacter ludwigii of the Bacillus genus, that is, the strain is Enterobacter ludwigii.

[0031] The microorganisms in this embodiment are preserved as follows:

[0032] Enterobacter ludwigii HHXJ1T1-1, taxonomically named Enterobacter ludwigii, is deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC) at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, on September 2, 2022, with accession number GDMCC No. 62758.

[0033] Example 2

[0034] This embodiment measures the degradation rate of pyrethroid pesticides by Enterobacter ludwigii HHXJ1T1-1 in Example 1 above. The specific measurement method is as follows:

[0035] (1) Preparation of Enterobacter luteoli seed culture: Take 5 mL of Enterobacter luteoli HHXJ1T1-1 bacterial culture and inoculate it into 50 mL of TSA medium. Incubate at 37℃ and 150 r / min. Take samples intermittently and measure the absorbance at 600 nm (A). 600 ) with OD 600 The bacterial culture at a concentration of ≈1 was used as the seed culture.

[0036] (2) Inoculation and Culture: The *Enterobacter lutei* seed culture was inoculated at a rate of 5% into 300 mL of MSM medium containing bifenthrin (50 mg / L), cypermethrin (50 mg / L), and deltamethrin (50 mg / L), respectively. On days 2, 4, 6, and 8, 5 mL of each culture was collected for pesticide residue determination. Simultaneously, A... 600 .

[0037] (3) Determination method: Referring to Part 9 of the "Standard Examination Methods for Drinking Water" (GB / T 5750.9-2023): Pesticide Indicators, pesticide residues were determined by gas chromatography-tandem mass spectrometry (CC-MS / MS), and the degradation rate (ρ) of pesticides by Enterobacter leucis HHXJ1T1-1 was calculated. The calculation formula is as follows:

[0038] ρ = (1 - Al / A0) × 100%

[0039] In the formula, A1 is the pesticide residue concentration (mg / L) after treatment with Enterobacter leuciscus HHXJ1T1-1, and A0 is the pesticide residue concentration (mg / L) of the control group.

[0040] The measurement results are shown in Table 1 below.

[0041] Table 1. Degradation rate (%) of pyrethroid pesticides by Enterobacter leucobacter HHXJ1T1-1

[0042]

[0043] As shown in Table 1, the degradation rate of pyrethroid pesticides by Enterobacter ludwigii HHXJ1T1-1 of the present invention reached over 80% after 8 days of culture, with the highest reaching 87.43%.

[0044] Example 3

[0045] This embodiment tests the heavy metal resistance of Enterobacter ludwigii HHXJ1T1-1 in Example 1 above. The specific test method is as follows:

[0046] (1) Activation of Enterobacter luteolyticus strain HHXJ1T1-1 was activated. 100 μL of bacterial suspension was spread on a plate and incubated at 37 °C for 24 h. Single colonies were picked from the solid plate for liquid culture. The operation was repeated twice.

[0047] (2) Activity of the strain in a medium containing heavy metal ions

[0048] Enterobacter leucovorin strain HHXJ1T1-1 was inoculated at a 2% inoculum into culture media containing different cadmium ion solutions (0 mg / L, 50 mg / L, 100 mg / L, 200 mg / L, 300 mg / L) and different chromium ion solutions (0 mg / L, 50 mg / L, 100 mg / L, 200 mg / L, 300 mg / L), with a heavy metal ion-free medium as a control. The culture was incubated at 37°C for 24 h, and the OD was measured. 600 The value was calculated, and the survival rate (S) in the medium containing heavy metal ions was determined. The calculation formula is as follows:

[0049] S(%) = (OD2 - OD1) × 100%

[0050] In the formula: OD1: OD of the strain after inoculation into a culture medium containing different heavy metal ions for 0 h. 600OD2: OD value of the strain after 24 hours of inoculation into a medium containing different heavy metal ions. 600 value.

[0051] The results are shown in Table 2 below.

[0052] Table 2. Activity of Enterobacter leuciscus in heavy metals cadmium and chromium.

[0053]

[0054] Table 2 shows that cadmium had no significant effect on the activity of *Enterobacter luciferae* HHXJ1T1-1 within the concentration range of 0-300 mg / L, with survival rates consistently above 90% and showing no significant difference. Chromium also had no significant effect on the activity of *Enterobacter luciferae* HHXJ1T1-1 within the concentration range of 0-100 mg / L, with survival rates consistently above 90%, and a good survival rate of over 80% was also observed within the concentration range of 200-300 mg / L. This indicates that the *Enterobacter luciferae* HHXJ1T1-1 of this invention exhibits strong tolerance to chromium and cadmium.

[0055] Example 4

[0056] This embodiment measures the adsorption rate of cadmium and chromium by Enterobacter leukemia HHXJ1T1-1 in Example 1 above. The specific measurement method is as follows:

[0057] A suspension of *Enterobacter leukemia* HHXJ1T1-1 was added to a solution containing 50 mg / L chromium and 50 mg / L cadmium to achieve a final bacterial suspension concentration of 5 g / L. The suspension was incubated at 37°C and 150 rpm for 60 min with shaking. After centrifugation, the supernatant was collected, and the concentration of heavy metal ions was determined using an atomic absorption spectrophotometer. The adsorption rate (R) was calculated. Three parallel experiments were conducted. The calculation formula is as follows:

[0058] R(%) = (M0 - M1) / M0) * 100%

[0059] In the formula: M0 is the concentration of heavy metals in the initial solution; M1 is the concentration of heavy metals in the solution after adsorption.

[0060] The results are shown in Table 3 below.

[0061] Table 3 Adsorption effect of Enterobacter leuciscus on heavy metals cadmium and chromium

[0062]

[0063] As shown in Table 3, when the concentration of Enterobacter lutea HHXJ1T1-1 in this invention is 5 g / L, the adsorption rate of chromium and cadmium is above 90%, indicating that Enterobacter lutea HHXJ1T1-1 in this invention has a good adsorption effect on heavy metals cadmium and chromium.

Claims

1. Enterobacter ludwigii HHXJ1T1-1, characterized in that, The preservation number of the Enterobacter ludwigii HHXJ1T1-1 is GDMCC No.62758.

2. A microbial agent comprising the Enterobacter ludwigii HHXJ1T1-1 according to claim 1 and / or a fermentation product of the Enterobacter ludwigii HHXJ1T1-1 according to claim 1, wherein the fermentation product comprises the Enterobacter ludwigii HHXJ1T1-1 according to claim 1.

3. The Enterobacter ludwigii HHXJ1T1-1 of claim 1 or the microbial agent of claim 2 in the degradation of pesticides and / or adsorption of heavy metals, characterized in that, The pesticide is a pyrethroid pesticide, and the heavy metal is chromium and / or cadmium.

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