Pseudomonas caviae LH-Cr-2, its fermentation process and use

Abstract

The application discloses a kind of septal inflammation pseudomonas LH-Cr-2, belong to microbial technical field.The biological preservation number of septal inflammation pseudomonas LH-Cr-2 is CGMCC NO.27552.The application also discloses the fermentation culture method of septal inflammation pseudomonas LH-Cr-2 for use as a way.The septal inflammation pseudomonas LH-Cr-2 of the application can efficiently reduce different concentrations of soluble hexavalent chromium, provides experimental technology and theoretical basis for the application of microbial reduction technology in chromium-contaminated soil remediation.Six valence chromium is efficiently reduced, and in-situ remediation of chromium-contaminated soil or groundwater can be applied, and can also be used in other related fields that can solve problems by reducing hexavalent chromium.

Description

Technical Field

[0001] This invention relates to microbial strains, and in particular to a strain of *Pseudomonas diaphragmatis* LH-Cr-2. The invention also relates to a fermentation culture method and uses of this *Pseudomonas diaphragmatis* LH-Cr-2. Background Technology

[0002] Reduction fixation is a widely used method for remediating Cr(VI) contaminated soil due to its advantages of rapid remediation, high efficiency, and simple operation. However, chemical reduction methods consume large amounts of reducing agents and are costly. Furthermore, the addition of various materials can alter the physicochemical and microbiological properties of the soil, potentially affecting its usability.

[0003] Chemical reduction of Cr(VI) to Cr(III) using reducing agents is currently the most widely used technology for remediating Cr(VI) contaminated soil. It is highly efficient and adaptable. More importantly, in engineering practice, Cr(VI) contaminated soil generally follows the strategy of in-situ Cr(VI) reduction and reduction precipitation / immobilization. Chemical reduction methods consume large amounts of reducing agents and are costly. The addition of various materials can alter the physicochemical and microbiological properties of the soil, potentially affecting its usability.

[0004] In the prior art, Chinese patent document CN 114657090 A discloses a strain of microbacterium and a microbial reduction method for remediating chromium-contaminated soil. It provides a microbacterium strain, classified as *Microbacterium sp. GRINML SWG1*, with accession number CCTCC NO: M2021992. This microbacterium can reduce soluble hexavalent chromium of varying concentrations. Its drawbacks include a relatively long reduction time and a low reduction rate.

[0005] Therefore, it is extremely important to find a new strain of Pseudomonas diaphragmatis for the reduction of soluble hexavalent chromium Cr(VI). Summary of the Invention

[0006] The technical problem this invention aims to solve is to address the shortcomings of existing technologies by providing a novel *Pseudomonas diaphragmatis* strain. The purpose of this invention is to provide a microorganism capable of efficiently reducing soluble hexavalent chromium at different concentrations, thus providing experimental techniques and theoretical basis for the application of microbial reduction technology in the remediation of chromium-contaminated soil. It achieves highly efficient reduction of hexavalent chromium. This invention belongs to the field of microbial technology, specifically involving a microbacterial strain. Potential applications include in-situ remediation of chromium-contaminated soil or groundwater, and it can also be used in other related fields where the reduction of hexavalent chromium can solve the problem.

[0007] To achieve the above objectives, this invention provides a *Pseudomonas phragmitis* bacterium, classified and named as *Pseudomonas phragmitis*, deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 33, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, on June 5, 2023, with accession number CGMCC NO. 27552.

[0008] The present invention also discloses a fermentation culture method for the aforementioned *Pseudomonas diaphragmatis* LH-Cr-2, characterized in that: the fermentation culture medium formula is as follows: per 1 liter of fermentation broth, there are 3 grams of glucose, 3 grams of corn flour, 5 grams of soybean meal, 2 grams of yeast extract, 8 grams of sodium chloride, 2 grams of dipotassium hydrogen phosphate, and 0.03 grams of manganese sulfate.

[0009] The fermentation conditions were: pH 7.2, fermentation temperature: 37℃.

[0010] The present invention also discloses the use of the aforementioned *Pseudomonas diaphragmatis* LH-Cr-2, characterized in that the use is to use the strain or its fermentation broth for the reduction of soluble hexavalent chromium Cr(VI).

[0011] A further preferred technical solution for the use of *Pseudomonas diaphragmatis* LH-Cr-2 described above in this invention is:

[0012] 1. The intended use is to apply the strain or its fermentation broth to the remediation of hexavalent chromium (Cr(VI)) contaminated soil.

[0013] 2. The intended use is to apply the strain or its fermentation broth to the treatment of groundwater contaminated with hexavalent chromium (Cr(VI)). Prior to treatment, it is preferable to control the concentration of hexavalent chromium (Cr(VI)) below 1000 mg / L; more preferably, the concentration of hexavalent chromium (Cr(VI)) below 100 mg / L.

[0014] Compared with existing technologies, the present invention has the following advantages:

[0015] 1. The microbial Pseudomonas diaphragmaticis LH-Cr-2 provided by this invention has a strong reducing ability and significant repair effect at a concentration of hexavalent chromium Cr(VI) (0-500 mg / L).

[0016] 2. The microorganisms provided by this invention are inexpensive, green and economical, easy to prepare, have a short remediation time, and good long-term stability, making them particularly suitable for the remediation of hexavalent chromium contaminated soil. Attached Figure Description

[0017] Figure 1 Phylogenetic tree of the 16S rDNA sequence of strain LH-Cr-2;

[0018] Figure 2 This is a colony morphology diagram of strain LH-Cr-2;

[0019] Figure 3 Electron micrograph of strain LH-Cr-2;

[0020] Figure 4 The graph shows the reduction rate of hexavalent chromium by strain LH-Cr-2 under high concentrations of hexavalent chromium.

[0021] Figure 5 The growth curve of LH-Cr-2 strain under high concentration of hexavalent chromium is shown.

[0022] Figure 6 The graph shows the reduction rate of hexavalent chromium by strain LH-Cr-2 under low concentrations of hexavalent chromium.

[0023] Figure 7 The growth curve of LH-Cr-2 strain under low concentration of hexavalent chromium is shown.

[0024] Figure 8 The reduction rate curves of water-soluble chromium of strain LH-Cr-2 at different concentrations are shown.

[0025] Figure 9 The graph shows the reduction rate of water-soluble chromium by different dosages and reaction times for strain LH-Cr-2. Detailed Implementation

[0026] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below.

[0027] Example 1, Screening method for LH-Cr-2 strains:

[0028] 1. Strain Screening: Cr-contaminated soil from Shihezi, Xinjiang, and Cr-contaminated sites in Hebei were collected. 1g of soil was weighed and inoculated into Erlenmeyer flasks containing LB medium. A certain concentration of potassium dichromate was added for acclimatization and enrichment culture. The initial Cr(VI) concentration was 100mg / L, and subsequently increased by 100mg / L each time, with each acclimatization period lasting 20-30 days, until the final Cr(VI) concentration reached 1000mg / L. The culture was carried out at a constant temperature of 35℃, and the entire acclimatization process lasted approximately one year.

[0029] After acclimatization, a small amount of bacterial suspension was taken with an inoculation needle and streaked onto LB solid medium containing 250 mg / L Cr(VI). After approximately 5 days of growth, single colonies were picked and further isolated and purified using the same method for at least five generations until the colonies exhibited consistent morphology, thus obtaining a pure LH-Cr-2 strain. Its morphology and electron micrographs are shown in the reference [reference needed]. Figure 2 and3 .

[0030] 2. Identification of strains

[0031] The LH-Cr-2 strain was identified, and its phylogenetic tree was referenced. Figure 1 Through identification, strain LH-Cr-2 was confirmed to be *Pseudomonas phragmitis*. Its gene sequence is shown in SEQ NO.1.

[0032] 3. The utilization of the sole carbon source by strain LH-Cr-2 is shown in Table 1 below:

[0033] Table 1. Determination of the unique carbon source for strain LH-Cr-2 using BIOLOG GENIII reagent strips.

[0034]

[0035]

[0036]

[0037]

[0038] Note: + indicates positive; - indicates negative; w indicates weak positive.

[0039] 4. Reduction test of LH-Cr-2 strain

[0040] Single colonies of pure bacteria were picked and added to 100 mL of LB liquid medium containing a certain concentration of Cr(VI). The culture was incubated at 30 °C and 150 rpm on a shaker. At regular intervals, 10 mL of the test solution was drawn into a 10 mL centrifuge tube using a sterile syringe and centrifuged at 4000 rpm for 10 minutes. The supernatant was collected and the concentration of hexavalent chromium (VI) was determined. A blank control experiment was performed using sterile medium.

[0041] Depend on Figure 4As shown in Figure 5, at a hexavalent chromium concentration of 250 mg / L, compared to the control at 0 mg / L, the growth of strain LH-Cr-2 was somewhat inhibited in the early stages, but the growth of the strain was better in the later stages, indicating a stronger Cr(VI) removal capacity. At 12 days, the strain could reduce 37.65% of Cr(VI), and at 21 days, the reduction rate reached 48.51%. At hexavalent chromium concentrations of 500 mg / L and 1000 mg / L, the growth of the strain was greatly inhibited, especially at 1000 mg / L, where the corresponding reduction capacity was weak. However, at 500 mg / L, the later-stage effect was smaller, and 500 mg / L Cr(VI) could be reduced to about 161 mg / L, with a reduction rate of 67.67%. At a hexavalent chromium concentration of 1000 mg / L, compared to 500 mg / L, the reduction rate of Cr(VI) by the strain was significantly inhibited, with a reduction rate of only 24.53% at 21 days. This indicates that high concentrations of hexavalent chromium inhibit the reduction ability of strain LH-Cr-2 to Cr(VI) and its growth.

[0042] Depend on Figure 6 As shown in Figure 7, strain LH-Cr-2 exhibits good growth under low concentrations of hexavalent chromium, ranging from 3 to 100 mg / L, with almost no effect from increased hexavalent chromium concentration. However, growth is somewhat inhibited under quiescent conditions, indicating that this strain grows better under sufficient oxygen supply. Although the growth curves show some differences, under these test conditions, strain LH-Cr-2 achieved a Cr(VI) reduction rate of over 83% at 12 days; at all tested concentrations, over 76% of the hexavalent chromium was reduced within 4 days; compared to hexavalent chromium concentrations of 250 mg / L and above, strain LH-Cr-2 demonstrates a strong reducing ability at low concentrations.

[0043] Depend on Figure 8 It can be seen that the reduction rate of water-soluble chromium by the LH-Cr-2 strain is approximately 70% at different concentrations. Even with increasing hexavalent chromium concentration in the soil, LH-Cr-2 still exhibits good reduction performance. Figure 8 It can be seen that under low hexavalent chromium concentration (50-150 mg / kg), the reduction rate of LH-Cr-2 after 17 days of reaction is better than that after 7 days of reaction. However, under high hexavalent chromium concentration (>200 mg / kg), the reduction rate after 7 days of reaction is better, but the difference is not significant, indicating that the reduction rate of LH-Cr-2 can remain stable to a certain extent.

[0044] Depend on Figure 9It can be seen that the reduction rate of water-soluble chromium in the soil generally increased with the increase of the dosage of LH-Cr-2 strain. When the dosage of LH-Cr-2 strain solution was 1% to 5%, the reduction effect was 70%-80%, but when the dosage increased to 7% to 9%, the reduction rate of water-soluble chromium increased to over 90%. Figure 9 It can also be seen that the reaction time (7d and 17d) had no significant effect on the reduction rate of water-soluble chromium, indicating that the LH-Cr-2 strain has a relatively stable reducing ability at a hexavalent chromium concentration of 200 mg / kg. The bacterial concentration in the added bacterial solution was 5-10 billion cells / mL.

[0045] Experiments have shown that the optimal fermentation medium and production fermentation process for strain LH-Cr-2 are as follows:

[0046] 1L optimized culture medium formula: 3g glucose, 3g corn flour, 5g soybean meal, 2g yeast extract, 8g sodium chloride, 2g dipotassium hydrogen phosphate, 0.03g manganese sulfate, pH 7.2, temperature: 37℃.

[0047] Experiments showed that at high Cr(VI) concentrations (250-1000 mg / L), the growth of the strain was severely inhibited and slowed at concentrations of 1000 and 500 mg / L. Regarding reductive degradation, the strain reduced Cr(VI) from 273 mg / L to 121 mg / L within 21 days. At low Cr(VI) concentrations (3-100 mg / L), the final bacterial concentration was similar and almost unaffected by Cr(VI) concentration, with the concentration under anaerobic conditions being slightly lower than under aerobic conditions. Regarding reductive degradation, concentrations of 50 mg / L and below were reduced to near 0 mg / L within 5 days, and 100 mg / L was reduced to near 0 mg / L after 10 days.

[0048] In this invention, when the LH-Cr-2 strain reduces Cr(VI), the optimal concentration range for hexavalent chromium treatment is 0-500 mg / L. When the concentration is higher than 500 mg / L, it needs to be diluted to the appropriate range of 0-500 mg / L.

Claims

1. A type of *Pseudomonas diaphragmatis* ( Pseudomonas phragmitis LH-Cr-2, characterized in that: Its biological accession number is: CGMCC NO.27552.

2. A fermentation culture method for *Pseudomonas diaphragmatis* LH-Cr-2 as described in claim 1, characterized in that: The fermentation medium formula is as follows: per liter of fermentation broth, there are 3 grams of glucose, 3 grams of corn flour, 5 grams of soybean meal, 2 grams of yeast extract, 8 grams of sodium chloride, 2 grams of dipotassium hydrogen phosphate, and 0.03 grams of manganese sulfate. The fermentation conditions were: pH 7.2, fermentation temperature: 37 ℃.

3. The use of *Pseudomonas diaphragmatis* LH-Cr-2 according to claim 1, characterized in that, The stated use is to use the strain or its fermentation broth to reduce soluble hexavalent chromium Cr(VI).

4. The use of *Pseudomonas diaphragmatis* LH-Cr-2 according to claim 1, characterized in that, The intended use is to apply the strain or its fermentation broth to the remediation of soil contaminated with hexavalent chromium (Cr(VI)).

5. The use of *Pseudomonas diaphragmatis* LH-Cr-2 according to claim 1, characterized in that, The intended use is to apply the strain or its fermentation broth to the treatment of groundwater contaminated with hexavalent chromium (Cr(VI)).

6. The use of *Pseudomonas diaphragmatis* LH-Cr-2 according to claim 5, characterized in that: Before treatment, the concentration of hexavalent chromium Cr(VI) was controlled to be below 500 mg / L.

7. The use of *Pseudomonas diaphragmatis* LH-Cr-2 according to claim 5 or 6, characterized in that: Before treatment, the concentration of hexavalent chromium Cr(VI) was controlled to be below 100 mg / L.

8. The use of *Pseudomonas diaphragmatis* LH-Cr-2 according to claim 3, 4, or 5, characterized in that: The dosage of LH-Cr-2 is 7%–9%.

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