Rhodococcus and application thereof

Abstract

The application belongs to the technical field of environmental microorganisms, and particularly relates to a strain of Rhodococcus and application thereof. Rhodococcus qingshengii The strain is Rhodococcus qingshengii (abbreviated as strain R), which has been preserved in the China General Microbiological Culture Collection Center on May 13, 2025, and the strain preservation number is CGMCC No.34527. The high-efficiency fermentation iron reduction ability of the strain under low-temperature (4 DEG C) and strict anaerobic conditions can not only be applied to the repair of contaminated sites in cold regions, especially those deep anaerobic environments with low-temperature stress, such as high-latitude groundwater, polar soil and even glacial sediment environments, but also help to understand the influence of extreme low temperature on the fermentation iron reduction process of microorganisms. This study helps to develop innovative in-situ bioremediation technology to solve a series of trivalent iron pollution problems in high-cold anaerobic environments.

Description

Technical Field

[0001] This invention belongs to the field of environmental microbiology technology, and relates to a strain of Rhodococcus and its application, specifically to a novel low-temperature resistant Rhodococcus fanningeri isolated from Antarctica. Rhodococcus qingshengii (and its application in fermentative iron reduction and bioremediation under low temperature and anaerobic conditions.) Background Technology

[0002] Iron is the most abundant variable-valence metallic element in the Earth's crust and a crucial trace element essential for living systems. As a common heavy metal, iron is present in industrial wastewater. The presence of iron in wastewater not only causes changes in water color but can also have long-term negative impacts on the ecological environment and human health. Excessive accumulation of iron ions in water can react with elements such as phosphorus and nitrogen, promoting eutrophication, leading to rapid algal growth and subsequent oxygen depletion. Iron pollution has a significant negative impact on aquatic life, especially fish and aquatic plants. Iron ions or iron oxide particles can directly affect the respiration and nutrient absorption of aquatic organisms, reducing their reproductive capacity. For humans, iron pollution in wastewater can lead to the deterioration of drinking water sources. Excessive iron concentration not only affects the taste of water but can also cause long-term harm to human health. In particular, long-term ingestion of water containing high concentrations of iron can lead to chronic poisoning, especially affecting organs such as the liver and kidneys. For the treatment of iron-containing wastewater, especially in cold regions, deep groundwater, and glacial sediments in low-temperature (<10℃) and anaerobic environments, existing remediation technologies face significant bottlenecks. Traditional physicochemical precipitation methods suffer from high reagent costs and the generation of secondary sludge; while existing bioremediation technologies mostly rely on mesophilic microorganisms (optimal temperature 25-30℃). At low temperatures around 4℃, the enzyme activity of these microorganisms is severely inhibited, making it difficult to drive the reduction and transformation of ferric iron (Fe(III)). Furthermore, organically complexed iron (such as ferric citrate) commonly found in the environment is highly stable and more difficult to biodegrade and utilize than free iron. Therefore, screening for microbial strains that can tolerate extreme low temperatures and possess highly efficient fermentative iron reduction capabilities is urgently needed to overcome the technical limitations of iron pollution treatment and ecological restoration in cold environments.

[0003] Microbial-driven iron reduction (EMR) is not only a key engine of geochemical cycles but also demonstrates significant potential in environmental remediation. The biogenic ferrous iron continuously generated in this process has a higher specific surface area and reactivity than chemical iron. It can directly remove physical iron blockage in aquifers and also serve as a powerful, broad-spectrum electron donor, driving various non-biochemical reduction reactions in the underground environment, such as heavy metal fixation or organic pollutant degradation. Among the various mechanisms of microbial EMR, "fermentative EMR" has attracted considerable attention in recent years as a unique metabolic strategy. Unlike obligate iron respiration, fermentative EMR bacteria primarily obtain energy through the fermentation of organic substrates such as peptone and glucose, with Fe(III) acting as an "auxiliary electron sink" in this process. Studies have shown that when highly bioavailable soluble iron, such as ferric citrate, is present in the environment, Fe(III) can rapidly divert excess reducing equivalents (such as NADH) generated during fermentation, thereby relieving thermodynamic constraints and significantly promoting cell growth and deep transformation of fermentation substrates. Furthermore, this mechanism also provides a feasible pathway to alleviate the common "acidification inhibition" problem in anaerobic fermentation. The organic acids accumulated during fermentation often lead to a sharp drop in environmental pH, inhibiting microbial metabolism. The reduction process of Fe(III) is accompanied by ligand dissociation and proton consumption. This natural "acid-consuming effect" can theoretically buffer the acidification pressure of the fermentation system and maintain the pH stability of the anaerobic microenvironment.

[0004] Rhodococcus spp. ( Rhodococcus This genus is renowned for its remarkable environmental adaptability and metabolic plasticity. However, current research suggests that most strains of this genus are mesophilic aerobic bacteria, with optimal growth temperatures generally between 28–32°C. Although recent studies have found that some *Rhodococcus fanningeri* strains exhibit certain tolerance and metabolic potential—for example, strain PM1 has been reported to reduce selenite to elemental selenium at 25–30°C (Peng et al., 2024), and strain S10107 can assist in the biotransformation of heavy metals within the range of 15–25°C (Suyal et al., 2019)—these existing technologies are still limited to mesophilic or slightly hypothermic (>15°C) aerobic or microaerobic environments. Currently, there are no reports of this species performing fermentative iron reduction under hypothermic and strictly anaerobic conditions at 4°C. Facing the low-temperature environment (<10°C) that covers more than 70% of the Earth's surface, the decline in microbial enzyme activity and the stagnation of anaerobic metabolism pose a dual challenge to bioremediation practices. Currently, there is a lack of systematic research on whether Rhodococcus can perform efficient fermentative iron reduction at an extreme low temperature of 4°C, thereby overcoming the limitations of low-temperature anaerobic metabolism. Therefore, discovering cold-resistant strains with such functions from extreme habitats such as Antarctica is of great significance for expanding the application range of Rhodococcus and developing pollution remediation technologies in high-altitude and cold regions. Summary of the Invention

[0005] The purpose of this invention is to provide a strain of Rhodococcus and its applications, specifically a novel low-temperature resistant Rhodococcus fanningeri isolated from Antarctica. Rhodococcus qingshengii (and its application in fermentative iron reduction and bioremediation under low temperature and anaerobic conditions.)

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

[0007] A strain of Rhodococcus, specifically Rhodococcus fannings ( Rhodococcus qingshengii (hereinafter referred to as strain R), which was deposited on May 13, 2025 at the China General Microbiological Culture Collection Center, with the accession number CGMCC No. 34527, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0008] An application of the aforementioned Rhodococcus, characterized in that: the application of the strain R in anaerobic iron reduction.

[0009] Application of strain R in anaerobic iron reduction under low temperature (4-10℃) conditions.

[0010] Anaerobic iron reduction refers to the process of reducing ferric iron (Fe(III)) to ferrous iron (Fe(II)) through anaerobic fermentation.

[0011] An application of the aforementioned Rhodococcus, specifically the application of strain R in the remediation of iron-contaminated sites.

[0012] An iron contamination remediation microbial agent, the microbial agent containing the aforementioned Rhodococcus (abbreviated as strain R).

[0013] The bacterial agent is one or more of the following: culture, bacterial suspension, concentrate, and separation liquid containing the bacterial strain.

[0014] The strain was cultured by adding fermentation substrate and water-soluble ferric complex to an anaerobic inorganic salt medium, and then inoculating the anaerobic iron-reducing bacteria R with the medium and culturing it at pH 7.2, 30 ℃, and in the dark; wherein the fermentation substrate was peptone.

[0015] An application of the aforementioned iron contamination remediation microbial agent, wherein the microbial agent is applied to iron-contaminated sites for the remediation of iron contamination.

[0016] A method for low-temperature anaerobic iron reduction involves inoculating the strain into the system to be reduced under low-temperature and anaerobic conditions of 4-10℃, thereby achieving the reduction of ferric iron in the system.

[0017] Advantages of this invention:

[0018] The strain R obtained in this invention is a low-temperature resistant fermentation-reduced Rhodococcus genus that was screened from Antarctic sediments.

[0019] Strain R is a Gram-positive bacterium. Through 16S rRNA gene sequence similarity comparison, it shows a species similarity of 99.93% with strain JCM 15477 of the genus Rhodococcus, proving that strain R belongs to the genus Rhodococcus.

[0020] This strain can reduce Fe(III) to Fe(II) at 4-30 °C and pH 7.2. Its iron reduction capability under low-temperature conditions not only allows for the remediation of iron-contaminated sites, especially those with low-temperature anaerobic conditions such as polar and permafrost environments, but also contributes to understanding the effects of temperature on the microbial fermentation process of iron reduction and the molecular mechanisms of microbial cold tolerance. This research contributes to the development of innovative bioremediation technologies to address a range of iron contamination problems in low-temperature environments. Attached Figure Description

[0021] Figure 1 This is a scanning electron microscope image of strain R of the present invention.

[0022] Figure 2 A phylogenetic tree was constructed by homology comparison of the 16S rRNA gene sequences of strain R of the present invention and representative strains of the genus Rhodococcus.

[0023] Figure 3 The reduction curve of Fe(III) by strain R of the present invention is shown.

[0024] Figure 4 The reduction curve of Fe(III) by strain R of the present invention at 4°C is shown. Detailed Implementation

[0025] The following examples further illustrate specific embodiments of the present invention. It should be noted that the specific embodiments described herein are merely for illustration and explanation and are not intended to limit the scope of the present invention.

[0026] This invention yields a novel *Rhodococcus fanningeri* strain isolated from Antarctica, capable of efficiently inducing iron reduction through anaerobic fermentation at a low temperature of 4°C. Rhodococcus qingshengii The strain can reduce Fe(III) to Fe(II) under low temperature conditions, thus achieving iron remediation in a low-temperature anaerobic environment.

[0027] Unless otherwise specified, the experimental methods, materials and reagents used in the following examples are all conventional methods.

[0028] Example 1: Isolation, purification and identification of strains

[0029] (1) Prepare the basic culture medium:

[0030] Each liter of inorganic salt anaerobic culture medium contains: NaCl 1.0 g, MgCl2·6H2O 0.5 g, KH2PO4 0.2 g, NH4Cl 0.3 g, KCl 0.3 g, CaCl2·2H2O 0.015 g, FeCl2·4H2O 1.5 mg, CoCl2·6H2O 190 μg, MnCl2·4H2O 100 μg, ZnCl2 70 μg, H3BO3 6 μg, Na2MoO4·2H2O 36 μg, NiCl2·6H2O 24 μg, CuCl2·2H2O 2 μg, Na2SeO3·5H2O 6 μg, Na2WO4·2H2O 8 μg, and sodium resazurin 0.025% (w / v) as an oxygen indicator. The headspace of the culture medium was heated to boiling under high-purity N2 and then cooled to room temperature in an ice bath. 24 mg of cysteine, 48 mg of Na2S·9H2O, and 77 mg of dithiothreitol (DTT) were added as reducing agents to remove oxygen. 2.52 g (30 mM) of NaHCO3 was added as a buffer. The pH of the medium was adjusted to 7.2-7.3 with CO2. The medium was then aliquoted into serum bottles and autoclaved at 121°C for 30 minutes. A multivitamin was then added. The final concentrations of each vitamin in the medium were as follows: biotin 20 μg / L, folic acid 20 μg / L, pyridoxine hydrochloride 100 μg / L, riboflavin 50 μg / L, thiamine 50 μg / L, pantothenic acid 50 μg / L, nicotinic acid 50 μg / L, and vitamin B12. 12 50 μg / L, p-aminobenzoic acid 50 μg / L, thioctic acid 50 μg / L.

[0031] (2) Enrichment of iron-reducing bacteria during fermentation:

[0032] Dispense 80 mL of the anaerobic inorganic salt culture medium described in step (1) above into a 120 mL serum bottle. Add 0.5 g of peptone as a carbon source and 5 mM ferric citrate as an electron acceptor to the serum bottle. In an anaerobic glove box, inoculate ~5 g of Antarctic soil sediment (collected from Beaufort Island, Antarctica) and seal the serum bottle with a blue rubber stopper and aluminum cap to establish an enrichment culture system. Incubate statically at 30 ℃ in the dark. Monitor the iron reduction process periodically using the phenanthridine method. After Fe(III) is completely reduced to Fe(II), repeat the transfer to the same composition culture medium more than 5 times at an inoculation rate of 3% (v / v) to simplify the community structure and make the iron-reducing fermentation bacteria highly enriched.

[0033] (3) Isolation of iron-reducing bacteria during fermentation:

[0034] Dispense 9 mL of the inorganic culture medium described in step (1) into a 20 mL culture flask, replace the headspace gas with N2 / CO2 (80 / 20, v / v), seal with a blue rubber stopper and aluminum cap, and add 0.2 g of peptone and 5 mM ferric citrate through a microsyringe. Transfer 1 mL of the above-mentioned iron-reduced fermentation culture from the 120 mL serum bottle in step (2) into the above culture flask to establish a 10-year culture. -1 Dilute bottles, and so on, repeat the above 10-fold serial dilution operation until a 10-fold serial dilution is established. -10 Dilution bottles were prepared. Subsequently, a solid culture medium containing the same basic components, plus 0.5 g of peptone, 5 mM ferric citrate, and 2% agar powder, was prepared in disposable sterile culture dishes. After Fe(III) in the diluted culture medium was completely reduced to Fe(II), approximately 50 μL of bacterial suspension was dropped onto the surface of the culture dish, streaked, and then the dish was transferred to an anaerobic glove box and incubated at 30°C in the dark for approximately 30 days. After the formation of distinct single colonies, a single colony was picked and inoculated into a 120 mL serum bottle containing 80 mL of liquid culture medium with 0.5 g of peptone and 5 mM ferric citrate. The culture was then incubated at 30°C in the dark. The growth of the culture medium was observed daily, and the presence of contaminating bacteria was determined by the visually perceptible turbidity of the culture medium over two weeks. Finally, a pure culture of strain R was obtained.

[0035] (4) Identification of strains:

[0036] Morphological identification:

[0037] The obtained strain R culture was centrifuged at 4°C and 15000 xg for 10 minutes to collect cells in the logarithmic growth phase. The pellet was washed with phosphate-buffered saline (PBS) and fixed overnight with 2.5% glutaraldehyde solution. After gradient dehydration with ethanol, critical point drying, and gold sputtering, the bacterial morphology was observed under a scanning electron microscope (see [link to sample]). Figure 1 The cells of strain R exhibit a typical short rod-shaped morphology, with a slightly rough surface and no flagella or other appendages observed. The cells are relatively uniform in size; measured by electron microscopy, the cell width is approximately 0.4–0.6 μm and the length is approximately 1.0–1.6 μm. This morphological characteristic is consistent with the genus *Rhodococcus*. Rhodococcus Typical characteristics of microorganisms in the logarithmic growth phase.

[0038] 16S rRNA sequence homology analysis:

[0039] Total DNA was extracted from strain R as a template for amplification. PCR amplification was performed using universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGT TACGACTT-3') for bacterial 16S rRNA. The amplified products were submitted to a sequencing company for Sanger sequencing. Sequencing results showed that the 16S rRNA gene fragment obtained by PCR amplification was 1403 bp in length, with a G+C mol% of 59.8%. Sequence alignment analysis indicated that strain R is related to *Rhodococcus* spp. (…). Rhodococcus The type strain of Rhodococcus fanqingsheng ( under) Rhodococcus qingshengii The JCM 15477 sequence showed the highest homology, with a similarity of 99.93%. Figure 2 Despite their high similarity in genetic background, the two strains differ significantly in their physiological and biochemical characteristics: the model strain JCM 15477 is a typical mesophilic bacterium (25–37℃); while the strain R of this invention can grow at a low temperature of 4℃ and can efficiently reduce ferric iron (Fe(III)) through fermentation under anaerobic conditions, a unique physiological function not possessed by the model strain of this species. The 16S rRNA sequence of strain R is as follows:

[0040]

[0041] The strain is *Rhodococcus fanningsense*, which was deposited on May 13, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 34527, hereinafter referred to as R.

[0042] Example 2: Identification of the reducing performance of strain R on ferric iron

[0043] Add 0.5g peptone as a fermentation substrate and 5mM ferric citrate as an iron source to the 80 mL anaerobic inorganic salt basal medium described in step (1) above, and inoculate the purified strain R at 3% (v / v) and culture it statically at pH 7.2 and 30 ℃ in the dark.

[0044] The concentrations of ferrous ions (Fe(II)) and total iron (Total Fe) in the system were determined using the ferrozine colorimetric method. The specific operating steps are as follows.

[0045] Sample pretreatment: To prevent Fe(II) from being oxidized in air, the sampling process was carried out in an anaerobic chamber. Immediately after sample removal, hydrochloric acid (HCl) was added for acidification to a final HCl concentration of 0.5 M, and the sample was allowed to stand overnight to fully extract iron ions.

[0046] Determination of Fe(II) concentration: Take 100 μL of the acidified sample and add it to a cuvette. Then add 750 μL of deionized water, 100 μL of phenoxyazine solution (0.01 M, dissolved in 0.1 M ammonium acetate), and 150 μL of buffer solution (5 M ammonium acetate, pH 9.5). Mix thoroughly and react for 2-3 minutes. Measure the absorbance at 562 nm using a UV spectrophotometer.

[0047] Determination of Total Iron (Total Fe) concentration: Take 100 μL of acidified sample and add 150 μL of reducing agent (1.4 M hydroxylamine hydrochloride solution) to completely reduce Fe(III) to Fe(II). Then add 750 μL of deionized water and let it stand for 30 minutes. After the reaction is complete, add 100 μL of phenoxyazine solution and 150 μL of buffer solution, react for 2-3 minutes, and then measure the absorbance at 562 nm.

[0048] Standard curve construction and calculation: A standard curve was constructed by measuring the absorbance of ferrous ion standard solutions of different known concentrations at a wavelength of 562 nm. The absorbance of the samples was then converted to the corresponding concentration based on the standard curve. The concentration of ferric iron was calculated using the formula: Fe(III) concentration = Total iron concentration - Fe(II) concentration.

[0049] Quantitative analysis was performed based on absorbance values. Figure 3 ).

[0050] The analysis results showed that strain R could reduce Fe(III) through peptone fermentation. At the start of the culture, the Fe(III) concentration in the system was approximately 5 mM (about 280 mg / L). With the extension of the culture time, the Fe(III) concentration continuously decreased, while the Fe(II) concentration showed a corresponding linear increasing trend. By day 40 of the culture, the Fe(III) in the system was completely reduced to below the detection limit, and at the same time, an isostoichiometric amount of Fe(II) was generated, with a reduction rate of 7.54 mg / (L·d), demonstrating that strain R can efficiently drive the reduction process of ferric iron by utilizing the reducing power generated by the fermentation substrate.

[0051] Example 3: Identification of the iron reduction performance of strain R during fermentation at 4 °C

[0052] Add 0.5 g peptone as a fermentation substrate and 5 mM ferric citrate as an iron source to the 80 mL anaerobic inorganic salt basal medium described in step (1) above. Inoculate the purified strain R at 3% (v / v) and incubate statically at pH 7.2 and 4 ℃ in the dark. The iron content was determined using the Ferrozine colorimetric method. Figure 4 ).

[0053] The analysis results showed that strain R could completely reduce Fe(III) to Fe(II) at 4 ℃, with a reduction cycle of about 77 days and an average of about 3.08 mg / (L·d).

[0054] Prepare the bacterial agent according to the above description. For example, add peptone as a fermentation substrate and 5-10 mM ferric citrate as an electron acceptor to an anaerobic inorganic salt medium. Inoculate the strain R at 3% (v / v) and incubate at pH 7.0-7.2, 30℃, and in the dark for 3-5 days. Centrifuge the obtained culture, collect the precipitate, and resuspend it to obtain a resuspended solution. The liquid phase is the separation liquid. Concentrate the culture to obtain a concentrate, which is the bacterial agent. Apply it to low-temperature anaerobic environments such as groundwater in cold regions, polar soils, or deep sediments; and allow it to react with Rhodococcus spp. (… RhodococcusUnlike commonly reported obligate aerobic and mesophilic organic pollutant degrading strains, the strain R of this invention maintains unique fermentative iron reduction activity under low temperature (4℃) and strictly anaerobic conditions, continuously producing highly reducing ferrous iron (Fe(II)) through metabolism. The biogenic ferrous iron produced by microbial reduction is a broad-spectrum electron donor driving the reduction and fixation of various high-risk pollutants. It can reduce and fix highly toxic heavy metals in the environment (such as hexavalent chromium and uranium) into low-toxicity precipitates, or synergistically degrade oxidized pollutants such as nitroaromatics through non-biological pathways. This strain can specifically destroy highly stable organic complexed iron (such as ferric citrate), efficiently converting clogging and ecotoxicologically hazardous ferric iron into highly soluble ferrous iron, thereby eliminating physical blockage at the source and reducing ecological risks. This provides a breakthrough microbial germplasm resource for solving iron pollution control and in-situ ecological restoration in high-latitude and deep groundwater environments. It has good application prospects in the fields of heavy metal pollution remediation in cold regions and in-situ regulation of groundwater geochemical environment.

[0055] In summary, the highly efficient fermentation iron reduction capacity of the strain of this invention under low temperature (4°C) and strictly anaerobic conditions not only enables its application in the remediation of contaminated sites in cold regions, particularly deep anaerobic environments under low-temperature stress, such as high-latitude groundwater, polar soils, and even glacial sediment environments, but also contributes to understanding the impact of extreme low temperatures on the microbial fermentation iron reduction process. This research contributes to the development of innovative in-situ bioremediation technologies to address a range of challenges related to ferric iron contamination in high-altitude anaerobic environments.

[0056] The above-described embodiments are preferred application examples of this invention, but do not constitute any limitation on this invention. In practical applications, without departing from the scope of the technical solution of this invention, some modifications or alterations can be made to the disclosed technical content to create equivalent embodiments.

Claims

1. A strain of Rhodococcus, characterized by: The strain was Rhodococcus fannings ( Rhodococcus qingshengii This strain was deposited at the China General Microbiological Culture Collection Center (CGMCC) on May 13, 2025, with the accession number CGMCC No. 34527.

2. An application of Rhodococcus according to claim 1, characterized in that: Application of the strain in anaerobic iron reduction; Anaerobic iron reduction refers to the process of reducing ferric iron (Fe(III)) to ferrous iron (Fe(II)) through anaerobic fermentation.

3. The application of Rhodococcus according to claim 2, characterized in that: Application of the strain in anaerobic iron reduction at low temperatures of 4-10℃.

4. An application of Rhodococcus according to claim 1, characterized in that: Application of the strain in the remediation of iron-contaminated sites; The iron pollution remediation process involves reducing ferric iron (Fe(III)) to ferrous iron (Fe(II)) through anaerobic fermentation.

5. An iron contamination remediation microbial agent, characterized in that: The bacterial agent contains the Rhodococcus as described in claim 1.

6. The iron contamination remediation microbial agent according to claim 5, characterized in that: The bacterial agent is one or more of the following: a culture, a bacterial suspension, or a concentrate containing the bacterial strain.

7. The iron contamination remediation microbial agent according to claim 6, characterized in that: The strain is cultured by adding fermentation substrate and water-soluble ferric complex to an anaerobic inorganic salt medium, inoculating the strain of claim 1, and culturing at pH 7.2, 30°C, and in the dark; wherein the fermentation substrate is peptone.

8. The application of the iron contamination remediation microbial agent according to claim 5, characterized in that: The application of the bacterial agent in the remediation of iron contamination in sites contaminated with ferric iron; The iron pollution remediation process involves reducing ferric iron (Fe(III)) to ferrous iron (Fe(II)) through anaerobic fermentation.

9. A method for low-temperature anaerobic iron reduction, characterized in that: The strain described in claim 1 is inoculated into the reduction system under low temperature and anaerobic conditions of 4-10℃, thereby reducing the trivalent iron in the system to divalent iron.

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