A thermofilum and applications

Abstract

The application discloses a geothrix and application, and relates to the technical field of microorganisms.The geothrix SGZ-164 is classified as Geothrix sp., has a preservation number of GDMCC No: 63128, a preservation date of January 2, 2023, and a preservation address of the 5th floor of No. 59 building in the courtyard, No. 100, Middle Martyrs Road, Guangzhou City, Guangdong Microbial Culture Collection Center.The geothrix Geothrix sp.SGZ-164 of the application can utilize sodium fumarate and trivalent iron as electron acceptors under anaerobic conditions, and can accelerate the degradation of organic matter.Using the new strain of the application, a high-efficiency soil in-situ remediation microbial preparation can be prepared.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and more particularly to a microbial culture and its application. Background Art

[0002] Iron is the fourth most abundant element in the Earth's crust and is found in high abundance in soils and sediments. Iron reduction in soils and sediments is driven by microorganisms. Under anaerobic conditions, iron-reducing microorganisms oxidize the substrate (electron donor) within their bodies and use external Fe(III) as the terminal electron acceptor of the respiratory chain to transfer electrons along the respiratory chain, forming a transmembrane proton concentration potential gradient that is then converted into energy required for their metabolism, thereby reducing Fe(III) to Fe(II). Dissimilatory iron reduction is an important process in the decomposition of organic matter in certain soils and sediments. It plays an important role in the degradation of organic pollution and also plays an important role in the bioremediation of anaerobic formations.

[0003] Rice is my country's top food crop. Paddy field ecosystems, naturally alternating between aerobic and anaerobic conditions, have long attracted research attention as a model system for studying redox processes, with iron redox playing a crucial role. Therefore, systematic research on the characteristics of Fe(III)-reducing microorganisms has important theoretical and practical implications for elucidating the microbial reduction mechanisms of iron oxides in soil, understanding the crucial role of dissimilatory iron reduction in paddy field ecosystems, and promoting the transformation and remediation of pollutants in paddy soils.

[0004] Geothrix is ​​a genus of strictly anaerobic, Gram-negative bacteria in the Holophagaceae family. Currently, Geothrix contains only one known species, and few reports exist on its resources. Preliminary research on this strain revealed its ability to reduce iron. While pure cultures of anaerobic microorganisms account for less than 0.1% of the world's culturable microorganisms, the majority of anaerobic microorganisms remain uncultured. Therefore, discovering new species of microorganisms with iron-reducing capabilities is a pressing issue for those skilled in the art. Summary of the Invention

[0005] In view of this, the present invention provides a ground fungus and application;

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] A geothrix sp., the geothrix sp. SGZ-164, is classified and named Geothrix sp., with a preservation number of GDMCC No: 63128, a preservation date of January 2, 2023, and a preservation address of Guangdong Provincial Microbial Culture Collection Center, 5th Floor, Building 59, Compound 100, Xianlie Middle Road, Guangzhou.

[0008] The invention discloses an application of a fungus in the preparation of an iron-reducing biological agent.

[0009] The invention discloses an application of a ground fungus in preparing an in-situ soil remediation agent.

[0010] A microbial preparation for soil pollution remediation, comprising the terrestris SGZ-164 according to claim 1.

[0011] A method for remediating contaminated soil, comprising inoculating the ground fungus SGZ-164 described in claim 1 into the contaminated soil to remediate the contaminated soil.

[0012] A method for reducing residual organic pollutants in paddy soil, comprising inoculating the ground fungus SGZ-164 described in claim 1 into the paddy soil.

[0013] It can be seen from the above technical solution that, compared with the prior art, the beneficial effects of the present invention are:

[0014] The Geothrix sp. SGZ-164 of the present invention can utilize sodium fumarate and ferric iron as electron acceptors under anaerobic conditions to accelerate the degradation of organic matter. Using the new strain of the present invention, a highly efficient soil in-situ remediation microbial preparation can be prepared.

[0015] In addition, the optimal growth temperature of the Geothrix sp. SGZ-164 of the present invention is 30° C., and when inoculated into paddy soil, it can effectively remove the refractory organic pollutants therein. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are merely embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying any creative work.

[0017] Figure 1 This is a transmission electron micrograph of strain Geothrix sp. SGZ-164 cells;

[0018] Figure 2 The phylogenetic tree diagram based on the 16S rRNA gene using the maximum likelihood method provided by the present invention;

[0019] Figure 3 The full wavelength absorption spectrum of cytochrome c provided by the present invention;

[0020] Figure 4 Schematic diagram of the iron reducing ability provided by the present invention. DETAILED DESCRIPTION

[0021] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0022] Example 1

[0023] 1. Isolation and enrichment culture of strain SGZ-164

[0024] 1) Take 200 μL of a serially diluted fresh soil suspension (soil sampled from the experimental rice field of Fujian Agriculture and Forestry University, No. 15, Shangxiadian Road, Cangshan District, Fuzhou City, Fujian Province (26.9450N, 119.3771E), diluted with anaerobic sterile water to obtain a suspension), spread it on an R2A plate containing 40 mM sodium fumarate, and incubate under strict anaerobism at 30°C for 10 days.

[0025] R2A medium (L-1): 0.25 g tryptone, 0.5 g acid-hydrolyzed casein, 0.5 g yeast extract powder, 0.5 g soluble starch, 0.3 g potassium dihydrogen phosphate, 0.1 g magnesium sulfate, 0.3 g sodium pyruvate, 0.25 g peptone, 0.5 g glucose, pH 7.0. 100 μL of the serially diluted fresh soil suspension and enrichment solution was spread onto modified R2A plates (supplemented with 40 mM sodium fumarate) and incubated under strict anaerobism at 30°C for 10 days.

[0026] 2) Pick colonies from the plate and purify using the continuous streak method until a single pure culture is obtained.

[0027] 3) Repeat step 2) until pure bacteria SGZ-164 are obtained.

[0028] 2. Morphological characteristics of strain SGZ-164

[0029] 1) Morphological characteristics of bacteria

[0030] The strain SGZ-164 is a Gram-negative bacterium with a rod-shaped body and a flagellum at the end.

[0031] 2) Colony morphology characteristics

[0032] After activation, strain SGZ-164 was cultured on R2A plates containing sodium fumarate at 30°C under anaerobic conditions for 7 days to form reddish, opaque circular colonies.

[0033] 3. Taxonomic status of strain SGZ-164

[0034] The genomic DNA of the invented strain SGZ-164 was extracted. 1 μL of the extracted DNA was used as a template for amplification using the universal bacterial 16S rRNA gene primers 27F and 1492R.

[0035] 27F: 5′-GAGTTTGATCCTGGCTCAG-3′, see SEQ ID No. 1;

[0036] 1492R: 5′-ACGGCTACCTTGTTACGACTT-3′, see SEQ ID No. 2.

[0037] PCR reaction program: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 55°C for 60 s, and extension at 72°C for 90 s, for a total of 30 cycles; and final extension at 72°C for 10 min.

[0038] Take 5 μL of PCR product and spot it on 1% agarose gel. Use 2000 bp marker as standard molecular weight, 5 V / cm voltage, electrophoresis for 15 min, and observe the electrophoresis results with a gel imaging system.

[0039] PCR products from strains with detected bands were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. Sequencing of the 16S rRNA gene sequences was performed using Contig Express software, removing random bases at the beginning and end. The resulting valid sequences were submitted to EzBioCloud (https: / / www.ezbiocloud.net / ) for sequence alignment analysis and the 16S rRNA accession number of the strain was obtained from NCBI (https: / / www.ncbi.nlm.nih.gov / ). If the alignment results indicated a 16S rRNA gene similarity greater than 98.65%, the strain was preliminarily classified at the same taxonomic level as the closest strain; otherwise, it was identified as a potential new taxon.

[0040] Average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) were analyzed using the ANICalculator on the EzBioCloud website to calculate the ANI between bacteria and their model strains and the GGDC online software (Genome-to-Genome Distance Calculator) to calculate dDDH. Average amino acid identity (AAI) was analyzed using enveomics online software to calculate AAI.

[0041] The results showed that the 16S rRNA gene similarities of strain SGZ-164 with the similar type strains Geothrixfermentans DSM 14018T and Holophagafoetida DSM 6591T were 94.8% and 94.6%, respectively, which were lower than the prokaryotic species definition threshold of 98.65% and between the genus definition threshold of 94.5-95.5%, indicating that it may be a potential new genus under the Holophagaceae family.

[0042] The ANI and dDDH values ​​of strain SGZ-164 compared to its closest type strains, Geothrixfermentans DSM 14018T and Holophagafoetida DSM 6591T, were 78.8-79.2% and 19.7-19.9%, respectively, below the prokaryotic species definition thresholds of 96% and 70%, respectively. Therefore, strain SGZ-164 is distinct from Geothrixfermentans DSM 14018T as a new species.

[0043] The AAI of strain SGZ-164 was 60.9-62% compared with its closest type strains, Geothrixfermentans DSM 14018T and Holophagafoetida DSM 6591T, which is lower than the 65% threshold for prokaryotic genus delimitation, indicating that strain SGZ-164 is a new genus within the family Holophagaceae.

[0044] 4. Physiological and biochemical analysis of strain SGZ-164

[0045] To further understand the strain SGZ-164, the relevant physiological and biochemical characteristics of the strain were tested according to standard procedures.

[0046] In liquid R2A medium with sodium fumarate as the electron acceptor, SGZ-164 can grow at temperatures between 20°C and 37°C, with an optimum growth temperature of 30°C. It can grow at a pH of 6.0 to 8.0, with an optimum pH of 7.0. Its tolerance to NaCl ranges from 0% to 0.4%, with an optimum growth concentration of 0-0.2%. The electron donor / acceptor profile of SGZ-164 under anaerobic conditions is shown in Tables 1 and 2; a physiological and biochemical comparison of strain SGZ-164 and its related strains is shown in Table 3; and the results of the API ZYM reagent strip test for strain SGZ-164 are shown in Table 4.

[0047] Table 1 Electron donor spectrum experiment

[0048]

[0049] Table 2 Electron acceptor spectrum

[0050]

[0051] Table 3 Physiological and biochemical comparison of strain SGZ-164 and its closely related species

[0052]

[0053] +, positive; -, negative.

[0054] Table 4. Test results of strain SGZ-164 using API ZYM reagent strips

[0055]

[0056]

[0057] 5. Biochemical Analysis of Strain SGZ-164

[0058] The main fatty acids contained in strain SGZ-164 when cultured at 30℃ for 3 days were iso-C15:0, C16:0, iso-C13:03OH and iso-C11:0; see Table 5; the quinone of strain SGZ-164 was MK-8.

[0059] Table 5 Comparison of fatty acids between strain SGZ-164 and related strains

[0060]

[0061]

[0062] Phylogenetic analysis based on SGZ-16416s rRNA similarity, as well as a series of identification experiments including physiological and biochemical characteristics, showed that SGZ-164 has many important differences from its closest model bacteria:

[0063] 1. Different growth temperature ranges and optimal growth temperatures; 2. Different major fatty acid contents; 3. Different API ZYM enzyme activities.

[0064] Combined with the low similarity (94.8%) between SGZ-164 and the nearest strain in the 16s rRNA gene sequence, polyphasic taxonomic results identified SGZ-164 as a new genus under the Holophagaceae family in taxonomy, and the strain SGZ-164 of the present invention was named Geothrix sp. SGZ-164.

[0065] 6. Overview of the Characteristics of Strain SGZ-164

[0066] Based on the various distinguishing characteristics between strain SGZ-164 and existing strains, as well as the test results of strain SGZ-164 in other aspects, the characteristics of strain SGZ-164 are summarized as follows:

[0067] ① Morphological characteristics: Gram-negative, arcuate, motile, with terminal flagella. It forms reddish, round colonies on plates containing 40 mM sodium fumarate.

[0068] ② Growth characteristics: Anaerobic growth, can utilize different electron donors and electron acceptors. Growth range: 20-37°C (optimum 30°C), pH 6.0-8.0 (optimum pH 7.0), 0-0.4% (w / v) NaCl salinity (optimum 0-0.2%).

[0069] ③ Electron donors / acceptors: At 30°C, when ferric citrate is used as the electron acceptor, fumaric acid, lactic acid, malic acid, palmitic acid, propionic acid, pyruvic acid, succinic acid, ethanol, glucose, glycerol, methanol, toluene, and yeast extract can be used as electron donors. However, acetic acid, benzene, benzoic acid, formic acid, H2, and phenol cannot serve as electron donors. When sodium lactate is used as the electron donor, fumarate and AQDS can serve as electron acceptors, while sulfate, thiosulfate, nitrate, malate, sulfur, oxygen, and manganese (IV) cannot serve as electron acceptors.

[0070] ④ Chemical characteristics: The major fatty acids in strain SGZ-164 are iso-C15:0, C16:0, iso-C13:03OH, and iso-C11:0; the major quinone is MK-8. The strain exhibits alkaline phosphatase, esterase (C4), lipid esterase (C8), cystine arylaminase, trypsin, chymotrypsin, acid phosphatase, naphthol-AS-BI-phosphohydrolase, α-glucosidase, and α-mannosidase activities.

[0071] VII. Reduction of Ferric Citrate by Strain SGZ-164

[0072] ① Materials and methods

[0073] 1) Use freshwater basal culture medium: 2.5g NaHCO₃, 0.1g CaCl₂·2H₂O, 0.1g KCl, 1.5g NH₄Cl, 0.6g NaH₂PO₄·H₂O, 10.0mL vitamin solution, and 10.0mL trace element solution per liter of deionized water. Flush the culture medium with a nitrogen:carbon dioxide (80:20) mixture to remove oxygen. After sterilization, add 10mM sodium lactate as an electron donor and 10mM ferric citrate as an electron acceptor. Standard curve: Develop a standard curve with y = 2.0465x - 0.0028 and R² = 0.9999.

[0074] 2) Cultivate in modified R2A liquid medium at 30°C for 3 days to allow the bacteria to reach the exponential growth phase;

[0075] 3) Centrifuge at 6000 rpm for 10 min, remove the supernatant, resuspend in oxygen-free sterile water, and repeat the above steps twice;

[0076] 4) Add 1 ml of bacterial suspension as treatment and set up culture medium without strain SGZ-164 as control, and culture at 30°C in anaerobically static manner in triplicate;

[0077] 5) Immediately after inoculation, sample the culture medium to determine the initial Fe(II) concentration. Continue sampling every two days until the concentration stops increasing. Dissolve 0.1 mL of culture medium in 900 μL of 0.5 M HCl solution. Add 60 μL of this mixture to 2940 μL of ferroxine solution for color development. Measure the Fe(II) content using a UV spectrophotometer at 562 nm. Use uninoculated culture medium as a control to verify the strain's iron-reducing ability.

[0078] ② Results and Discussion

[0079] The strain SGZ-164 has a high reducing ability to ferric citrate, and the reduction amount reaches the highest level after 12 days of cultivation. Figure 4 .

[0080] 8. Cytochrome Determination of Strain SGZ-164

[0081] Strain SGZ-164 was grown in modified R2A medium for 3 days. 5 mL of the cultured bacteria was centrifuged, and then suspended in 5 mL of 20 mM PIPES buffer (pH 7.0) and 5 mL of 2 mM sodium dithionite in 20 mM PIPES buffer (pH 7.0). After reduction for 6 hours, the difference spectrum of the dithionite-reduced minus air-oxidized cells was obtained by scanning the wavelength of 400-800 nm using a UV-2600 (SHIMADZU, Japan) spectrophotometer.

[0082] Reference is made to Figure 3 Strain SGZ-164 detected cytochrome c absorption peaks at wavelengths of 423, 522, and 553 nm.

[0083] The above description of disclosed embodiments enables one skilled in the art to make or use the application. Numerous modifications to these embodiments will be apparent to those skilled in the art, and the generic principles defined herein can be applied to other embodiments without departing from the spirit or scope of the application. Therefore, the present application is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A ground fungus, characterized in that The ground fungus SGZ-164 is classified and named Geothrix sp. The deposit number is GDMCC No: 63128, the deposit date is January 2, 2023, and the deposit address is Guangdong Provincial Microbiological Culture Collection Center, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

2. Use of the fungus according to claim 1 in the preparation of an iron-reducing biological agent.

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