CN114686404A - Bacterial strain with iron reduction capacity and electricity generation capacity and application thereof - Google Patents

Abstract

The invention provides a strain with iron reduction capability and electricity generation capability and application thereof, belonging to the technical field of microbial system classification and environmental biology. The strain with iron reduction capability and electricity generation capability is named as Shewanella sp A3A, and is preserved in Guangdong province microbial culture collection center (GDMCC) of institute of microbiology of Guangdong province academy of sciences, No. 59, No. 5, No. 59, of Mirabilitum Tochu 100, Guangzhou city, in 2022, 17 th day, and the preservation number is CDMCC No. 62213. The bacterial strain with iron reduction capability and electrogenesis capability can grow in an anaerobic environment by using ferric citrate or ferrihydrite as an electron acceptor and sodium lactate as a unique electron donor, has a good electrogenesis effect in a bioelectrochemical system, and has wide application prospects in the aspects of environmental pollution remediation and microbial electrochemistry research.

Description

Bacterial strain with iron reduction capacity and electricity generation capacity and application thereof

Technical Field

The invention belongs to the technical field of microbial system classification and environmental biology, and relates to a strain with iron reduction capacity and electricity generation capacity and application thereof, in particular to Shewanella sp A3A with iron reduction capacity and electricity generation capacity and application thereof.

Background

Mangrove is a wetland woody plant community consisting of evergreen trees or shrubs which grow in intertidal zones of tropical and subtropical coasts and mainly comprise mangrove plants. The mangrove ecosystem is the most distinctive wetland ecosystem and has the characteristics of land ecology and marine ecology, natural aerobic/anaerobic alternate environment and rich extracellular respiration microorganism resources; in addition, the method plays an important role in purifying seawater, preventing wind and wave, fixing carbon and storing carbon, maintaining biological diversity and the like, has the reputations of coast guards, ocean green lung and the like, and is also an important habitat for rare or endangered waterfowls.

Shewanella (Shewanella) is a facultative anaerobic, extracellular respiratory strain. It is widely distributed in nature, and Shewanella members are separated from various sources such as activated sludge, seawater, sediments, invertebrates, fishes and the like. The Shewanella can utilize a plurality of electron acceptors to carry out extracellular electron transfer, firstly can participate in dissimilatory reduction of manganese and iron oxides and oxidize organic compounds, and meanwhile, iron is the fourth rich element in the crust and commonly exists in water body sediments, wetlands, soil and underground water, so the Shewanella has great application potential in the aspect of microbial treatment of environmental organic pollution; furthermore, Shewanella can generate current by using graphite plate as electron acceptor, and is widely used in bioelectricity research.

Mangrove ecosystem contains abundant strain resources with iron reduction capability and electrochemical activity, but has not been excavated in large quantities. Currently, studies on pure culture diversity of Shewanella in mangrove ecosystem are few, and Shewanella mangrovi obtained from mangrove is only a new species isolated from 1 Humicola humus reducing bacterium (Shewanella sp.) W3 and Liu et al (2015) found by Wupeng et al (2010). Therefore, there is a need to develop Shewanella having good iron-reducing ability and electricity-generating ability.

Disclosure of Invention

The invention aims to overcome the defects in the prior art and provide a strain with iron reduction capacity and electricity generation capacity.

The second purpose of the invention is to provide the application of the strain with iron reduction capability and electricity generation capability.

The purpose of the invention is realized by the following technical scheme:

a strain with iron reduction capability and electricity generation capability is named as Shewanella sp A3A, and is preserved in Guangdong province microbial culture collection (GDMCC) of institute of microbiology, Guangdong province institute of sciences, Guangzhou, No. 59, large institute of Middleyao, No. 100, Middleyao, Guangzhou city, at 17.01.2022, with the preservation number of CDMCC No. 62213.

The Shewanella sp A3A of the invention is separated and screened from mangrove forest sediment in the futian region of Shenzhen, Guangdong province. The strain is pink round on an LB solid culture medium, the edge is neat, and the surface is smooth and moist. The strain is rod-shaped, has a width of about 0.6-0.875 μm and a length of about 1.53-1.90 μm, and has periphytic flagella when observed by transmission electron microscopy. The Shewanella sp A3A is a facultative anaerobic gram-negative bacterium, and the activities of oxidase and catalase are negative. The genome of Shewanella (Shewanella sp.) A3A is used as a template, the 16SrRNA is amplified by using universal primers 27F and 1492R of the 16SrRNA, the obtained 16SrRNA sequences are subjected to homology comparison through an EzBioCloud database, and the result shows that the effective published strain with the highest homology with the strain is Shewanella mangrove YQH10^TAnd the similarity is 97.34%, a phylogenetic tree of the strain based on a 16s rRNA sequence is constructed by using MEGAX software, and the strain is preliminarily identified to belong to a new species of Shewanella. The strain can grow in an LB culture medium at the temperature of 4-40 ℃, and the optimal growth temperature is 28 ℃. The tolerant NaCl concentration range is 0-10% (w/v), the optimum growth NaCl concentration is 2% (w/v), the pH growth range is 5-10, and the optimum pH is7. Shewanella sp A3A of the present invention was inoculated into an anaerobic medium containing ferric citrate or ferrihydrite as an electron acceptor and sodium lactate as an electron donor, and the bacterium was found to have the ability to reduce Fe (III). The bacterium was introduced into a battery reaction apparatus having a graphite plate as an electron acceptor and sodium lactate as an electron donor, and it was found that the bacterium had a certain electricity-generating effect.

The LB culture medium comprises the following components in percentage by weight: 10g/L of tryptone, 5g/L of yeast extract, 10g/L of sodium chloride and 7.4 of pH. The solid medium was prepared by adding 2% (w/v) agar powder to the above fresh medium.

The strain with iron reduction capability and electricity generation capability is applied to iron reduction and/or microbial electricity generation.

The iron reduction is preferably iron reduction in ferric citrate or ferrihydrite.

The method for reducing iron by using the strain with iron reduction capability and electricity generation capability comprises the operation of inoculating the strain with iron reduction capability and electricity generation capability into a citric acid iron basal medium or a ferrihydrite medium with the addition of sodium lactate.

The strain having iron reducing ability and electricity generating ability is preferably a strain having iron reducing ability and electricity generating ability in the logarithmic phase.

The strain with the iron reduction capability and the electricity generation capability in the logarithmic phase is obtained by culturing the strain with the iron reduction capability and the electricity generation capability to OD on an LB liquid culture medium at 160-200 rpm and 28-32 DEG C₆₀₀0.8 to 1.0.

The preparation method of the iron citrate basal culture medium with the addition of sodium lactate comprises the following steps: pouring 13.7g of ferric citrate into 150mL of boiling ultrapure water, and stirring with a glass rod until the ferric citrate is completely dissolved to obtain a ferric citrate dissolved solution; adding the ferric citrate dissolved solution into 600mL of ultrapure water, cooling to room temperature, adjusting the pH value to 6.0-6.5 by using NaOH solution, and then sequentially adding: DL vitamin solution 10mL, DL mineral solution 10mL, NaHCO₃ 2.5g、NaH₂PO₄·H₂O 0.6g，NH₄Cl 0.25g，KCl 0.1g、1mM Na₂SeO₄Solutions of1mL, sodium lactate, and finally fixing the volume to 1L; wherein the final concentration of sodium lactate is 20 mM.

Composition of the DL vitamin solution: 0.002g/L of biotin, 0.002g/L of folic acid, 0.01g/L of pyridoxine hydrochloride, 0.005g/L of riboflavin, 0.005g/L of pantothenic acid, 0.0001g/L of cobalamin, 0.005g/L of p-aminobenzoic acid, 0.005g/L of lipoic acid, 0.005g/L of nicotinic acid, 0.005g/L of thiamine and the balance of ultrapure water.

DL mineral solution composition: nitrilotriacetic acid trisodium 1.5g/L, MgSO₄·7H₂O 3.0g/L、MnSO₄·H₂O 0.5g/L、NaCl 0.5g/L、FeSO₄·7H₂O 0.1g/L、CaCl₂ 0.1g/L、CoCl₂·6H₂O 0.1g/L、ZnSO₄·7H₂O 0.1g/L、CuSO₄·5H₂O 0.01g/L、AlK(SO₄)₂·12H₂O 0.01g/L、H₃BO₃ 0.01g/L、Na₂MoO₄0.025g/L、NiCl₂·6H₂O 0.024g/L、Na₂WO₄·2H₂O0.025 g/L and the balance of ultrapure water.

The formula of the ferrihydrite culture medium with the addition of sodium lactate is as follows: ferrihydrite, DL vitamin solution 10mL, No-sized DL Mineral Mix (NoNTA)10mL, NaHCO₃ 2.5g，NaH₂PO₄·H₂O 0.6g，NH₄Cl 0.25g，KCl 0.1g，1mM Na₂SeO₄1mL of solution, providing sodium lactate of an electron donor, and finally fixing the volume to 1L; wherein the final concentration of the sodium lactate is 20mM, and the final concentration of the ferrihydrite is 100 mM.

Composition of the DL vitamin solution: 0.002g/L of biotin, 0.002g/L of folic acid, 0.01g/L of pyridoxine hydrochloride, 0.005g/L of riboflavin, 0.005g/L of pantothenic acid, 0.0001g/L of cobalamin, 0.005g/L of p-aminobenzoic acid, 0.005g/L of lipoic acid, 0.005g/L of nicotinic acid, 0.005g/L of thiamine and the balance of ultrapure water.

No-sized DL Mineral Mix (NoNTA) composition: MgSO (MgSO)₄ 3.0g/L，MnSO₄·H₂O 0.5g/L，NaCl 1.0g/L，FeSO₄·7H₂O 0.1g/L，CaCl₂·6H₂O 0.1g/L，ZnCl₂ 0.13g/L，CuSO₄·5H₂O 0.01g/L，AlK(SO₄)₂·12H₂O 0.01g/L，H₃BO₃ 0.01g/L，Na₂MO₄·2H₂O 0.025g/L，NiCl₂·6H₂O 0.024g/L，Na₂WO₄·2H₂O0.025 g/L and the balance of ultrapure water.

The preparation method of the ferrihydrite is preferably as follows: 8.11g FeCl was weighed₃Placing the mixture in 500mL of ultrapure water and placing the mixture on a magnetic stirrer to stir vigorously until FeCl is formed₃All dissolved, and at the same time, NaOH solution is quickly dropped into FeCl₃In the solution, the pH value of the solution is 7.2-7.5; after balancing for 1h, dropwise adding NaOH again to stabilize the pH value of the solution at 7.2-7.5; after the pH is stabilized, the suspension is kept stand for 6 hours at the temperature of 4 ℃ in a dark place, and the precipitate is centrifugally rinsed repeatedly to remove Cl^-Until the conductivity in the supernatant is less than or equal to 10 mu S/cm, and the obtained precipitate is ferrihydrite; more preferably: 8.11g FeCl was weighed₃Placing the mixture in 500mL of ultrapure water and placing the mixture on a magnetic stirrer to stir vigorously (1200r/min) to FeCl₃All dissolved while freshly prepared NaOH solutions of different concentrations (5M, 1M) were quickly added dropwise (done within 10min to avoid gibbsite) FeCl₃In the solution, the pH of the solution is 7.2-7.5. After balancing for 1h, NaOH is added dropwise again to stabilize the pH value of the solution at 7.2-7.5. After the pH stabilized, the suspension was left to stand for 6h in the dark at 4 deg.C (low temperature to promote crystallization), and the precipitate was centrifuged (4500g, 5min) and rinsed repeatedly to remove Cl^-And obtaining the precipitate which is the ferrihydrite until the conductivity in the supernatant is less than or equal to 10 mu S/cm.

The method for generating electricity by microorganisms using the strain having iron reducing ability and electricity generating ability includes the operation of inoculating the strain having iron reducing ability and electricity generating ability in an electrolyte.

The strain with iron reduction capacity and electrogenesis capacity is a strain with iron reduction capacity and electrogenesis capacity in logarithmic phase.

The formula of the electrolyte is as follows: DL vitamin solution 10mL, DL mineral solution 10mL, NaHCO₃ 2.5g，NaH₂PO₄·H₂O 0.6g，NH₄Cl 0.25g，KCl 0.1g，1mM Na₂SeO₄Solution 1mLProviding electron donor sodium lactate, and finally fixing the volume to 1L; wherein the final concentration of sodium lactate is 20 mM.

Composition of the DL vitamin solution: 0.002g/L of biotin, 0.002g/L of folic acid, 0.01g/L of pyridoxine hydrochloride, 0.005g/L of riboflavin, 0.005g/L of pantothenic acid, 0.0001g/L of cobalamin, 0.005g/L of p-aminobenzoic acid, 0.005g/L of lipoic acid, 0.005g/L of nicotinic acid, 0.005g/L of thiamine and the balance of ultrapure water.

DL mineral solution composition: nitrilotriacetic acid trisodium 1.5g/L, MgSO₄·7H₂O 3.0g/L、MnSO₄·H₂O 0.5g/L、NaCl 0.5g/L、FeSO₄·7H₂O 0.1g/L、CaCl₂ 0.1g/L、CoCl₂·6H₂O 0.1g/L、ZnSO₄·7H₂O 0.1g/L、CuSO₄·5H₂O 0.01g/L、AlK(SO₄)₂·12H₂O 0.01g/L、H₃BO₃ 0.01g/L、Na₂MoO₄0.025g/L、NiCl₂·6H₂O 0.024g/L、Na₂WO₄·2H₂O0.025 g/L and the balance of ultrapure water.

Compared with the prior art, the invention has the following advantages and effects:

(1) the strain can grow in an anaerobic environment by using ferric citrate or ferrihydrite as an electron acceptor and sodium lactate as a unique electron donor, has a good electricity generation effect in a bioelectrochemical system, and has a wide application prospect in the aspect of environmental pollution remediation.

(2) The invention provides a Shewanella sp A3A with a certain electrogenesis capability, which has a wide application prospect in the aspect of microbial electrochemical research.

Drawings

FIG. 1is a colony morphology of the strain Shewanella sp A3A.

FIG. 2 is a transmission electron micrograph of the strain Shewanella sp A3A.

FIG. 3 is a phylogenetic tree diagram of the strain Shewanella sp A3A based on the 16S rRNA sequence.

FIG. 4 is a phylogenetic map of the strain Shewanella sp A3A based on the whole genome sequence.

FIG. 5 is a graph showing the results of the iron-reducing ability of the strain Shewanella sp A3A in a ferric citrate medium and a ferrihydrite medium; wherein (a) is a result graph of iron reduction capability of a strain Shewanella sp A3A in a ferric citrate culture medium; (b) results are shown for the iron reducing capacity of the strain Shewanella (Shewanella sp.) A3A in ferrihydrite medium.

FIG. 6 is a graph showing the changes in current monitored during constant potential cultivation of the strain Shewanella sp A3A in the electrochemical workstation CHI 1000C.

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.

Example 1: isolation and characterization of Shewanella sp A3A

1. Preparation of culture Medium

LB liquid medium: 10g/L of tryptone, 5g/L of yeast extract, 10g/L of sodium chloride and 7.4 of pH. The solid medium was prepared by adding 2% (w/v) agar powder to the above fresh medium.

2. Isolation of the Strain

Weighing 2g of a mangrove deposit sample collected in an mangrove ecological protection region (22 degrees 30 '-22 degrees 32' N, 113 degrees 56 '-114 degrees 3' E) in the Futian region of Guangdong Shenzhen, province, mixing with 50mL of sterile water, standing for precipitation, taking a supernatant, and using 0.9% of normal saline according to the proportion of 10^-1～10^-5Diluting with gradient, and mixing with 10^-3Diluted supernatant, 10%^-4Diluted supernatant, 10%^-5And (3) uniformly coating the diluted supernatant onto an LB culture medium plate by using a coating rod respectively, culturing for a week in a constant temperature incubator at the temperature of 30 ℃, observing the colony morphology on the plate every day, selecting out a strain with better growth vigor, scribing on a new plate, and further purifying. After culturing the strain on LB medium at 30 ℃ for 48 hours, the single colony formed on the plate appeared as a pink circle as shown in FIG. 1The edge is neat, and the surface is smooth and wet. The strain was observed to be rod-shaped under transmission electron microscope, about 0.6-0.875 μm wide and about 1.53-1.90 μm long, with periphytic flagella (FIG. 2). The separated strain can be inoculated in an LB liquid culture medium, so that subsequent experiments are convenient to carry out. Further, the bacterium was stored at-80 ℃ for a long period of time with 10% (v/v) glycerol.

3. 16S rDNA identification of Shewanella sp A3A

Genomic DNA of the strain was extracted using a DNA extraction kit (Takara, Japan). PCR amplification of the 16S rDNA sequence was performed using the entire genome of Shewanella sp A3A as a template, using the universal primers 27F (5'-AGAGTTTGATCCTGGCTCA-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3'). The amplification procedure was as follows: 1, circulating (pre-denaturation: 94 ℃, 5 min); ② 30 cycles (denaturation: 94 ℃, 30 s; annealing: 58 ℃, 30 s; extension: 65 ℃, 1.5 min); ③ 1 circulation (renaturation: 72 ℃, 10 min); fourthly, 1 circulation (preservation: 4 ℃). Then, the PCR product was subjected to sequencing by Kwangsu Gene Co., Ltd, Guangzhou, and the obtained 16SrRNA sequence was uploaded to an EzbioCloud database for strain homology comparison, and as a result, the strain was found to be the most homologous strain, Shewanella mangrove YQH10^TThe similarity of (c) was 97.34%, which is below the new species identification threshold of 98.7%. Based on the 16S rRNA sequences of the strain and all near-source strains, a phylogenetic tree is constructed by MEGA X software, and as shown in figure 3, the phylogenetic tree is distinguished from other Shewanella branches to form independent branches. Therefore, it was preliminarily identified as belonging to the genus Shewanella (Shewanella sp.) and named as Shewanella (Shewanella sp.) A3A.

4. Analysis of physiological and biochemical characteristics of the strain Shewanella sp A3A

Gram staining test uses gram staining kit (Baso, Zhuhai, China). Growth experiments of the strains were as follows: shewanella sp A3A, a strain cultured to logarithmic growth phase, was inoculated in LB medium at an inoculum size of 1%, and the strains were tested for one week growth at different temperatures (0, 4 ℃, 10 ℃, 15 ℃,20 ℃, 25 ℃, 28 ℃, 30 ℃, 37 ℃, 40 ℃, 42 ℃ and 45 ℃). Containing 0-10% (w/v) NaCl (NaCl concentration is 0, 1%2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%) of LB medium. The growth pH range of the strain was determined in LB medium and set at pH range 4-14( pH 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, respectively). 3 parallel samples are set in the experiment, and the OD is determined by taking the samples once every 12h₆₀₀. Hydrolysis experiments of the strains were performed on LB solid medium containing escin, gelatin, casein, tween 20, tween 40 and tween 80. Other physiological and biochemical characteristics were detected by the API 20NE kit (BioMerieux, France). The drug sensitivity test is carried out by a paper sheet method.

The identification results are as follows: the Shewanella sp A3A strain is a gram-negative bacterium which can grow at the temperature of 4-40 ℃, and the optimal growth temperature is 28 ℃; the tolerant NaCl concentration range is 0-10% (w/v), and the optimum growth NaCl concentration is 2% (w/v); the growth range of pH is 5-10, and the optimum pH is 7. The bacterium is free of oxidase and contact enzyme activity; can produce nitrate; can hydrolyze aescin, gelatin, tween 20, tween 40 and tween 80; can assimilate glucose, arabinose, N-acetyl-glucosamine, adipic acid, malic acid and citric acid. The results of drug sensitivity tests show that the bacterium has drug resistance to antibiotics kanamycin and carbenicillin. Shewanella (Shewanella sp.) A3A and its similar model strain Shewanella mangrovei YQH10^TThe morphological and physiological and biochemical characteristics of (A) are compared in Table 1.

Table 1: comparison table of morphological, physiological and biochemical characteristics of Shewanella sp A3A strain and its adjacent strain

Note that: + indicates positive, w indicates weak positive, -indicates negative, NR indicates not reported.

In addition, the strain Shewanella (Shewanella sp.) A3A and the model strain Shewanella mangrovei YQH10^TThe following characteristics were all positive: nitrate producing, hydrolyzing aescin, tween 40 and tween 80, assimilating glucose, arabinose,Malic acid. The following characteristics were all negative: gram stain, oxidase activity, indole production, assimilation of mannose, mannitol, maltose, gluconate, decanoic acid, phenylacetic acid. Are all sensitive to the following antibiotics: erythromycin, penicillin, gentamicin, polymyxin, rifampin, chloramphenicol, ofloxacin, norfloxacin, and ciprofloxacin.

5. Fatty acid characterization of the strain Shewanella sp A3A

The strains Shewanella (Shewanella sp.) A3A and Shewanella mangrovei YQH10^TEntrust mansion marine microorganism culture preservation management center to identify fatty acid content. The sample pretreatment comprises the following steps: culturing the strain (Shewanella sp) A3A in LB culture medium to late logarithmic phase, centrifuging at 6000g at room temperature for 10min to collect thallus, washing thallus with physiological saline for 1 time, freeze drying in a freeze dryer to obtain 1g of freeze-dried thallus, and testing. Fatty acid detection the fatty acids were subjected to the steps of saponification, methylation, extraction and washing according to the MIDI standard (Shorock microorganism identification System, version 6.0B). The laboratory instrument used was a gas chromatograph (Agilent6850, usa) and was identified by the TSBA6.0 database of the microbiological identification system.

Table 2: shewanella sp A3A and its similar Shewanella manggrovi YQH10^TThe fatty acid analysis of (2) compares the results.

In the table, the summary features indicate two or three fatty acid groups that cannot be separated by gas-liquid chromatography. Summary feature 1 included 15:1iso H/13: 03 OH, summary feature 2 included 12:0 aldehyde (unknown), summary feature 3 included 16:1 ω 7c/16:1 ω 6c, and summary feature 8 included 18:1 ω 7c/18:1 ω 6 c.

The main fatty acids (> 5%) of Shewanella sp A3A of the present strain were 15:0iso (24.15%), 17: 1. omega.8 c (16.09%), summary feature 3 (11.40%), 16:0 (5.66%) and 18: 1. omega.9 c (5.16%) as shown by fatty acid analysis. The inventionThe strain Shewanella (Shewanella sp.) A3A and the model strain Shewanella mangrovei YQH10^TIn contrast, the fatty acid content ratios were approximately similar, but the 18:1 ω 9c and 15:0iso content was significantly higher for the Shewanella sp A3A strain than for the model strain, while the 16:0 and generalized characteristic 3 content was significantly lower for the Shewanella mangrove YQH10 strain^T. As can be shown above, the strain Shewanella sp A3A of the present invention is different from the model strain Shewanella mangrovei YQH10^TThe novel one of (1).

6. Molecular taxonomic status of the strain Shewanella sp A3A

To further determine the molecular classification status of the strain Shewanella (Shewanella sp.) A3A, the genome of the strain Shewanella (Shewanella sp.) A3A was analyzed with other bacteria of the Shewanella genus. Analysis of genomic sequence-based genetic relationship of the strain Shewanella sp A3A using the TYGS platform revealed (FIG. 4) that the strain of the present invention is located in a branch different from other bacteria and belongs to the genus Shewanella. Calculations were performed on the digital DNA-DNA hybridization (dDDH) and the Average computational similarity (ANI) between the genome of the strain shiva (Shewanella sp.) A3A and its closely related species (see table 3), dDDH showing the results: between 18.3% and 21.0%, the values are all far lower than the same similarity threshold (70%); the ANI values were all below the threshold of 95%, which strongly reflects the fact that the strain Shewanella sp A3A is an independent new species.

Table 3: calculated values for dDDH and ANI between the strain Shewanella sp A3A and its closely related species

In conclusion, the Shewanella (Shewanella sp.) A3A can be determined as a new species of Shewanella by combining morphological, physiological and biochemical characteristics, chemical classification of fatty acids and molecular biological classification techniques.

Shewanella sp A3A was deposited at 17.01.2022 at the Guangdong province culture Collection (GDMCC) of institute for microorganisms of Guangdong province academy of sciences, No. 59 of large institute of Guangdong province, No. 59 of Ministry of sciences, Middleya, Middy, Guangzhou, with the deposit number CDMCC No. 62213.

Example 2: shewanella sp A3A iron reduction capacity under anaerobic conditions

The iron reduction ability of the new species Shewanella (Shewanella sp.) A3A was studied using sodium lactate (20mM) as an electron donor and ferric citrate (56mM) and ferrihydrite (100mM) as electron donors.

The strain Shewanella sp A3A was cultured on LB liquid medium in a constant temperature shaker (180rpm, 30 ℃) to logarithmic growth phase (OD)₆₀₀(0.8-1.0), the cells were harvested by high speed centrifugation (5000 Xg, 15min), centrifuged with 1 XPBS buffer, repeated three times, and resuspended to OD with 1 XPBS buffer₆₀₀The obtained bacterial solution was aerated with nitrogen for about 20min, and a citrate iron basal medium and a ferrihydrite medium to which 20mM sodium lactate (electron donor) was added were inoculated according to an inoculum size of 10% (v/v). Three culture media are arranged in parallel, placed in a constant-temperature incubator at 30 ℃ and cultured in the dark, samples are taken at intervals, and the iron reduction condition of the Shewanella sp A3A strain is monitored by adopting a phenazine method.

The preparation method of the iron citrate basal medium with the addition of 20mM sodium lactate (electron donor) comprises the following steps: pouring 13.7g of ferric citrate into 150mL of boiling ultrapure water, and stirring with a glass rod until the ferric citrate is completely dissolved to obtain a ferric citrate dissolved solution; adding the ferric citrate dissolved solution into 600mL of ultrapure water, cooling to room temperature, adjusting the pH to 6.0-6.5 by using a NaOH solution, and then sequentially adding: DL vitamin solution 10mL, DL mineral solution 10mL, NaHCO₃ 2.5g、NaH₂PO₄·H₂O 0.6g，NH₄Cl 0.25g，KCl 0.1g、1mM Na₂SeO₄1mL of solution and sodium lactate, and finally, fixing the volume to 1L; wherein the final concentration of sodium lactate is 20 mM. Subpackaging the citric acid iron base culture medium with 20mM sodium lactate (electron donor) into anaerobic tubes or anaerobic bottles, and charging CO₂：N₂(20:80) of mixed gas to removeRemoving dissolved oxygen in the culture medium. After the inflation, the tube mouth was sealed with a rubber plug and an aluminum cap, and sterilized at 121 ℃ for 20 min.

Composition of the DL vitamin solution: 0.002g/L of biotin, 0.002g/L of folic acid, 0.01g/L of pyridoxine hydrochloride, 0.005g/L of riboflavin, 0.005g/L of pantothenic acid, 0.0001g/L of cobalamin, 0.005g/L of p-aminobenzoic acid, 0.005g/L of lipoic acid, 0.005g/L of nicotinic acid, 0.005g/L of thiamine and the balance of ultrapure water.

DL mineral solution composition: nitrilotriacetic acid trisodium 1.5g/L, MgSO₄·7H₂O 3.0g/L、MnSO₄·H₂O 0.5g/L、NaCl 0.5g/L、FeSO₄·7H₂O 0.1g/L、CaCl₂ 0.1g/L、CoCl₂·6H₂O 0.1g/L、ZnSO₄·7H₂O 0.1g/L、CuSO₄·5H₂O 0.01g/L、AlK(SO₄)₂·12H₂O 0.01g/L、H₃BO₃ 0.01g/L、Na₂MoO₄0.025g/L、NiCl₂·6H₂O 0.024g/L、Na₂WO₄·2H₂O0.025 g/L and the balance of ultrapure water.

Ferrihydrite medium formulation with the addition of 20mM sodium lactate (electron donor): ferrihydrite, DL vitamin solution 10mL, No-sized DL Mineral Mix (NoNTA)10mL, NaHCO₃ 2.5g，NaH₂PO₄·H₂O 0.6g，NH₄Cl 0.25g，KCl 0.1g，1mM Na₂SeO₄1mL of solution, providing sodium lactate for an electron donor, and finally metering the volume to 1L; wherein the final concentration of sodium lactate is 20mM, and the final concentration of ferrihydrite is 100 mM. Subpackaging ferrihydrite culture medium with 20mM sodium lactate (electron donor) into anaerobic bottles, and charging CO₂：N₂(20:80) to remove dissolved oxygen from the medium. After the inflation, the tube mouth was sealed with a rubber plug and an aluminum cap, and sterilized at 121 ℃ for 20 min.

Composition of the DL vitamin solution: 0.002g/L of biotin, 0.002g/L of folic acid, 0.01g/L of pyridoxine hydrochloride, 0.005g/L of riboflavin, 0.005g/L of pantothenic acid, 0.0001g/L of cobalamin, 0.005g/L of p-aminobenzoic acid, 0.005g/L of lipoic acid, 0.005g/L of nicotinic acid, 0.005g/L of thiamine and the balance of ultrapure water.

No-sized DL Mineral Mix (NoNTA) composition: MgSO (MgSO)₄ 3.0g/L，MnSO₄·H₂O 0.5g/L，NaCl 1.0g/L，FeSO₄·7H₂O 0.1g/L，CaCl₂·6H₂O 0.1g/L，ZnCl₂ 0.13g/L，CuSO₄·5H₂O 0.01g/L，AlK(SO₄)₂·12H₂O 0.01g/L，H₃BO₃ 0.01g/L，Na₂MO₄·2H₂O 0.025g/L，NiCl₂·6H₂O 0.024g/L，Na₂WO₄·2H₂O0.025 g/L and the balance of ultrapure water.

The synthetic method of the ferrihydrite comprises the following steps: 8.11g FeCl was weighed₃Placing the mixture in 500mL of ultrapure water and placing the mixture on a magnetic stirrer to stir vigorously (1200r/min) to FeCl₃All dissolved while freshly prepared NaOH solutions of different concentrations (5M, 1M) were quickly added dropwise (done within 10min to avoid gibbsite) FeCl₃In the solution, the pH of the solution is 7.2-7.5. After balancing for 1h, NaOH is added dropwise again to stabilize the pH value of the solution at 7.2-7.5. After the pH stabilized, the suspension was left to stand for 6h in the dark at 4 deg.C (low temperature to promote crystallization), and the precipitate was centrifuged (4500g, 5min) and rinsed repeatedly to remove Cl^-And obtaining the precipitate which is the ferrihydrite until the conductivity in the supernatant is less than or equal to 10 mu S/cm. Resuspending the resulting pellet in ultrapure water and charging with high purity N₂And storing at 4 ℃ in the dark.

The iron Fe (II) and Fe (III) concentrations were measured as follows:

(1) preparing a feloxazine solution:

dissolving 11.96g N- (2-hydroxyethyl) piperazine-N' -2 sulfonic acid (HEPES, molecular weight 238.3) in 800mL of ultrapure water, adding 1.0g of feloxazine, fully mixing, adjusting the pH to 7.0, and metering to 1L with the ultrapure water to obtain a feloxazine solution, which is stored at 4 ℃ in a dark place.

(2) Fe (ii) standard curve configuration:

1) ammonium ferrous sulfate was formulated to the following series of concentrations: 0.1 mM, 5mM, 10mM, 20mM, 40mM, 80 mM;

2) diluting each concentration with 0.5M dilute hydrochloric acid by 50 times and digesting for 15min to obtain digestion solution;

3) sucking 0.05mL of digestion solution, adding 2.45mL of phenazine solution, and uniformly mixing;

4) measuring the light absorption value by using an enzyme-labeling instrument at the wavelength of 562 nm;

5) making a standard curve of Fe (II) diluted by 50 times;

(3) determination of the Fe (II) concentration in the sample:

sucking 0.1mL of sample, adding the sample into 4.9mL of 0.5M hydrochloric acid, uniformly mixing, and digesting for 15min to obtain a digestion solution; and then 0.05mL of digestion solution and 2.45mL of phenanthroline solution are sucked and mixed uniformly, the light absorption value is measured at the wavelength of 562nm, then a Fe (II) standard curve is brought in, and the concentration of Fe (II) in the sample is calculated.

(4) Determination of total iron:

1) adding 0.2mL of 6.25M hydroxylamine hydrochloride into a test tube containing 4.7mL of 0.5M hydrochloric acid, and uniformly mixing to obtain a mixed solution of hydrochloric acid and hydroxylamine hydrochloride;

2) quickly sucking 0.1mL of sample, adding the sample into the mixed solution of hydrochloric acid and hydroxylamine hydrochloride, uniformly mixing, and then placing the mixture in a dark place for digestion for 2 hours to obtain a digestion solution; the ferrihydrite needs to be digested for 12 hours;

3) sucking 0.05mL of digestion solution and 2.45mL of felazine solution, and mixing uniformly to obtain a mixed solution;

4) if any precipitate exists, the mixed solution needs to be filtered;

5) measuring the absorbance at 562nm wavelength;

6) the calculation was carried out with the Fe (II) standard curve.

The iron reducing capacity of the Shewanella strain (Shewanella sp.) A3A in a ferric citrate culture medium and a ferrihydrite culture medium can be used as an electron acceptor, and the iron reducing capacity of the Shewanella strain (Shewanella sp.) A3A in the ferric citrate culture medium and the ferrihydrite culture medium is shown in figure 5, and the result shows that the Shewanella strain A3A enters a logarithmic growth phase for 13-24h, and ferric citrate is reduced by 50mM after 36h reaction, and the reducing efficiency can reach 89% (figure 5 (a)); as shown in FIG. 5(b), the Shewanella sp A3A strain reacts slowly in the ferrihydrite medium for 10 days, generates 18mM Fe (II), and reduces ferrihydrite to 25mM with a reduction efficiency of 25% after 15 days of oxidation-reduction.

Shewanella oneidensis MR-1 described in Han R, Liu T, Li F, et al, dependence of secondary minor formation on Fe (II) production from ferrihydrite reduction by Shewanella oneidensis MR-1[ J ]. ACS Earth and Space Chemistry,2018,2(4): 399-. It can be seen that Shewanella sp A3A of the present invention has outstanding ferrihydrite reducing ability.

Example 3: electricity-producing effect of Shewanella sp A3A

The experiment adopts a single-chamber three-electrode system and adopts a 100mL transparent reagent bottle as a cell reactor. The bottle cap contains a working electrode, a reference electrode and a counter electrode. The working electrode and the counter electrode are both graphite plates with the area of 4.5cm²(3cm is multiplied by 1.5cm) and the thickness is 0.5cm, and all electrodes are penetrated into the inner part by titanium wires and do not contact the bottom of the bottle. All potentials were referenced to Hg/HgCl electrodes (saturated KCl solution), a three-electrode system was constructed and three cell reactors were assembled, 80mL of electrolyte was added to the reactor, and after deoxygenation and sterilization, Shewanella sp A3A in logarithmic growth phase was inoculated into the reactor in an inoculum size of 10% (v/v). The three electrodes were connected to an electrochemical workstation CHI1000C and the current generated by the microorganisms was monitored in real time using the time-current (i-t) mode. All experiments were performed at 30 ℃.

The electrolyte formula comprises: DL vitamin solution 10mL, DL mineral solution 10mL, NaHCO₃ 2.5g，NaH₂PO₄·H₂O 0.6g，NH₄Cl 0.25g，KCl 0.1g，1mM Na₂SeO₄1mL of solution, providing electron donor sodium lactate, and finally metering the volume to 1L; wherein the final concentration of sodium lactate is 20 mM.

Composition of the DL vitamin solution: 0.002g/L of biotin, 0.002g/L of folic acid, 0.01g/L of pyridoxine hydrochloride, 0.005g/L of riboflavin, 0.005g/L of pantothenic acid, 0.0001g/L of cobalamin, 0.005g/L of p-aminobenzoic acid, 0.005g/L of lipoic acid, 0.005g/L of nicotinic acid, 0.005g/L of thiamine and the balance of ultrapure water.

DL mineral solution composition: nitrilotriacetic acid trisodium 1.5g/L, MgSO₄·7H₂O 3.0g/L、MnSO₄·H₂O 0.5g/L、NaCl 0.5g/L、FeSO₄·7H₂O 0.1g/L、CaCl₂ 0.1g/L、CoCl₂·6H₂O 0.1g/L、ZnSO₄·7H₂O 0.1g/L、CuSO₄·5H₂O 0.01g/L、AlK(SO₄)₂·12H₂O 0.01g/L、H₃BO₃ 0.01g/L、Na₂MoO₄0.025g/L、NiCl₂·6H₂O 0.024g/L、Na₂WO₄·2H₂O0.025 g/L and the balance of ultrapure water.

The current change monitored during the constant potential culture process of the electrochemical workstation CHI1000C is shown in FIG. 6, and it can be seen from FIG. 6 that Shewanella sp A3A strain rapidly increases at 0-1 d, reaches the highest peak at 1d, is about 0.6mA, and then slowly decreases. In the process, the Shewanella sp A3A strain uses sodium lactate in electrolyte as electron donor, graphite plate electrode as electron acceptor, microorganism is attached to the electrode, red biomembrane is formed on the graphite plate after enrichment, and the electrogenesis density reaches 0.044mA/cm²Is obviously higher than the prior documents Bretscheger O, A Obraztsova, C A Sturm, et al]Applied environmental microbiology,2007,73(21):7003-12, Shewanella oneidensis MR-1^TThe power generation density (0.014 mA/cm)²)。

The above results indicate that the Shewanella sp A3A of the present invention has good electricity generating ability.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Sequence listing

<110> river-south university

<120> strain with iron reduction capability and electricity generation capability and application thereof

<160> 3

<170> SIPOSequenceListing 1.0

<210> 1

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<223> Forward primer 27F

<400> 1

agagtttgat cctggctca 19

<210> 2

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<223> reverse primer 1492R

<400> 2

ggttaccttg ttacgactt 19

<210> 3

<211> 1512

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<223> 16S rDNA sequence of Shewanella sp A3A

<400> 3

gagtttgatc ctggctcaga ttgaacgctg gcggcaggcc taacacatgc aagtcgagcg 60

gcagcgggga gtagcttgct actctgccgg cgagcggcgg acgggtgagt aatgtctggg 120

aacttgccca ttcgaggggg ataacagttg gaaacgactg ctaataccgc atacgcccta 180

cgggggaaag caggggaact tcggtccttg cgcgaatgga taggcccaga tgggattagc 240

taattggtaa ggtaacggct taccaaggcg acgatctcta gctggtttga gaggatgatc 300

agccacactg gaactgagac acggtccaga ctcctacggg aggcagcagt ggggaatatt 360

gcacaatggg ggaaaccctg atgcagccgt gccgcgtgtg tgaagaaggc cttcgggttg 420

taaagcactt tcagcgagga ggaaagggtg tgtgctaata ccatacatct gtgacgttac 480

tcgcagaaga agcaccggct aactccgtgc cagcagccgc ggtaatacgg agggtgcgag 540

cgttaatcgg aattactggg cgtaaagcgt gcgcaggcgg tttgttaagc gagatgtgaa 600

agccccgggc tcaacctggg aattgcattt cgaactggca agctagagtc ttgtagaggg 660

gggtagaatt tcaggtgtag cggtgaaatg cgtagagatc tgaaggaata ccggtggcga 720

aggcggcccc ctggacaaag actgacgctc atgcacgaaa gcgtggggag caaacaggat 780

tagataccct ggtagtccac gccgtaaacg atgtctactc ggagtttggt gtcttgaaca 840

ctgggctctc aagctaacgc attaagtaga ccgcctgggg agtacggccg caaggttaaa 900

actcaaatga attgacgggg gcccgcacaa gcggtggagc atgtggttta attcgatgca 960

acgcgaagaa ccttacctac tcttgacatc cacagaatct ggtagagata cctcagtgcc 1020

ttcgggaact gtgagacagg tgctgcatgg ctgtcgtcag ctcgtgttgt gaaatgttgg 1080

gttaagtccc gcaacgagcg caacccttat ccttacttgc cagcgggtaa tgccgggaac 1140

tttagggaga ctgccggtga taaaccggag gaaggtgggg acgacgtcaa gtcatcatgg 1200

cccttacgag tagggctaca cacgtgctac aatggtcagt acagagggaa gcgaagcagc 1260

gatgtggagc gaatctctta aagctggtcg tagtccggat cggagtctgc gactcgactc 1320

cgtgaagtcg gaatcgctag taatcgcaaa tcagaatgct gcggtgaata cgttcccggg 1380

ccttgtacac accgcccgtc acaccatggg agtgggttgc accagaagta gatagcttaa 1440

ccttcggggg ggcgtttacc acggtgtgat tcatgactgg ggtgaagtcg taacaaggta 1500

accaatcgtc ga 1512

Claims (10)

1. A strain with iron reduction capability and electricity generation capability is characterized in that: is named as Shewanella sp A3A, and is preserved in Guangdong province microorganism strain preservation center of the institute for microorganisms of Guangdong province academy of sciences, Guangzhou, No. 59 building, No. 5 building, Mirabilite 100, located in Guangzhou city, with the preservation number of CDMCC No. 62213 in 17 th 2022.

2. Use of the strain having iron-reducing ability and electricity-generating ability according to claim 1 for iron reduction and/or microbial power generation.

3. Use according to claim 2, wherein the iron reduction is iron citrate or iron reduction in ferrihydrite.

4. The method for reducing iron using the strain having iron-reducing ability and electricity-generating ability according to claim 1, comprising the operation of inoculating the strain having iron-reducing ability and electricity-generating ability according to claim 1 in a citrate iron basal medium or a ferrihydrite medium with the addition of sodium lactate.

5. The method according to claim 4, wherein the strain having iron-reducing ability and electricity-generating ability is a strain having iron-reducing ability and electricity-generating ability in the logarithmic phase.

6. The method of claim 4, wherein the iron citrate basal medium with the addition of sodium lactate is prepared by: pouring 13.7g of ferric citrate into 150mL of boiling ultrapure water, and stirring with a glass rod until the ferric citrate is completely dissolved to obtain a ferric citrate dissolved solution; adding the ferric citrate dissolved solution into 600mL of ultrapure water, cooling to room temperature, adjusting the pH to 6.0-6.5 by using a NaOH solution, and then sequentially adding: DL vitamin solution 10mL, DL mineral solution 10mL, NaHCO₃ 2.5g、NaH₂PO₄·H₂O 0.6g，NH₄Cl 0.25g，KCl 0.1g、1mM Na₂SeO₄1mL of solution and sodium lactate, and finally, fixing the volume to 1L; wherein the final concentration of sodium lactate is 20 mM;

composition of the DL vitamin solution: 0.002g/L of biotin, 0.002g/L of folic acid, 0.01g/L of pyridoxine hydrochloride, 0.005g/L of riboflavin, 0.005g/L of pantothenic acid, 0.0001g/L of cobalamin, 0.005g/L of p-aminobenzoic acid, 0.005g/L of lipoic acid, 0.005g/L of nicotinic acid, 0.005g/L of thiamine and the balance of ultrapure water;

DL mineral solution composition: nitrilotriacetic acid trisodium 1.5g/L, MgSO₄·7H₂O 3.0g/L、MnSO₄·H₂O 0.5g/L、NaCl 0.5g/L、FeSO₄·7H₂O 0.1g/L、CaCl₂ 0.1g/L、CoCl₂·6H₂O 0.1g/L、ZnSO₄·7H₂O 0.1g/L、CuSO₄·5H₂O 0.01g/L、AlK(SO₄)₂·12H₂O 0.01g/L、H₃BO₃ 0.01g/L、Na₂MoO₄0.025g/L、NiCl₂·6H₂O 0.024g/L、Na₂WO₄·2H₂O0.025 g/L and the balance of ultrapure water.

7. The method according to claim 4, wherein the formula of the ferrihydrite medium with the addition of sodium lactate is as follows: ferrihydrite, DL vitamin solution 10mL, No-sized DL Mineral Mix 10mL, NaHCO₃ 2.5g，NaH₂PO₄·H₂O 0.6g，NH₄Cl 0.25g，KCl 0.1g，1mM Na₂SeO₄1mL of solution, providing sodium lactate for an electron donor, and finally metering the volume to 1L; wherein the final concentration of the sodium lactate is 20mM, and the final concentration of the ferrihydrite is 100 mM;

composition of the DL vitamin solution: 0.002g/L of biotin, 0.002g/L of folic acid, 0.01g/L of pyridoxine hydrochloride, 0.005g/L of riboflavin, 0.005g/L of pantothenic acid, 0.0001g/L of cobalamin, 0.005g/L of p-aminobenzoic acid, 0.005g/L of lipoic acid, 0.005g/L of nicotinic acid, 0.005g/L of thiamine and the balance of ultrapure water;

no-sized DL Mineral Mix composition: MgSO (MgSO)₄ 3.0g/L，MnSO₄·H₂O 0.5g/L，NaCl 1.0g/L，FeSO₄·7H₂O 0.1g/L，CaCl₂·6H₂O 0.1g/L，ZnCl₂ 0.13g/L，CuSO₄·5H₂O 0.01g/L，AlK(SO₄)₂·12H₂O 0.01g/L，H₃BO₃ 0.01g/L，Na₂MO₄·2H₂O 0.025g/L，NiCl₂·6H₂O 0.024g/L，Na₂WO₄·2H₂O0.025 g/L and the balance of ultrapure water.

8. The method according to claim 7, characterized in that the method for preparing ferrihydrite is: 8.11g FeCl was weighed₃Placing the mixture in 500mL of ultrapure water and placing the mixture on a magnetic stirrer to stir vigorously until FeCl is formed₃All are dissolved, and at the same time, NaOH solution is quickly dropped into FeCl₃In the solution, the pH value of the solution is 7.2-7.5; after balancing for 1h, dropwise adding NaOH again to stabilize the pH value of the solution at 7.2-7.5; after the pH is stabilized, the suspension is kept stand for 6 hours at the temperature of 4 ℃ in a dark place, and the precipitate is centrifugally rinsed repeatedly to remove Cl^-And obtaining the precipitate which is the ferrihydrite until the conductivity in the supernatant is less than or equal to 10 mu S/cm.

9. A method for microbial power generation using the strain having iron-reducing ability and power-generating ability according to claim 1, comprising the operation of inoculating the strain having iron-reducing ability and power-generating ability according to claim 1 in an electrolytic solution.

10. The method according to claim 9, wherein the strain having iron-reducing ability and electricity-generating ability is a strain having iron-reducing ability and electricity-generating ability in the logarithmic phase;

the formula of the electrolyte is as follows: DL vitamin solution 10mL, DL mineral solution 10mL, NaHCO₃ 2.5g，NaH₂PO₄·H₂O 0.6g，NH₄Cl 0.25g，KCl 0.1g，1mM Na₂SeO₄1mL of solution, providing electron donor sodium lactate, and finally metering the volume to 1L; wherein the final concentration of sodium lactate is 20 mM;

composition of the DL vitamin solution: 0.002g/L of biotin, 0.002g/L of folic acid, 0.01g/L of pyridoxine hydrochloride, 0.005g/L of riboflavin, 0.005g/L of pantothenic acid, 0.0001g/L of cobalamin, 0.005g/L of p-aminobenzoic acid, 0.005g/L of lipoic acid, 0.005g/L of nicotinic acid, 0.005g/L of thiamine and the balance of ultrapure water;

DL mineral solution composition: nitrilotriacetic acid trisodium 1.5g/L, MgSO₄·7H₂O 3.0g/L、MnSO₄·H₂O 0.5g/L、NaCl 0.5g/L、FeSO₄·7H₂O 0.1g/L、CaCl₂ 0.1g/L、CoCl₂·6H₂O 0.1g/L、ZnSO₄·7H₂O 0.1g/L、CuSO₄·5H₂O 0.01g/L、AlK(SO₄)₂·12H₂O 0.01g/L、H₃BO₃ 0.01g/L、Na₂MoO₄0.025g/L、NiCl₂·6H₂O 0.024g/L、Na₂WO₄·2H₂O0.025 g/L and the balance of ultrapure water.

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