Perchlorate-degrading bacterium strain hc7-4 and application thereof

Abstract

The application discloses a strain HC7-4 capable of degrading perchlorate and coupling oxygen production and an application thereof, and belongs to the technical field of biotechnology.The strain HC7-4 is preserved in the China General Microbiological Culture Collection Center on October 11, 2022; the address of the preservation unit is No.3, Xibei Road, Beichen, Chaoyang District, Beijing; and the preservation number is CGMCC No.25888.The strain CH4-7 has strong adaptability, is easy to cultivate, has strong resistance to stress environments such as low temperature, drought and ultraviolet radiation, and the strain and its metabolic polymers can efficiently degrade perchlorate and simultaneously couple oxygen production, and have a degrading effect on perchlorate with a mass concentration of 0.10% to 2.00%.The strain can be applied to the bioremediation of perchlorate-polluted water bodies and other contaminated sites, and can be used as an alternative germplasm resource for perchlorate biodegradation and coupling oxygen production in the creation of a habitable environment on Mars.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a strain HC7-4 that degrades perchlorate and generates oxygen via coupling, and its applications. Background Technology

[0002] Mars is the most Earth-like planet in the solar system, and many countries have proposed plans for human landing on Mars. China and the United States plan to achieve a manned Mars landing in 2033.

[0003] The Martian surface contains 0.50%-1.00% perchlorates, which are highly oxidizing and corrosive. The strong oxidizing properties of perchlorates and the extreme lack of oxygen on the Martian surface create a harsh environment highly unfavorable to human survival, posing a significant threat to human landing on Mars and future colonization.

[0004] Meanwhile, perchlorate is also a persistent and difficult-to-degrade inorganic pollutant in the Earth's environment. It is frequently detected in surface water, groundwater, milk, crops, meat, and other foods, making the perchlorate pollution problem increasingly prominent.

[0005] Perchlorate is neurotoxic, affecting hippocampal synaptic function and causing irreversible damage to synaptic neurotransmitters. Perchlorate can also affect thyroid function, has reproductive and immunotoxic effects, and may even impair the intellectual development and central nervous system of infants, thus having an adverse impact on human health.

[0006] Perchlorate removal methods include physical methods, chemical methods, and biodegradation methods. Among them, biodegradation methods have advantages such as high efficiency, low consumption, and environmental friendliness, making them a promising treatment method with broad application prospects. Currently, there are studies on perchlorate degradation in the Earth's environment both domestically and internationally, but these mainly focus on the degradation of low-concentration perchlorate by microbial strains, while research reports on the degradation of high-concentration perchlorate (0.50%-1.00%) are extremely rare.

[0007] Therefore, this invention will provide a microbial strain that has the ability to degrade low, medium and high concentrations of perchlorate and has strong stress resistance. It can be applied to the bioremediation of perchlorate-contaminated water bodies and other polluted sites, and in particular, it can be used as a candidate strain for the harmless treatment and coupling oxygen production of perchlorate on the surface of Mars. Summary of the Invention

[0008] The purpose of this invention is to provide a strain HC7-4 that degrades perchlorate and generates oxygen, and its applications, to solve the problems existing in the prior art. Strain HC7-4 exhibits strong resistance, surviving freezing at -20℃ for 12 hours, UV irradiation at 20 W for 120 minutes, and in soil culture medium with a moisture content of 2.86%. It can degrade perchlorate at concentrations of 0.10%-2.00% while simultaneously generating oxygen.

[0009] To achieve the above objectives, the present invention provides the following solution:

[0010] This invention provides a strain of *Lysinibacillus fusiformis* HC7-4 that degrades perchlorate and generates oxygen via coupling. *Lysinibacillus fusiformis* HC7-4 was deposited on October 11, 2022, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 25888.

[0011] The present invention also provides a polymer, which is a metabolite of the spindle-shaped lysine spore bacillus HC7-4, which can degrade perchlorate, degrading perchlorate ions into chloride ions and generating oxygen in the process.

[0012] Furthermore, the polymer is a polysaccharide or a protein.

[0013] The present invention also provides the application of the strains / substances described in (1) or (2) in the degradation of perchlorate and its coupled oxygen production:

[0014] (1) Bacillus spindleii HC7-4 with accession number CGMCC No. 25888;

[0015] (2) The polymer secreted by the spindle-shaped lysine spore bacillus HC7-4, wherein the polymer is a polysaccharide or a protein.

[0016] Furthermore, the degradation of perchlorate involves degrading perchlorate ions into chloride ions while simultaneously producing oxygen, and also reducing the redox potential of the perchlorate degradation system.

[0017] Furthermore, the perchlorate includes sodium perchlorate.

[0018] The present invention also provides a method for degrading perchlorate, comprising a system and steps for degrading perchlorate using the aforementioned Bacillus fusiformis HC7-4 or the aforementioned polymer.

[0019] Furthermore, the degradation of perchlorate is carried out under static or shaking conditions at a temperature of 25-40°C.

[0020] Furthermore, the system and process for degrading perchlorate also include an electron donor, a carbon source, and an inorganic salt. The electron donor is one of riboflavin, reduced iron powder, and pyrite; the carbon source is one of sodium acetate, sodium carbonate, and sodium bicarbonate; and the inorganic salt is a component of an inorganic salt culture medium, the composition of which is as follows:

[0021] (1) Mother liquor of macro elements I: 0.130 g of dipotassium hydrogen phosphate, 0.060 g of potassium dihydrogen phosphate, 0.060 g of magnesium sulfate, 1.000 g of anhydrous sodium acetate, 0.180 g of ammonium nitrate, and water added to 1000 mL;

[0022] (2) Trace element mother liquor II: 0.110 g zinc sulfate, 0.275 g calcium sulfate, 0.133 g manganese sulfate, 0.1055 g sodium molybdate, 0.0785 g copper sulfate, add water to 100 mL;

[0023] (3) Iron salt mother liquor solution: 0.125 g of ferrous sulfate heptahydrate, 0.171 g of EDTA, add water to 100 mL.

[0024] This invention also provides an application of Bacillus fusiformis HC7-4 or its polymer in the degradation of perchlorate and coupling oxygen production on the Martian surface. The Bacillus fusiformis HC7-4 can serve as a pioneer species for Martian environmental terraforming.

[0025] The present invention also provides the application of the aforementioned spindle-shaped lysine-containing Bacillus HC7-4 or the aforementioned polymer in the bioremediation of perchlorate-contaminated water bodies and other contaminated sites.

[0026] Compared with the prior art, the beneficial effects achieved by the present invention are as follows:

[0027] (1) The sodium perchlorate degrading bacterium HC7-4 obtained by screening in this invention was identified as Lysinibacillus fusiformis. This strain is easy to culture and can grow under both static and shake-flask culture conditions. It can also grow in beef extract peptone medium, LB medium and inorganic salt medium.

[0028] (2) The strain HC7-4 has strong resistance. It can tolerate and survive freezing treatment at -20℃ for 12h, UV irradiation at 20 W for 120min, and soil culture medium with a water content of 2.86%.

[0029] (3) The HC7-4 strain has a wide degradation range for perchlorate concentrations, capable of degrading sodium perchlorate at concentrations ranging from 0.10% to 2.00%. Specifically, the half-life for degrading sodium perchlorate at a mass concentration of 0.50% is 3.89 days, with a degradation rate reaching 97.53%. It can be applied to the bioremediation of Earth's aquatic environment and other perchlorate-contaminated environments, and can also be used as a candidate strain for the degradation and harmless treatment of perchlorate on the surface of Mars.

[0030] (4) The polymers metabolized by strain HC7-4 have a high efficiency in degrading perchlorate and can be applied to the degradation of perchlorate under extreme environmental conditions on Mars / Earth.

[0031] (5) HC7-4 produces oxygen by degrading perchlorate, providing pioneer germplasm resources for the terraforming of Mars, where perchlorate content is high and oxygen is extremely low. Attached Figure Description

[0032] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0033] Figure 1 The colony morphology of strain HC7-4;

[0034] Figure 2 The microscopic morphology of strain HC7-4 under a scanning electron microscope;

[0035] Figure 3 Phylogenetic tree of strain HC7-4;

[0036] Figure 4 The growth curves of the strain in shake flask (A) and static (B) culture in beef extract peptone medium are shown.

[0037] Figure 5 The growth of strain HC7-4 in 0.5% perchlorate at different temperatures;

[0038] Figure 6 The effect of adding electron donors on the degradation of sodium perchlorate by strain HC7-4 was investigated, where A was riboflavin; B was sodium anthraquinone-2,6-disulfonate; C was reduced iron powder; and D was pyrite.

[0039] Figure 7 The degradation of sodium perchlorate by strain HC7-4 at different concentrations;

[0040] Figure 8 The dissolved oxygen (left) and redox potential changes (right) generated in the degradation system of strain HC7-4. Detailed Implementation

[0041] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0042] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0043] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0044] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0045] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0046] Experimental reagents and culture media used in the examples:

[0047] The main chemical reagents used in this invention are commercially available conventional biochemical reagents or analytical grade reagents.

[0048] Beef extract peptone medium: 5.00 g beef extract, 10.00 g peptone, 5.00 g NaCl, 1 L water, and agar added to solid medium at a concentration of 15.00 g / L-20.00 g / L.

[0049] LB medium: 5.00 g yeast extract, 10.00 g peptone, 5.00 g NaCl, 1 L water, and agar added to solid medium at a concentration of 15.00 g / L-20.00 g / L.

[0050] Inorganic salt culture medium: Take 1000 mL of stock solution I, 2 mL of stock solution II, and 2 mL of iron salt stock solution. Add 15.00 g / L to 20.00 g / L agar to the solid culture medium.

[0051] (1) Mother liquor of macro elements I: 0.130 g of dipotassium hydrogen phosphate, 0.060 g of potassium dihydrogen phosphate, 0.060 g of magnesium sulfate, 1.00 g of anhydrous sodium acetate, 0.180 g of ammonium nitrate, and water added to 1000 mL.

[0052] (2) Trace element mother liquor II (1:500): Zinc sulfate 0.110 g, calcium sulfate 0.275 g, manganese sulfate 0.133 g, sodium molybdate 0.1055 g, copper sulfate 0.0785 g, add water to 100 mL.

[0053] (3) Iron salt mother liquor (1:500): 0.125 g of ferrous sulfate heptahydrate, 0.171 g of EDTA, add water to 100 mL.

[0054] Different concentrations of perchlorate were added according to the experimental requirements.

[0055] Example 1 Screening and Identification of Perchlorate-Degrading Strains

[0056] 1. Screening of perchlorate-degrading strains

[0057] (1) Strains domestication and enrichment culture

[0058] Multiple soil samples, sludge, and landscape water samples were collected. After mixing the different samples evenly, they were added to inorganic salt liquid culture media with sodium perchlorate contents of 0.50%, 1.00%, 2.00%, 3.00%, 4.00%, 5.00%, 6.00%, 7.00%, 8.00%, 9.00%, and 10.00%, respectively. The samples were then placed at 20-37℃ for acclimatization for one month to obtain the first acclimatization bacterial solution.

[0059] Take 10 mL of the first acclimatization solution and transfer it to a new sodium perchlorate inorganic salt liquid culture medium of the same concentration. Place it in a acclimatization culture at 20-37℃ and continue to acclimatize. This process will yield the second and third acclimatization enrichment solutions.

[0060] (2) Isolation and purification of perchlorate-degrading strains

[0061] The second and third enrichment bacterial suspensions were taken and spread onto inorganic salt solid culture media containing 0.50% and 1.00% sodium perchlorate, respectively, using the dilution plating method. The media were incubated at 20-37℃ for 3-10 days to obtain different colonies. Single colonies were picked and streaked onto the aforementioned culture media for further isolation and purification until single colonies were obtained, resulting in multiple strains with different morphologies.

[0062] (3) Screening of perchlorate-degrading strains

[0063] Preparation of bacterial suspension: The strains isolated and purified above were activated and cultured in beef extract peptone liquid medium or LB liquid medium for 24 h. Then, they were centrifuged at 8000-10000 rpm / min for 5 min, the supernatant was discarded, and the suspension was washed twice with sterile distilled water. The OD of the bacterial suspension was adjusted with inorganic salt medium. 600 It is 1.50.

[0064] Inoculation and cultivation: Prepare inorganic salt culture media containing 0.50%, 1.00%, 1.50%, and 2.00% sodium perchlorate, respectively. Inoculate the bacterial suspension at a rate of 10% and incubate statically at 20-37 ℃. Observe the changes in turbidity of the culture medium regularly.

[0065] Strain screening: Starting from day 0, bacterial culture samples were taken every 2 days for 10 consecutive days, and the turbidity (OD) of the bacterial culture was measured. 600 The chloride ion production was determined according to the national standard GB / T15453-2018, and the perchlorate degradation rate was calculated according to formula (1).

[0066] De (%) = P t / P0×100% (1)

[0067] Wherein, P0 is the Cl in the culture medium after the complete degradation of perchlorate ions in theory. - mass concentration; P t The Cl in the culture medium at time t - The mass concentration.

[0068] By comparing the test results of different purified strains, strains with high degradation rate and large growth were screened out. A total of 4 strains were obtained and named as follows: HC7-4, P351-1, P372-2 and GT4.

[0069] Four perchlorate-degrading bacterial strains were re-screened. Strain HC7-4 was cultured in an inorganic salt medium with a sodium perchlorate concentration of 0.50%. OD 600 The value can reach 0.4815, indicating a relatively high degradation rate, thus the strain HC7-4 was obtained through screening.

[0070] 2. Identification of sodium perchlorate-degrading strains

[0071] The bacterial strains were identified by combining colony morphology and molecular biology.

[0072] (1) Identification of strain morphology

[0073] Strawberry strain HC7-4 was streaked onto beef extract peptone agar and incubated at 30 °C for 24 h. Colonies were pale yellow, opaque, 0.1-2.0 mm in diameter, smooth, moist, and glossy, with irregular, flat edges. See attached image for colony morphology. Figure 1 As shown.

[0074] The microscopic morphology of strain HC7-4 cells was observed using scanning electron microscopy. Figure 2 As shown.

[0075] (2) Molecular biological identification of strain HC7-4

[0076] a) Genome extraction, PCR amplification, and sequencing

[0077] Genomic DNA was extracted from strain HC7-4 using the Tiangen Bacterial Genomic DNA Extraction Kit and sent to Beijing Sangon Biotech Co., Ltd. for PCR amplification and sequencing. The 16S rDNA gene of the strain was amplified by PCR using universal bacterial primers 27F and 1492R. The primer sequences were: forward 27F: 5'-AGAGTTTGATCCTGGCTCA G-3', reverse 1492R: 5'-GGTTACCTTGTTACGACTT-3'.

[0078] b) Phylogenetic tree construction

[0079] The obtained sequencing sequences were subjected to BLAST homology alignment on EZBiocloud, revealing that the 16S rDNA sequence of strain HC7-4 shared 99.45% homology with *Lysinibacillus fusiformis*. Homology alignment was performed using ClustalX 2.0 software, and a phylogenetic tree was constructed using MEGA 6.0 software. The results are shown (see...). Figure 3 The strain HC7-4 showed the highest homology with Lysinibacillus fusiformis. Based on the colony morphology characteristics, strain HC7-4 was identified as Lysinibacillus fusiformis.

[0080] (3) Strain HC7-4 was identified as a strain with novel functions.

[0081] Based on morphological observation and phylogenetic tree construction analysis, strain HC7-4 was identified as *Lysinibacillus fusiformis*. This strain was deposited on October 11, 2022, at the China General Microbiological Culture Collection Center (CGMCC), accession number CGMCC No. 25888.

[0082] Currently, there are no reports, either domestically or internationally, of *Lysinibacillus fusiformis* possessing the ability to degrade perchlorate. Therefore, this strain is a novel strain with perchlorate-degrading capabilities. It can be used for the degradation and bioremediation of perchlorate-contaminated environments, including water, soil, manufacturing industries, and other environments.

[0083] Example 2: Growth characteristics and stress resistance of strain HC7-4

[0084] 1. Effects of different culture media and cultivation methods on strain growth

[0085] Strain strain HC7-4 was inoculated into LB medium, beef extract peptone medium, and inorganic salt liquid medium, respectively, and placed in a shaker at 25-30 ℃ and 160-180 rpm / min for static culture at 25-30 ℃. Samples were taken every 5 hours starting from 0 h, for a total of 10 consecutive samples, to determine the growth of the strain under different culture media and different culture conditions.

[0086] The results showed that strain HC7-4 could grow in LB medium, beef extract peptone medium, and inorganic salt liquid medium, using both shake-flask and static cultures. The growth curves of the strain in beef extract medium under shake-flask (A) and static (B) cultures are shown below. Figure 4 The biomass of shake flask culture and static culture can reach OD values ​​of 100%. 600 =2.9360 and OD 600 =2.4445. This shows that strain HC7-4 is easy to culture, with a variety of available culture media, and can be cultured using both shaking and static methods.

[0087] 2. Strain HC7-4 exhibits strong tolerance to low temperature and drought stress.

[0088] Preparation of HC7-4 bacterial suspension: Activate strain HC7-4 using beef extract peptone liquid medium, centrifuge at 8000-10000 rpm / min for 5 min, discard the supernatant, wash the precipitate twice with sterile physiological saline, and suspend it in sterile physiological saline to obtain the bacterial suspension.

[0089] Preparation of soil-containing petri dishes: Select glass petri dishes with a diameter of 90 mm, grind the newly created soil from Yan'an and pass it through a 60-mesh sieve, add it to the petri dishes, add 20.00 g of Yan'an soil sample to each petri dish, and sterilize at 121 ℃ for 20-30 min.

[0090] Low-temperature tolerance test of the strain: Take 10 mL of bacterial suspension and spray it evenly onto the surface of a petri dish containing soil, then cover the dish. Place it in a -20℃ refrigerator. Remove the petri dish from the refrigerator at 3 h, 6 h, 9 h, and 12 h, and incubate it at 25℃-30℃. Take samples every 10 days and investigate the growth of the strain. Take a sample with an area of ​​1 cm². 2 Soil samples were cultured in inorganic salt medium. 200 μL of the culture solution was spread onto the surface of beef extract peptone solid medium and incubated at room temperature (25-35℃). The low-temperature tolerance of the strain was determined based on the presence and number of colonies on the plates. The results are shown in Table 1.

[0091] Table 1. Effects of low-temperature treatment on strain growth

[0092]

[0093] Note: "+" indicates 5-30 colonies have grown, and "++" indicates more than 30 colonies have grown.

[0094] Low temperature studies have shown that strain HC7-4 can still grow after being treated at -20℃ for 12 h, indicating that strain HC7-4 has strong resistance to low temperatures.

[0095] After the low-temperature resistance experiment of the above-mentioned strains was completed, the moisture content of the soil sample in the culture plate was measured, and the result was 2.86%. A 1cm sample was taken. 2 The topsoil sample was inoculated into an inorganic salt medium and allowed to stand for 10-15 days. The inorganic salt medium became turbid. The bacterial solution was then spread onto beef extract peptone solid medium and cultured for 24 hours. The bacterial strain grew on the medium, indicating that the strain could survive in the dry soil sample with a water content of 2.86%, revealing that strain HC7-4 has strong drought resistance.

[0096] In summary, strain HC7-4 exhibits strong resistance to low temperature and drought stress, making it suitable for use in low temperature and drought environments, as well as for the degradation of perchlorate in such environments.

[0097] 3. Strain HC7-4 has strong resistance to ultraviolet radiation.

[0098] Take 10 mL of bacterial suspension and spray it evenly onto the surface of the soil sample in a soil culture dish. Place the dish in a clean bench and irradiate it with a 20 W UV lamp for 30 min, 60 min, 90 min, and 120 min respectively. Then cover the dish and incubate it at 25-30℃. Take a 1 cm² area sample every 10 days. 2Soil samples were cultured in inorganic salt medium. 200 μL of the culture solution was spread onto the surface of beef extract peptone solid medium and incubated at 20-35℃ for 24 h. The presence and number of colonies on the plates were used to determine the strength of the strain's resistance to ultraviolet radiation. The experimental results are shown in Table 2.

[0099] Experimental results showed that the strain survived irradiation with a 20 W UV lamp for 120 min. This indicates that strain HC7-4 has a strong tolerance to UV radiation. Therefore, this strain can be used in environments with high radiation, especially for the degradation of perchlorate in such environments.

[0100] Table 2 Effects of UV irradiation treatment on bacterial growth

[0101]

[0102] Note: "+" indicates the presence of 10-30 colonies.

[0103] Example 3: Factors affecting the degradation of perchlorate by strain HC7-4 and the degradation system

[0104] 1. Effect of temperature on bacterial growth in perchlorate degradation system

[0105] HC7-4 bacterial suspension was inoculated at a 10% inoculum into inorganic salt medium containing 0.50% sodium perchlorate and incubated statically at 25 ℃, 30 ℃, 35 ℃, and 40 ℃, with each experiment performed in triplicate. Samples were taken on days 0, 2, 4, 6, 8, and 10, and OD values ​​were measured sequentially. 600 The values ​​of chloride ions and perchlorate degradation rates were calculated. The experimental results on the effect of temperature on the growth of strain HC7-4 are as follows: Figure 5 As shown.

[0106] Experimental results showed that strain HC7-4 could grow and degrade sodium perchlorate at temperatures of 25 ℃, 30 ℃, 35 ℃, and 40 ℃. Among these, when cultured at 35 ℃ for 6 days, the OD of the bacterial culture was [missing value]. 600 The degradation rate can reach 0.821; the degradation rate is higher when the culture temperature is 30℃-35℃.

[0107] 2. Effects of different carbon sources on the degradation of perchlorate by the strain

[0108] Sodium acetate, sodium carbonate, and sodium bicarbonate were selected as carbon sources and added at a concentration of 1.7 g / L. The mixture was incubated statically in a 25-30 ℃ incubator. Samples were taken on days 0, 2, 4, 6, 8, and 10, and OD values ​​were measured sequentially. 600 Chloride ion content and dissolved oxygen were measured, and the perchlorate degradation rate was calculated.

[0109] The test results show that the addition of different carbon sources, such as sodium acetate, sodium carbonate, and sodium bicarbonate, all promote the degradation of perchlorate, with sodium acetate and sodium carbonate showing higher degradation rates when used as carbon sources.

[0110] Based on the above experiments, sodium acetate was selected as the carbon source, and different concentrations of sodium acetate (0.20 g / L, 0.40 g / L, 0.60 g / L, 0.80 g / L, 1.00 g / L, 1.20 g / L, and 1.40 g / L) were added to study the effect of different concentrations of sodium acetate on the degradation of sodium perchlorate. The study found that the addition of sodium acetate at concentrations of 0.40–1.40 g / L promoted the degradation of sodium perchlorate. Under the conditions of sodium acetate addition concentrations of 0.8–1.2 g / L, strain HC7-4 exhibited a higher degradation rate, reaching 73.67% after 8 days of cultivation.

[0111] 3. The effect of electron donors on perchlorate degradation

[0112] Four different electron donors were selected: riboflavin, sodium anthraquinone-2,6-disulfonate, reduced iron powder, and pyrite, with electron donor concentrations of 0.12 mM, 0.24 mM, 0.48 mM, and 0.96 mM, respectively. The bacterial suspension was inoculated at a 10% inoculum into inorganic salt media containing electron donors and 0.50% sodium perchlorate, and cultured statically. The degradation rate of sodium perchlorate and dissolved oxygen production by strain HC7-4 were measured. The degradation rate of sodium perchlorate by strain HC7-4 is shown in […]. Figure 6 .

[0113] Depend on Figure 6 It was found that the addition of four different electron donors had varying effects on the degradation of sodium perchlorate by strain HC7-4. The addition of reduced iron powder and riboflavin showed better degradation effects, while the addition of anthraquinone-2,6-disulfonate sodium showed relatively poor effects. It was also found that the ionic composition of the inorganic salt culture medium also played a role in the growth of the strain and the degradation of sodium perchlorate. Therefore, the type and concentration of added electron donors have a significant impact on the degradation of perchlorate. Under the conditions of 0.12-0.24 mM reduced iron powder, this strain showed the best degradation effect on perchlorate, with a degradation rate of 87.66% and dissolved oxygen of 8.45 mg / L on day 8.

[0114] 4. Degradation of sodium perchlorate by different concentrations by the strain

[0115] The bacterial suspension of strain HC7-4 was inoculated at a rate of 10% into inorganic salt media with sodium perchlorate concentrations of 0.10%, 0.50%, 1.00%, 1.50%, and 2.00%, respectively, and incubated statically in a constant temperature incubator at 25-30℃. Samples were taken at 0, 2, 4, 6, 8, and 10 days to determine the OD value. 600Values, chloride ions, dissolved oxygen, and redox potential were measured. Dissolved oxygen was measured using a JPB-607A dissolved oxygen meter, and redox potential was measured using a FE20 ORP meter.

[0116] The results showed that within the sodium perchlorate concentration range of 0.10%–2.00%, the degradation rate of sodium perchlorate by the strain initially increased and then decreased. The results of the strain's degradation of sodium perchlorate at concentrations of 0.10%–1.00% are shown in [the table below]. Figure 7 and Figure 8 When the sodium perchlorate concentration is 0.50%, the strain can degrade sodium perchlorate by 97.53%.

[0117] Experiments showed that the degradation of sodium perchlorate by strain HC7-4 follows first-order kinetics, and the degradation kinetic equation and half-life are shown in Table 3. The half-life of strain HC7-4 for the degradation of 0.50% sodium perchlorate is 3.89 days.

[0118] Table 3. Degradation kinetic equations of strain HC7-4 under different sodium perchlorate concentrations.

[0119]

[0120] By measuring dissolved oxygen and redox potential, it was found that the dissolved oxygen production in the 0.50% sodium perchlorate degradation solution can reach 7.80 mg / L. The strain decomposes the highly oxidizing perchlorate ions into harmless chloride ions, which promotes the reduction of redox potential in the perchlorate degradation system. In other words, the oxidizing power of perchlorate can be gradually removed as it is degraded.

[0121] Analysis of the degradation rate of strain HC7-4 in relation to dissolved oxygen and OD 600 The correlation between the indicators revealed that the degradation rate was related to dissolved oxygen and OD. 600 The values ​​showed a highly significant correlation (P < 0.01), and the results are shown in Table 4.

[0122] Table 4 Perchlorate degradation rate and dissolved oxygen and OD 600 Correlation analysis

[0123]

[0124] Note: **At the 0.01 level, the correlation is significant.

[0125] The above research reveals that strain HC7-4 can degrade 0.10-2.00% perchlorate, and can be applied to the degradation and coupled oxygen production of perchlorate in different environments, as well as to the removal of the strong oxidizing properties of perchlorate. Strain HC7-4 can be applied to the bioremediation of perchlorate-contaminated water bodies and other polluted sites, especially as a candidate germplasm resource for the biodegradation and coupled oxygen production of perchlorate on the Martian surface, for the creation of a habitable environment on Mars.

[0126] Example 4: Degradation of perchlorate by polymers metabolized by strain HC7-4

[0127] 1. Distribution of EPS (Extractive Polymers) from Strains' Metabolism

[0128] To investigate the degradation effect of the strain's metabolic polymers on perchlorate, the distribution of the strain's metabolic polymers was first determined, and the polysaccharide and protein contents in the extracellular, intracellular, and membrane polymers were measured.

[0129] Methods for obtaining extracellular, intracellular, and membrane polymers:

[0130] The bacterial suspension of strain HC-7-4 was inoculated at a rate of 10% into an inorganic salt medium with a sodium perchlorate concentration of 0.50%, and then incubated statically at 25-30℃ to obtain the fermentation broth.

[0131] Take 40 mL of fermentation broth sample into a 50 mL centrifuge tube, centrifuge at 8000-10000 r / min for 5 min, and retain the supernatant as the extracellular polymer extract. Collect the precipitate and resuspend it in 50 mL of Tris-HCl buffer solution. Sonicate at 800W for 5 s with a 2 s interval for 30 min at low temperature. Centrifuge the lysate at 4℃ and 10000-12000 rpm / min for 10 min. Collect the supernatant as the intracellular polymer extract. Resuspend the centrifuged precipitate in Tris-HCl buffer solution as the membrane polymer extract.

[0132] The protein and polysaccharide contents in the extracellular, intracellular, and membrane polymer extracts were determined. Protein content was determined using the Coomassie brilliant blue method, and polysaccharide content was determined using the anthrone colorimetric method. The results showed that polymers were present in the extracellular, intracellular, and membrane regions, with higher contents in the extracellular polymers than in the intracellular and membrane regions.

[0133] 2. EPS secreted by strain HC7-4 degrades perchlorate.

[0134] To investigate the effect of EPS secreted by the strain on perchlorate degradation, EPS was extracted from strain HC7-4 and used to degrade sodium perchlorate in inorganic salt liquid culture medium and sandy soil culture medium, respectively. Samples were taken at 0, 1, 2, 3 and 4 days to determine the chloride ion content. A control group without added EPS was also set up.

[0135] Studies have found that the extracellular polymers produced by the strain have a highly efficient degradation effect on perchlorate. The degradation rate of sodium perchlorate in inorganic salt culture medium can reach 91.2% on the 3rd day, and the degradation rate of sodium perchlorate in sandy soil culture medium can reach 91.2% on the 4th day.

[0136] Further determination of the degradation rate of 0.5% perchlorate by strain HC7-4 and the EPS generated during the degradation process were performed, and the correlation between degradation rate and EPS was analyzed.

[0137] Table 5. Correlation analysis between degradation rate and protein / polysaccharide content.

[0138]

[0139] Note: **The correlation is significant at the 0.01 level, and *the correlation is significant at the 0.05 level.

[0140] The results show that the changes in the content of extracellular polysaccharides and extracellular proteins in EPS are consistent with the degradation rate. The study also found that the degradation rate is significantly correlated with extracellular proteins and extracellular polysaccharides (P < 0.01), as shown in Table 5. This reveals that EPS plays an important role in the degradation of perchlorate and can be applied to the degradation of perchlorate under extreme environmental conditions.

[0141] In summary, strain HC7-4 and its metabolized EPS have a high efficiency in degrading perchlorate and can be applied to the bioremediation of perchlorate-contaminated water bodies and other polluted sites. In particular, it can serve as a candidate germplasm resource for the biodegradation of perchlorate and coupled oxygen production on the Martian surface, and can be used to create a habitable environment on Mars.

[0142] The main physicochemical indicators were determined using the methods described in the above embodiments as follows:

[0143] (1) Chloride ion determination method

[0144] Ion chromatography or silver sulfate titration (GBT15453-2018) was used.

[0145] Silver sulfate titration method: Based on GBT15453-2018, an improved method is used to determine the chloride ion content.

[0146] Centrifuge 8 mL of the sample at 10000 rpm for 5 min, and collect the supernatant. Accurately pipette 5 mL of the supernatant into a 50 mL beaker and add 0.10 mL of potassium chromate indicator. Titrate with silver sulfate standard solution under a white background until a brick-red precipitate just appears and does not fade within half a minute. Simultaneously, take 5 mL of distilled water and titrate according to the above steps to perform a blank test.

[0147] Calculation results: The chloride ion content is expressed as mass concentration P, and the value is expressed in milligrams per liter (mg / L), calculated according to formula (4):

[0148] P = [(V1 - V0) × c × 35.45] ÷ v × 10 3 (4)

[0149] In the formula:

[0150] V1------The volume of silver sulfate standard titration solution consumed by the sample, in milliliters (mL);

[0151] V0------The volume of silver sulfate standard titration solution consumed in the blank test, in milliliters (mL);

[0152] C ------ The accurate value of the actual concentration of the silver sulfate standard titration solution, in moles per liter (mol / L);

[0153] M ------ The numerical value of the molar mass of chlorine, in grams per mole (g / mol) (M=35.45);

[0154] V ------ The numerical value of the volume of sample transferred, in milliliters (mL).

[0155] The amount of chloride ions generated in the culture medium was obtained from the standard curve of the silver chloride turbidimetric method. The degradation rate was calculated according to formula (1).

[0156] (2) Method for determining polysaccharide content

[0157] Reagent preparation: ① 0.20% Anthrone solution: Dissolve 0.5000 g of anthrone in 250 mL of 80% H2SO4, and dilute to volume in a 250 mL volumetric flask to obtain a 0.20% anthrone solution. Store protected from light. ② Standard glucose solution (0.1 mg / mL): Dissolve 10.00 mg of glucose in distilled water and dilute to volume in a 100 mL volumetric flask to obtain a 100 μg / mL standard glucose solution.

[0158] Standard curve plotting: Plot the polysaccharide determination standard curve according to the method in Table 6 below. Plot the standard curve with the concentration of standard glucose solution as the x-axis and the absorbance at 620 nm as the y-axis. Extracellular polysaccharides need to be extracted for sample determination.

[0159] Table 6. Standard curve of polysaccharide content plotted using the anthrone colorimetric method.

[0160]

[0161] The absorbance of the sample at 620 nm was determined according to the polysaccharide content standard curve method, and the sugar content of the sample was calculated according to formula (5).

[0162] S (mg / mL) = C1 * dilution factor (5)

[0163] in:

[0164] S------Glucose content in culture medium (mg / mL).

[0165] C1------The amount of glucose (mg / mL) obtained from the polysaccharide content standard curve.

[0166] (3) Methods for determining protein content

[0167] Reagent Preparation: ① Coomassie Brilliant Blue Solution: Weigh 0.0050 g of Coomassie G250, dissolve it in 5 mL of 95% ethanol solution, then add 10 mL of 85% phosphoric acid solution and dilute to 500 mL with deionized water. This yields a 0.10 g / L Coomassie Brilliant Blue solution, which should be stored protected from light. ② Standard Protein Solution: Weigh 0.0100 g of bovine serum albumin, dissolve it in 0.90% NaCl, and dilute to 100 mL. This yields a standard protein solution with a concentration of 0.1 mg / mL. Plot a protein assay standard curve according to the methods in Table 7 below.

[0168] Table 7. Standard curve of protein content plotted using the Coomassie Brilliant Blue G-250 colorimetric method.

[0169]

[0170] With OD 595nm A standard curve was plotted with absorbance values ​​on the ordinate and protein concentration on the abscissa.

[0171] OD was determined according to the protein content standard curve method. 595nm The extracellular protein content of the sample is calculated according to formula (6).

[0172] P (mg / mL) = C2 × dilution factor (6)

[0173] in:

[0174] P ------ Total protein content in culture medium (mg / mL);

[0175] C2------The protein content (mg / mL) obtained from the protein content standard curve.

[0176] Data processing methods:

[0177] Microsoft Excel 2010 was used to organize the data and create graphs, and SPSS 20.0 software was used to perform correlation tests.

[0178] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A spindle-shaped lysine-producing Bacillus that degrades perchlorate and couples with oxygen production ( Lysinibacillus fusiformis HC7-4, characterized in that, The spindle-shaped lysine spores HC7-4 were deposited on October 11, 2022, at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 25888.

2. A polymer, characterized in that, The polymer is a metabolite of the spindle-shaped lysine spore bacillus HC7-4 described in claim 1, which can degrade perchlorate, converting perchlorate ions into chloride ions and generating oxygen in the process. The polymer is a polysaccharide or a protein; The preparation method of the polymer is as follows: the bacterial suspension of strain HC7-4 is inoculated into an inorganic salt culture medium with a sodium perchlorate concentration of 0.50% at an inoculation amount of 10%, and then placed in a static culture at 25-30℃ to obtain a fermentation broth; take 40 mL of the fermentation broth sample into a 50 mL centrifuge tube, centrifuge at 8000-10000 r / min for 5 min, and retain the supernatant as the extracellular polymer extract.

3. The application of the strains / polymers described in (1) or (2) below in the degradation of perchlorate and its coupled oxygen production: (1) The spindle-shaped lysine-containing Bacillus HC7-4 as described in claim 1; (2) The polymer as described in claim 2.

4. The application according to claim 3, characterized in that, The degradation of perchlorate involves converting perchlorate ions into chloride ions while simultaneously producing oxygen, and also reducing the redox potential of the perchlorate degradation system.

5. A method for degrading perchlorate, characterized in that, The method includes the step of using the spindle-shaped lysine-containing Bacillus HC7-4 as described in claim 1 or the polymer as described in claim 2 to degrade perchlorate.

6. The method according to claim 5, characterized in that, The degradation of perchlorate is carried out under static or shaking conditions at a temperature of 25-40℃.

7. The method according to claim 5, characterized in that, The step of degrading perchlorate further includes the addition of an electron donor, a carbon source, and an inorganic salt. The electron donor is one of riboflavin, reduced iron powder, and pyrite. The carbon source is one of sodium acetate, sodium carbonate, and sodium bicarbonate. The inorganic salt is a component of an inorganic salt culture medium, and the composition of the inorganic salt culture medium is as follows: (1) Mother liquor of macro elements I: 0.130g of dipotassium hydrogen phosphate, 0.060g of potassium dihydrogen phosphate, 0.060g of magnesium sulfate, 1.000g of anhydrous sodium acetate, 0.180g of ammonium nitrate, add water to 1000mL; (2) Trace element mother liquor II: 0.110 g zinc sulfate, 0.275 g calcium sulfate, 0.133 g manganese sulfate, 0.1055 g sodium molybdate, 0.0785 g copper sulfate, add water to 100 mL; (3) Iron salt mother liquor solution: 0.125g ferrous sulfate heptahydrate, 0.171g EDTA, add water to 100mL.

8. The application of the spindle-shaped lysine-containing Bacillus HC7-4 as described in claim 1 or the polymer as described in claim 2 in the degradation of perchlorate and coupling oxygen production on the Martian surface, characterized in that, The spindle-shaped lysine-containing Bacillus HC7-4 can serve as a pioneer species for Martian environmental terraforming.

9. The application of the spindle-shaped lysine-containing Bacillus HC7-4 as described in claim 1 or the polymer as described in claim 2 in the bioremediation of perchlorate-contaminated water bodies and other perchlorate-contaminated sites.

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