Bioremediation method for high-efficiency removal of sodium selenite in water body by combined culture of aspergillus niger and bacillus

Abstract

The present application relates to the field of biotechnology, disclose a kind of biological remediation method for removing sodium selenite in water body by aspergillus niger and bacillus combined culture, comprising the following steps: S1.in YPD culture medium inoculation activated aspergillus niger, shake culture, form aspergillus niger ball;S2.in LB culture medium inoculation bacillus, shake culture, until the bacillus enters logarithmic growth phase;S3.the aspergillus niger ball is washed and then poured into the LB culture medium containing bacillus obtained in S2, combined culture, form aspergillus niger and bacillus combination;S4.the aspergillus niger and bacillus combination is placed in contaminated water body, filtration centrifugation, remove nano selenium from water phase, and the aspergillus niger and bacillus combination is filtered out, realize repeated water purification.The present application can achieve substantial effect on selenium contaminated water quality treatment, and reduction efficiency is high, and the selenium tolerance is strong.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a biological remediation method for removing sodium selenite in water by combined culture of Aspergillus niger and Bacillus. BACKGROUND

[0002] In recent years, selenium pollution in surface water and groundwater in many river basins has become a key problem worldwide. Although low concentrations of selenium are beneficial to our health. However, even at low concentrations in aquatic environments, selenium can be bioamplified through the food chain, leading to toxic levels in higher-level consumers in the food web, whether directly or indirectly consuming selenium, which can ultimately pose serious risks and harm to human health.

[0003] So far, scholars at home and abroad have developed many treatment technologies to alleviate the harmful effects of selenium to permanently remove selenium-containing anions in selenium-containing soil and water. Physical methods separate selenium from water without changing the chemical valence of selenium, such as membrane technology, activated carbon adsorption, coagulation, and coprecipitation, etc. Chemical methods such as oxidation-reduction reactions, electrocoagulation technology, and precipitation flocculation technology. Biological methods include biological transformation, using plants, microorganisms, or wetland systems to bioconcentrate and / or transform inorganic selenium into organic species (such as selenoproteins, methyl selenium, etc.) to reduce pollution. Traditional chemical or physical-chemical methods for removing selenium not only have high operating costs, but are also not suitable for cities with large amounts of sewage treatment. Biological selenium removal technology is widely used due to its low treatment cost, easy management, and small environmental side effects, so the use of biological remediation to study the transformation of selenium pollutants or water purification has many practical significance for protecting the ecological environment. SUMMARY

[0004] The present application provides a biological remediation method for removing sodium selenite in water by combined culture of Aspergillus niger and Bacillus, which can achieve the purpose of removing selenium-contaminated wastewater by combining Aspergillus niger and Bacillus for symbiotic growth to form a symbiotic body. The present application can achieve a substantial treatment effect on selenium-contaminated water quality, and has high reduction efficiency and strong selenium tolerance.

[0005] Technical scheme: The present application provides a biological remediation method for removing sodium selenite in water by combined culture of Aspergillus niger and Bacillus, comprising the following steps:

[0006] S1. Inoculate activated Aspergillus niger in YPD medium and shake culture to form Aspergillus niger pellets;

[0007] S2. Inoculate Bacillus in LB medium and shake culture until the Bacillus enters the logarithmic growth phase;

[0008] S3. Pour the washed Aspergillus niger into the LB medium containing Bacillus in S2, combine and culture to form Aspergillus niger and Bacillus combination;

[0009] S4. Place the Aspergillus niger and Bacillus combination in a contaminated water body, filter and centrifuge, remove nano selenium from the water phase, and filter out the Aspergillus niger and Bacillus combination to achieve repeated water purification.

[0010] Further, in S1, the specific conditions of the shaking culture are: the temperature is 35-40 ℃, and the rotation speed is 150-200 rpm. The present application explores the culture time of Aspergillus niger to find the best specific surface area. Under the length of time and culture conditions, Aspergillus niger can achieve the purpose of high-efficiency adsorption of Paenibacillus.

[0011] Preferably, in S1, the specific preparation method of the YPD culture medium is as follows:

[0012] Take 1 L of distilled water and mix 20 g of glucose, 20 g of tryptone and 10 g of yeast extract, and then divide and pack them into 100 ml conical bottles. Sterilize at 115 ℃ for 20 min, and then use.

[0013] Preferably, in S2, the specific preparation method of the LB culture medium is as follows:

[0014] Take 1 L of distilled water and mix 10 g of glucose, 10 g of tryptone and 5 g of yeast extract, and then divide and pack them into 100 ml conical bottles. Sterilize at 121 ℃ for 20 min, and then use.

[0015] Further, in S2, the specific conditions of the shaking culture are: the temperature is 35-40 ℃, and the rotation speed is 150-200 rpm. A fixed OD600 value is used as the growth time of Paenibacillus, which is convenient for subsequent calculation of the capture rate.

[0016] Further, in S3, the specific conditions of the combined culture are: the temperature is 35-40 ℃, and the rotation speed is 150-200 rpm. The present application finds the highest combination efficiency conditions and time period, so that the combination rate of Aspergillus niger and Bacillus reaches saturation, and the specific calculation formula is:

[0017]

[0018] OD0 is the initial absorbance, which is fixed as 1 for convenience of calculation. ODy is the absorbance of Bacillus, and ODx is the absorbance of the Aspergillus niger and Bacillus combination.

[0019] Preferably, in S3, 80-100 Aspergillus niger balls with uniform diameter are selected and poured into the LB medium containing bacillus obtained in S2 for combined culture.

[0020] Further, the bacillus is Paenibacillus Bacillus. paramycoides 24522.

[0021] The present application reduces pollution and accelerates the flow and circulation of selenium in nature by bioremediation, biotransformation, cultivation of a double-bacterium combination and conversion of tetravalent inorganic selenium into zero-valent elemental nanometer selenium. Bioremediation by microorganisms to convert toxic selenium species into non-toxic forms is considered an effective alternative treatment. The biosynthetic selenium nanoparticles can also be adsorbed by Aspergillus niger. It is very important to develop bioremediation for treating selenium-contaminated water and soil. In addition, due to the excellent adsorption capacity of the double-bacterium combination, the present application can also achieve the recycling and reprocessing of nanometer selenium, thereby achieving water purification.

[0022] The innovation of the present application lies in the organic combination of Aspergillus niger and bacillus for co-growth to form a symbiotic body, thereby achieving the purpose of removing selenium-contaminated wastewater. Traditional bioremediation mostly uses a single microbial strain. The use of a single microbial strain not only has to face the interference of various heavy metals and antibiotics in wastewater, but also can only remove a single pollutant. When there are multiple pollutants in wastewater, a single microorganism is difficult to resist the inhibition of environmental changes on selenium reduction. The present application selects the organic combination of Aspergillus niger and bacillus. The complex bacterial community can resist the impact of environmental changes on the microbial community. In addition, there is a synergistic effect between Aspergillus niger and bacillus. The long and thin mycelium of Aspergillus niger can fix bacillus to maximize the removal effect of the combination on sodium selenite, resist environmental changes, and improve the efficiency of selenium reduction. Therefore, it is crucial to select appropriate strains with selenium oxyanion reduction capacity under specific operating conditions and target wastewater. The technology can be used in high-density water treatment and other fields in the future, and has a wide application prospect.

[0023] Advantages: Compared with the prior art, the present application has the following advantages: the method for treating selenium-contaminated water by forming a symbiotic body through the organic combination of Aspergillus niger and bacillus is based on the analysis of the advantages and disadvantages of traditional microbial remediation, and is based on the real environment. The method can substantially treat selenium-contaminated water, has high reduction efficiency, strong selenium tolerance and other excellent properties, and is considered to be a sustainable technology for treating selenium pollution. The present application can be used in the fields of selenium-contaminated water treatment and water purification treatment in the future, and has a wide application prospect. BRIEF DESCRIPTION OF DRAWINGS:

[0024] Figure 1 The idea and operation process constructed for the present application;

[0025] Figure 2 The OD600 curve of Paenibacillus alvei;

[0026] Figure 3 The experimental result graph of sodium selenite removal by Aspergillus niger in Comparative Example 1;

[0027] Figure 4 The experimental result graph of sodium selenite removal by Aspergillus niger in Comparative Example 1;

[0028] Figure 5 The experimental result graph of sodium selenite removal by Paenibacillus alvei in Comparative Example 2;

[0029] Figure 6 The experimental result graph of sodium selenite removal by Paenibacillus alvei in Comparative Example 2;

[0030] Figure 7 The experimental result graph of sodium selenite removal by Aspergillus niger combined with Paenibacillus alvei prepared in Embodiment 1;

[0031] Figure 8 The experimental result graph of sodium selenite removal by Aspergillus niger combined with Paenibacillus alvei prepared in Embodiment 1;

[0032] Figure 9 The standard curve for measuring sodium selenite;

[0033] Figure 10 The curve of the combination time of Aspergillus niger and Paenibacillus alvei and the removal efficiency of sodium selenite. DETAILED DESCRIPTION

[0034] The present application discloses a biological remediation method of Aspergillus niger combined with Paenibacillus alvei for efficiently removing sodium selenite in water, Figure 1 The idea and operation process constructed for the present application. Among them, the Paenibacillus used in the present application is Paenibacillus alvei Bacillus. paramycoides 24522 (CGMCC NO. 24522), and Aspergillus niger Aspergillus niger (CGMCC NO. 40430). B. paramycoides 24522 shows strong selenium removal ability and can reduce the toxic and bioaccumulative sodium selenite into low-toxicity nano selenium. However, since B. paramycoides 24522 is in free state, the actual application of the free bacteria is prone to loss, resulting in low cell density, low survival rate, and low biological remediation potential.

[0035] To solve B. paramycoides The limitations of 24522, we A. niger The selenium tolerance and removal capacity were evaluated. The results showed that... A. niger The selenium removal efficiency is relatively low, but it has good immobilization ability. A. niger Slender hyphae can fix B. paramycoides 24522, just enough to make up for it. B. paramycoides The limitations of 24522 were addressed. Therefore, we combined these two bacteria to form a symbiotic organism and explored the optimal environment through pellet formation conditions to maximize removal efficiency. The separated nano-selenium can be removed through simple filtration and centrifugation, thus achieving highly efficient water remediation.

[0036] The present invention will now be described in detail with reference to the embodiments.

[0037] Implementation method 1:

[0038] This embodiment provides a method for reducing sodium selenite to nano-selenium by organically combining Aspergillus niger and Bacillus to form a conjugate, as detailed below:

[0039] (1) Preparation of culture medium and sterile physiological saline:

[0040] YPD medium contains 20 g glucose, 20 g tryptone, and 10 g yeast extract per 1 L of distilled water. After preparation, it is dispensed into 100 ml Erlenmeyer flasks and sterilized at 115 ℃ for 20 min for later use.

[0041] LB medium contains 10 g glucose, 10 g tryptone, and 5 g yeast extract per 1 L of distilled water. After preparation, it is dispensed into 100 ml Erlenmeyer flasks and sterilized at 121 °C for 20 min for later use.

[0042] Sterile physiological saline is prepared by mixing distilled water with a 0.9% NaCl solution.

[0043] (2) Inoculate Aspergillus niger into YPD medium and culture at 37 °C and 180 rpm for 48 h until it becomes spherical;

[0044] (3) Inoculate Bacillus paramyxoides into LB medium and culture it until OD600=1;

[0045] It should be noted that the *Bacillus parasiticus* in the cryopreservation tube was first activated once and then transferred to LB medium as a backup strain. The OD600 of the strain was measured at regular intervals, and the OD600=1 of *Bacillus parasiticus* was used as the subsequent growth point to facilitate the calculation of the capture rate.

[0046] Figure 2 The OD600 curve of Paenibacillus elyakovii is shown, and 5.5 h is selected as the subsequent fixed growth time, at which the OD600 is a fixed value, so as to calculate the growth condition with the maximum binding rate, so that Aspergillus niger can adsorb Paenibacillus elyakovii more, and the combination of the two bacteria is as saturated as possible.

[0047] The Aspergillus niger balls cultured to be spherical in step (2) are taken out to a plate, sterile normal saline is added, and a glass rod is used for stirring to wash away the surface metabolites and medium components, and the balls are washed to be white, and then 100 Aspergillus niger balls with uniform diameters are selected and poured into the LB medium inoculated with Paenibacillus elyakovii and cultured for a certain time, and the culture is continuously cultured in a shaking incubator at 180 rpm until the combination rate of the two bacteria reaches saturation, and the combination of Aspergillus niger and Paenibacillus elyakovii is formed;

[0048] Here, the combination rate calculation formula of the combination of Aspergillus niger and Paenibacillus elyakovii is as follows:

[0049]

[0050] OD0 is the initial absorbance, which is fixed as 1 for convenience of calculation. ODy is the absorbance of Paenibacillus elyakovii, and ODx is the absorbance of the combination of Aspergillus niger and Paenibacillus elyakovii.

[0051] (5) The combination of Aspergillus niger and Paenibacillus elyakovii is taken out into selenium sewage containing sodium selenite, and the sodium selenite is removed. After the combination wrapped with red nanometer selenium is taken out, nanometer selenium can be recovered through simple centrifugal filtration, so as to achieve the purpose of water purification.

[0052] Comparative Example 1:

[0053] (1) Aspergillus niger previously stored in a -80 ℃ refrigerator is taken out, and the Aspergillus niger is inoculated and activated in YPD medium, and cultured in a shaking incubator at 37 ℃ and 180 rpm until the Aspergillus niger balls are formed.

[0054] (2) Sodium selenite is added to the Aspergillus niger balls to a concentration of 2 mmol / L, and the removal rate and removal rate of 2 mmol / L sodium selenite by the Aspergillus niger balls are measured.

[0055] Comparative Example 2:

[0056] (1) Paenibacillus elyakovii previously stored in a -80 ℃ refrigerator is taken out, and the Paenibacillus elyakovii is inoculated in LB medium, and cultured in a shaking incubator at 37 ℃ and 180 rpm until the logarithmic growth phase.

[0057] (2) In the Paenibacillus, add sodium selenite to 2 mmol / L, measure the removal rate of Paenibacillus to 2 mmol / L concentration of sodium selenite and removal rate.

[0058] In the above experiment, the method for measuring the residual amount of sodium selenite is as follows:

[0059] (1) After centrifuging the bacteria liquid containing sodium selenite in the centrifuge at high speed for 10 min, 1 ml of supernatant was taken out for standby;

[0060] (2) A certain concentration of hydrochloric acid and a certain concentration of ascorbic acid were added to the supernatant, mixed uniformly, and then placed at room temperature in the dark for 10 min;

[0061] (3) The absorbance value was measured under OD500 using an enzyme marker, and the content of residual sodium selenite was calculated by substituting the absorbance value into the formula of OD500 and sodium selenite.

[0062] Analysis of experimental results:

[0063] Figure 3 The experimental results of Aspergillus niger alone removing sodium selenite are shown in the figure. In the LB medium, Aspergillus niger has a fast removal effect on 2 mmol / L sodium selenite in the early stage, reaching 30% in 4 h, and reaching 95% in 24 h, which has a certain removal effect on sodium selenite, but the removal rate is slow and the time is long. Figure 4 The state of Aspergillus niger alone adding sodium selenite is shown in the figure. Aspergillus niger has weak ability to remove sodium selenite and low removal efficiency. After adding sodium selenite, it cannot adsorb and wrap the removed nano selenium.

[0064] Figure 5 The experimental results of Bacillus alone removing sodium selenite are shown in the figure. In the LB medium, Bacillus has a fast removal effect on 2 mmol / L sodium selenite, reaching more than 95% in 16 h, which has a obvious removal effect on sodium selenite, high removal efficiency and short time spent, but it is difficult to separate the bacteria from the nano selenium after combination. Figure 6 The state of Bacillus alone adding sodium selenite is shown in the figure. Bacillus has strong ability to remove sodium selenite and high removal efficiency. After adding sodium selenite, it reduces sodium selenite (SeO3 2- ) to nano selenium particles (Se 0 ) and mixes with the bacteria in the aqueous phase, which is difficult to remove from the aqueous phase and reuse.

[0065] Figure 7It is shown that after the organic combination of Aspergillus niger and Bacillus under appropriate conditions, the removal effect of sodium selenite is obvious, the removal efficiency is high, and the time spent is short, more than 98% of sodium selenite can be removed within 4 hours, and the removal efficiency is more than 6 times faster than that of the original Aspergillus niger, and more than 4 times faster than that of the original Bacillus. Figure 8 It is shown that after the organic combination of Aspergillus niger and Bacillus, the picture of sodium selenite added has been reduced to nano selenium, which is wrapped in a layer of red nano selenium. The removal efficiency is significantly improved, and nano selenium can be removed from the water phase by simple filtration centrifugation, and the combination can be filtered out to realize repeated water purification.

[0066] Experimental accuracy verification:

[0067] Draw the standard curve of sodium selenite measurement:

[0068] (1) Prepare a sodium selenite mother liquor of 1 mol / L, use a volumetric flask to make up to 100 ml, and dilute it to 0-300 mg / L;

[0069] (2) Take 1 ml of the diluted sodium selenite solution, add a certain concentration of hydrochloric acid and ascorbic acid, mix well, and stand at room temperature for 10 min in the dark;

[0070] (3) Take 1 ml of the reacted solution, take 200 μl in the enzyme label strip, and measure the absorbance under the condition of OD500 to draw the standard curve of absorbance and sodium selenite. ( Figure 9 )

[0071] Figure 9 It is shown that the standard curve of sodium selenite measurement is measured, and the OD500 value is calculated by the formula. The residual sodium selenite content is compared with the known sodium selenite addition amount, so as to calculate the removal rate of sodium selenite. The relationship between OD500 and sodium selenite is OD500=0.00185*residual sodium selenite content+0.03895, R²=0.99869, the experimental data and the curve fitting degree are high, and the experimental results are accurate.

[0072] In summary, in the system of the combination of Aspergillus niger and Bacillus constructed in the present application, the immobilization ability of Aspergillus niger and the high deselenization ability of Bacillus are organically combined, and the advantages of the two bacteria are utilized. The combination of Paraburkholderia and Aspergillus niger solves the problems faced by free microbial repair of selenium-containing wastewater. The free Bacillus is not diluted by the treated polluted water, which causes the problems of low cell density, low survival rate, and low biological repair potential. Through the reaction process: , the problems of low cell density, low survival rate, and low biological repair potential are solved. B. paramycoides24552 While reducing sodium selenite, sodium selenite is reduced to red elemental nanoseelenium. The immobilization technology can not only face the complex selenium pollution environment, but also realize the recovery of nanoseelenium and heavy metal pollutants. Water quality is greatly improved. It has a wide application prospect in the field of selenium-containing wastewater treatment.

[0073] The above embodiments are only for illustrating the technical concept and characteristics of the present application, and the purpose is to enable those skilled in the art to understand the content of the present application and to implement it, and cannot limit the protection scope of the present application. Any equivalent transformation or modification made according to the spirit and essence of the present application shall be covered within the protection scope of the present application.

Claims

1. A bioremediation method for efficiently removing sodium selenite from water by co-culturing Aspergillus niger and Bacillus, characterized in that, Includes the following steps: S1. Inoculate activated Aspergillus niger into YPD medium, shake and culture to form Aspergillus niger spheres; S2. Inoculate Bacillus in LB medium, shake and culture until the Bacillus enters the logarithmic growth phase; S3. After washing the Aspergillus niger balls, pour them into the LB medium containing Bacillus spores obtained in S2, and culture them together to form a conjugate of Aspergillus niger and Bacillus spores; S4. The Aspergillus niger and Bacillus conjugate is placed in polluted water, filtered and centrifuged to remove nano-selenium from the aqueous phase, and the Aspergillus niger and Bacillus conjugate is filtered out to achieve repeated water purification; The Bacillus is *Bacillus paramyophyte*. Bacillus paramycoides 24522, with accession number CGMCC NO.24522; the aforementioned Aspergillus niger, with accession number CGMCC NO.40430.

2. The bioremediation method for efficiently removing sodium selenite from water by co-culturing Aspergillus niger and Bacillus spp. according to claim 1, characterized in that: In S1, the specific conditions for the shaking culture are: temperature 35-40 ℃, rotation speed 150-200 rpm.

3. The bioremediation method for efficiently removing sodium selenite from water by co-culturing Aspergillus niger and Bacillus as described in claim 1, characterized in that: In S1, the specific preparation method of the YPD culture medium is as follows: Mix 1 L of distilled water with 20 g of glucose, 20 g of tryptone and 10 g of yeast extract, dispense into 100 ml Erlenmeyer flasks, and sterilize at 115 ℃ for 20 min before use.

4. The bioremediation method for efficiently removing sodium selenite from water by co-culturing Aspergillus niger and Bacillus as described in claim 1, characterized in that: In S2, the specific preparation method of the LB culture medium is as follows: Mix 1L of distilled water with 10g of glucose, 10g of tryptone, and 5g of yeast extract, dispense into 100ml Erlenmeyer flasks, and sterilize at 121℃ for 20min before use.

5. The bioremediation method for efficiently removing sodium selenite from water by co-culturing Aspergillus niger and Bacillus spp. according to claim 1, characterized in that: In S2, the specific conditions for the shaking culture are: temperature 35-40 ℃ and rotation speed 150-200 rpm.

6. The bioremediation method for efficiently removing sodium selenite from water by co-culturing Aspergillus niger and Bacillus as described in claim 1, characterized in that: In S3, the specific conditions for the combined culture are: temperature of 35-40 ℃ and rotation speed of 150-200 rpm.

7. The bioremediation method for efficiently removing sodium selenite from water by co-culturing Aspergillus niger and Bacillus as described in claim 1, characterized in that: In S3, select 80-100 Aspergillus niger balls of uniform diameter and pour them into the LB medium containing Bacillus spores obtained in S2 for incubation.

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