Application of Lysinibacillus fusiformis in removing hexavalent chromium from eutrophic wastewater

Abstract

Application of Lysinibacillus fusiformis in removing hexavalent chromium from eutrophic wastewater, belonging to the field of microbial technology. The strain is preserved in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms, with the preservation address being No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, the preservation number being CGMCC No. 10053, and the preservation name being Lysinibacillus fusiformis WTXJ1-4, and the preservation date is November 25, 2014. Through single-factor experiments and orthogonal experiments for optimization, the suitable biological adsorption conditions of this strain for hexavalent chromium in eutrophic wastewater were determined as follows: pH 2.0, initial potassium dichromate concentration 50 mg / L, adsorption time 24 h, wet cell mass dosage 1.0 g / L, adsorption temperature 34 °C, and rotation speed 160 r / min. When reaching equilibrium under these optimized adsorption conditions, the removal rates of hexavalent chromium and total chromium in eutrophic wastewater by viable cells of WTXJ1-4 reached 91.6% - 94.8% and 42.2% - 43.8% respectively. Lysinibacillus fusiformis Viable cells of WTXJ1-4 have strong adsorption-reduction ability, tolerance and resistance to hexavalent chromium in eutrophic wastewater, and their effective ranges are potassium dichromate concentrations of 0 - 100 mg / L, 100 - 300 mg / L, and 300 - 500 mg / L respectively.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to an application of lysinophilic Bacillus to remove hexavalent chromium from nutrient-rich wastewater. Background Art

[0002] Compared to traditional chemical and physical techniques for removing or recovering metals from waste, biosorption is more economically and environmentally feasible and has become a promising, environmentally friendly, and energy-saving technology for the treatment of heavy metal-containing wastewater and waste residues. Studies have shown that a variety of microorganisms exhibit good bioremoval effects on a variety of single metals or combinations of metals. Numerous factors may affect the adsorption efficiency of metal ions on the microbial cells being tested. The adsorption mechanism may be due to electrostatic attraction, the formation of surface complexes, and chemical reactions between metal ions and functional groups (primarily hydroxyl, acetylamino, or amino groups). Generally, when large amounts of heavy metals enter cells, they will cause a series of physiological and biochemical processes in the cells to become unbalanced, disrupting the cell's redox balance and causing oxidative damage and physiological toxicity to the cells. However, in the presence of high concentrations of metals, some microorganisms can still survive or grow, demonstrating resistance to metals. Some microorganisms also convert metals from a highly toxic state to a less toxic state through biotransformation or physiological metabolic activities.

[0003] Cr(VI) has a higher chemical toxicity than Cr(III), and Cr(VI) is often used as a typical representative of heavy metals. To date, there have been many reports on the removal of Cr(VI) from aqueous solutions using different biomasses as adsorbents. The mechanism of chromium biosorption is generally based on the reduction of Cr(VI). The amount of Cr(III) produced can be obtained by measuring the reduction of total Cr and Cr(VI) in the solution and biomass before and after adsorption. If only total Cr is measured in the solution and biomass, but no Cr(VI), it means that the total Cr is only Cr(III) and the Cr(VI) in the biomass is completely reduced to Cr(III). Conversely, if both Cr(III) and Cr(VI) are present on the biomass, it means that both Cr(VI) adsorption and Cr(VI) reduction contribute to the removal of Cr(VI) from aqueous solutions.

[0004] CN 104593296 B: Screening of typical bacteria in papermaking wastewater, sewage irrigation and reed fields - fusiform lysinibacillus and its use provides a fusiform lysinibacillus (Lysinibacillus fusiformis WTXJ1-4, CGMCC No.10053, Genbank No.KP150574, ZL 201410853541.9). The strain is a typical growth bacterium screened from papermaking wastewater, sewage irrigation and reed fields, and can advantageously degrade COD in papermaking wastewater from sewage irrigation and reed fields. CrAnd AOX, showing good ability to remove organochlorine pollutants. The complete genomic information of a Lysinibacillus fusiformis strain ZC1 isolated from chromium (Cr)-contaminated metal electroplating wastewater by He Minyan et al. from Huazhong Agricultural University indicates that this strain has the ability to reduce toxic hexavalent chromium Cr(VI) to less toxic trivalent chromium Cr(III). Therefore, Lysinibacillus fusiformis WTXJ1-4 with similar genomic information can theoretically be screened and cultured for the treatment of hexavalent chromium pollution or the co-pollution of hexavalent chromium and organochlorine. However, how this strain is actually applied to remove hexavalent chromium and its biosorption, tolerance, and resistance have not been explored yet. Summary of the Invention

[0005] Technical problems to be solved: In view of the above technical problems, the present invention provides an application of Lysinibacillus fusiformis in removing hexavalent chromium from eutrophic wastewater. This strain has found a new idea and method for treating hexavalent chromium pollution or the co-pollution of hexavalent chromium and organochlorine in eutrophic wastewater. The removal rates of hexavalent chromium and total chromium in eutrophic wastewater by its live cells are as high as 91.6% - 94.8% and 42.2% - 43.8% respectively.

[0006] Technical solution: An application of Lysinibacillus fusiformis in removing hexavalent chromium from eutrophic wastewater, wherein the preservation name of the Lysinibacillus fusiformis is Lysinibacillus fusiformis WTXJ1-4, which is preserved in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms, with the preservation address at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the preservation number CGMCC No. 10053, and the preservation date is November 25, 2014. The eutrophic wastewater is an LB liquid medium containing hexavalent chromium, and the composition of the LB liquid medium is 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, and pH 7.0.

[0007] Preferably, the application is as follows: Take 0.5 - 3.0 g / L of WTXJ1-4 live cells, add them to the eutrophic wastewater containing hexavalent chromium, and culture for 0 - 24 h under the conditions of pH 2.0 - pH 7.0, adsorption temperature 26 - 38 °C, and rotation speed 0 - 200 r / min.

[0008] Preferably, the dosage of the WTXJ1-4 live cells is 0.5 - 1.0 g / L.

[0009] Preferably, the eutrophic wastewater containing hexavalent chromium is an LB liquid medium containing 0 - 500 mg / L of potassium dichromate.

[0010] Furthermore, the eutrophic wastewater containing hexavalent chromium is an LB liquid medium containing 40 - 60 mg / L of potassium dichromate.

[0011] Preferably, the pH is 2.0.

[0012] Preferably, the adsorption temperature is 32 - 34 °C.

[0013] Preferably, the rotation speed is 140 - 160 r / min.

[0014] Preferably, the culturing time is 20 - 24 h.

[0015] Beneficial effects: Single - factor experiments prove that the viable cells of Lysinibacillus fusiformis WTXJ1 - 4 have extremely strong adsorption and reduction effects on hexavalent chromium in eutrophic wastewater. Under optimized conditions, the removal rates of hexavalent chromium and total chromium by the viable cells of Lysinibacillus fusiformis WTXJ1 - 4 in eutrophic wastewater reach 91.6% - 94.8% and 42.2% - 43.8% respectively, indicating that this strain has extremely strong potential for treating hexavalent chromium pollution or the co - pollution of hexavalent chromium and organochlorine. The Lysinibacillus fusiformis also has strong adsorption - reduction ability, tolerance and resistance to hexavalent chromium pollution. When the potassium dichromate concentration is 0 - 100 mg / L, it mainly shows bio - adsorption - reduction; when the potassium dichromate concentration is 100 - 300 mg / L, it mainly shows tolerance; when the potassium dichromate concentration is 300 - 500 mg / L, it mainly shows resistance. Description of the Drawings

[0016] Figure 1 Changes in the removal rate of hexavalent chromium by viable cells of WTXJ1 - 4 under different cell dosages and different adsorption times;

[0017] Figure 2 Changes in the removal rate of hexavalent chromium by viable cells of WTXJ1 - 4 under different potassium dichromate concentrations and different adsorption times;

[0018] Figure 3 Changes in the removal rate of hexavalent chromium by viable cells of WTXJ1 - 4 under different adsorption pH values and different adsorption times;

[0019] Figure 4 Changes in the removal rate of hexavalent chromium by viable cells of WTXJ1 - 4 under different adsorption temperatures and different adsorption times;

[0020] Figure 5 Changes in the removal rate of hexavalent chromium by viable cells of WTXJ1 - 4 under different adsorption rotation speeds and different adsorption times;

[0021] Figure 6 Adsorption kinetics of hexavalent chromium and total chromium by viable cells of WTXJ1 - 4 under optimal adsorption conditions;

[0022] Figure 7 The adsorption rate and antibacterial rate changes of live WTXJ1-4 bacteria under optimal adsorption conditions for different potassium dichromate concentrations. Specific implementation manners

[0023] Combined with the specific content of the present invention below, the technical solutions in the embodiments of the present invention are clearly and completely described. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.

[0024] I. Preservation of strains

[0025] Lysinibacillus fusiformis was preserved in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on November 25, 2014. The preservation address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the preservation number is CGMCC No. 10053. For the isolation and identification methods of this strain, please refer to the invention patent ZL 201410853541.9.

[0026] II. Biosorption, tolerance and resistance of the strain to hexavalent chromium in eutrophic wastewater

[0027] Example 1 Biosorption-reduction test of Lysinibacillus fusiformis on Cr(VI)

[0028] 1. Preparation of bacterial cells

[0029] Take 1000 mL of Lysinibacillus fusiformis LB culture solution in the logarithmic phase, centrifuge at 4°C and 13,000 r / min for 10 min, repeat centrifugation 3 times with sterile water, and weigh the wet cells to a constant weight as live bacteria, and store them at 4°C for later use.

[0030] 2. Single-factor test of biosorption-reduction test of live WTXJ1-4 bacteria

[0031] Take 0.5 - 3.0 g / L of live WTXJ1-4 bacteria respectively, add them to 100 mL of sterile aqueous solution containing potassium dichromate at a concentration of 0 - 500 mg / L, and culture for 0 - 24 h under the conditions of pH 2.0 - pH 7.0, adsorption temperature of 26 - 38 °C, and rotation speed of 0 - 200 r / min to explore the suitable single-factor conditions for the removal of hexavalent chromium pollutants by live WTXJ1-4 bacteria in eutrophic wastewater. The central values of each preset single factor are as follows: the dosage of live bacteria is 2.0 g / L, the concentration of potassium dichromate is 100 mg / L, the adsorption pH is 5.0, the adsorption temperature is 32 °C, and the adsorption rotation speed is 160 r / min. The residual Cr(VI) and total Cr concentrations in the solution are measured by diphenylcarbazide spectrophotometry respectively, and the biological removal rates of Cr(VI) and total Cr by the WTXJ1-4 strain are calculated.

[0032] (1) With the central values of other single factors unchanged, change the dosage of live bacteria to 0.5 g / L, 1.0 g / L, 1.5 g / L, 2.0 g / L, 2.5 g / L, and 3.0 g / L respectively. The results are as Figure 1 shown;

[0033] (2) With the central values of other single factors unchanged, change the concentration of potassium dichromate to 0 mg / L, 50 mg / L, 100 mg / L, 200 mg / L, 300 mg / L, 400 mg / L, and 500 mg / L respectively. The results are as Figure 2 shown;

[0034] (3) With the central values of other single factors unchanged, change the adsorption pH to pH 2.0, pH 3.0, pH 4.0, pH 5.0, pH 6.0, and pH 7.0 respectively. The results are as Figure 3 shown;

[0035] (4) With the central values of other single factors unchanged, change the adsorption temperature to 26 °C, 28 °C, 30 °C, 32 °C, 34 °C, 36 °C, and 38 °C mg / L respectively. The results are as Figure 4 shown;

[0036] (5) With the central values of other single factors unchanged, change the adsorption rotation speed to 0 r / min, 120 r / min, 140 r / min, 160 r / min, 180 r / min, and 200 r / min respectively. The results are as Figure 5 shown.

[0037] 3. Orthogonal experiment on biosorption-reduction test of live WTXJ1-4 bacteria

[0038] Select the three factors of adsorption pH, adsorption time, and potassium dichromate concentration that have the most significant influence on the biological removal rate of Cr(VI) in the single-factor experiment. Select the appropriate range levels of these three factors, design an orthogonal experiment, and complete the orthogonal experiment by controlling different factors in each experiment. The results are shown in Table 1.

[0039] Table 1 Orthogonal experiment table and results of biological adsorption-reduction of live bacteria WTXJ1-4

[0040]

[0041]

[0042] As can be seen from Table 1, the primary and secondary relationships of the main factors affecting the live bacteria WTXJ1-4 on hexavalent chromium are adsorption pH > potassium dichromate concentration > adsorption time. The determined optimal adsorption conditions are pH 2.0, potassium dichromate concentration 50 mg / L, and adsorption time 24 h. Since this group of experiments does not exist in Table 1, verification experiments were supplemented under the conditions of pH 2.0, potassium dichromate concentration 50 mg / L, culture time 24 h, 34 °C, and 160 r / min. The removal rate of hexavalent chromium under the verification experiment conditions reached 97.0%, which is very close to the No. 6 experimental combination corresponding to the highest removal rate in Table 1.

[0043] 4. Biological adsorption-reduction kinetics of live bacteria WTXJ1-4 on eutrophic wastewater containing hexavalent chromium under optimal conditions

[0044] Under the optimal adsorption conditions of live bacteria, namely pH 2.0, potassium dichromate concentration 50 mg / L, 34 °C, 160 r / min, and adsorption time 24 h, samples were taken at different times to measure the total chromium and hexavalent chromium concentrations in the reaction system, calculate the corresponding removal rates, and obtain the corresponding adsorption kinetic equations. The results are as Figure 6 shown.

[0045] Under the optimized conditions, the removal rate curves of hexavalent chromium and total chromium in the eutrophic wastewater by live bacteria WTXJ1-4 both showed a trend of rising rapidly first and then gradually approaching equilibrium, reaching equilibrium at 14 h and 16 h respectively. When reaching equilibrium, the removal rates of hexavalent chromium and total chromium in the eutrophic wastewater by live bacteria WTXJ1-4 were 91.6% - 94.8% and 42.2% - 43.8% respectively. The biological adsorption kinetic equations were y = -0.2525x 2 + 9.5155x + 10.752 (R 2 = 0.9626) and y = -0.1009x 2 + 4.2573x - 0.6433 (R 2= 0.9898), which conforms to the second-order kinetic model. Starting from 4 h of adsorption, the removal rates of hexavalent chromium and total chromium differed by 40.3% - 52.5% at the same time, indicating that the removal of hexavalent chromium is due to both the partial biosorption of WTXJ1-4 cells and the reduction of hexavalent chromium to trivalent chromium by WTXJ1-4 cells.

[0046] Example 2 Tolerance and resistance characteristics of Lysinibacillus fusiformis to Cr(VI)

[0047] Potassium dichromate with concentrations of 0 mg / L, 20 mg / L, 40 mg / L, 60 mg / L, 80 mg / L, 100 mg / L, 200 mg / L, 300 mg / L, 400 mg / L, and 500 mg / L were added to 100 mL of LB medium respectively. Then 10 mL of Lysinibacillus fusiformis culture solution in the logarithmic phase was added successively. Three parallels were set for each concentration. Samples were randomly taken at regular intervals to measure the cell biomass, residual Cr(VI), and total Cr in the solution, and the time relationship curves of the cell biomass, Cr(VI) removal rate, and total Cr removal rate of Lysinibacillus fusiformis were established respectively, as Figure 7 shown.

[0048] It can be seen from Figure 7 that as the concentration of potassium dichromate increases, the antibacterial rate of microorganisms first increases rapidly and then levels off, indicating that potassium dichromate has an obvious inhibitory effect on the growth of microorganisms. Since the number of microorganisms has a certain growth compared with the initial inoculum, this strain has good adsorption, tolerance, and resistance to potassium dichromate. When the concentration of potassium dichromate is within 100 mg / L, the total chromium adsorption rate and hexavalent chromium adsorption rate change little, and the WTXJ1-4 strain shows good adsorption. When the concentration of potassium dichromate is 100 - 300 mg / L, the total chromium adsorption rate and hexavalent chromium adsorption rate both show a gentle downward trend, and the WTXJ1-4 strain shows good tolerance. When the concentration of potassium dichromate is 300 - 500 mg / L, the total chromium adsorption rate and hexavalent chromium adsorption rate both decrease rapidly, and the WTXJ1-4 strain shows strong resistance.

[0049] The above examples are only to illustrate the technical concept and characteristics of the present invention, and their purpose is to enable those familiar with this technology to understand the content of the present invention and implement it accordingly, and it cannot be used to limit the protection scope of the present invention. Any equivalent transformation or modification made according to the spirit and essence of the present invention should be covered within the protection scope of the present invention.

Claims

1. Application of Lysinibacillus fusiformis in removing hexavalent chromium from eutrophic wastewater, characterized in that, The preservation name of the spindle-shaped Lysinibacillus sp. is Lysinibacillus fusiformis WTXJ1-4, which is preserved in the China General Microbiological Culture Collection Center. The preservation address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The preservation number is CGMCC No. 10053, and the preservation date is November 25, 2014. The eutrophic wastewater is an LB liquid medium containing 300-500 mg / L of potassium dichromate. The composition of the LB liquid medium is 10 g / L of peptone, 5 g / L of yeast extract, 10 g / L of sodium chloride, and pH 7.

0. The application is as follows: 0.5-1.0 g / L of live WTXJ1-4 cells are taken and added to the eutrophic wastewater, and cultured under the conditions of pH 2.0, adsorption temperature of 32-34 °C, and rotation speed of 140-160 r / min for 20-24 h.

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