CN108359621A - One plant height imitates rhizobium and its application of adsorption of Low Concentration copper ion - Google Patents

Abstract

The invention discloses a strain of rhizobium ( Rhizobium sp.) S2 that efficiently adsorbs low-concentration copper ions. It is characterized in that the strain was preserved in the Guangdong Microbial Culture Collection Center on September 24, 2017, and the strain preservation number is For GDMCC NO: 60247. The bacterial strain is obtained by screening and separating from the activated sludge of Guangzhou Lijiao Sewage Plant by means of bacterial separation medium, which can efficiently and rapidly absorb low-concentration copper ions, and can absorb copper ions with a concentration of 2mg/L, The removal rate can reach 90% in 15 minutes, and the maximum adsorption capacity of the bacteria on copper ions is 7.6mg/g wet cells. It can be used for the treatment of low-concentration copper-containing wastewater to meet the wastewater discharge standard, and has great application prospects.

Description

A strain of rhizobia capable of efficiently adsorbing low-concentration copper ions and its application

technical field

The invention belongs to the technical field of environmental microorganisms. More specifically, it relates to a rhizobia strain capable of efficiently adsorbing low-concentration copper ions and its application.

Background technique

With the development of cities and industries, the pollution of metal-containing wastewater to the environment is becoming more and more serious. Refractory and highly toxic heavy metal pollutants in industrial wastewater accumulate in water bodies, causing serious harm to aquatic plants and animal systems in water bodies, and can affect human health through the food chain. Although the heavy metal wastewater discharged by industry can greatly reduce its content after treatment, it is difficult for the concentration of heavy metal ions in the heavy metal wastewater discharged by many industries to fully meet the discharge standard. Among them, copper is an important heavy metal pollutant.

Copper is an essential element for animals and plants. The lack of copper in the human body can cause anemia, abnormal hair, abnormal bones and arteries, and even brain disorders. Although copper is an important and essential trace element, improper application can easily cause poisoning reactions. Excessive intake of copper can cause damage to liver cells and red blood cells, further causing liver cirrhosis, diarrhea, vomiting, movement disorders and sensory nerve disorders.

At present, the control methods of heavy metal pollution mainly include three categories: physical method, chemical method and biological method. Physical and chemical methods are effective relatively quickly, but the cost is high, and secondary pollution is easy to occur. The biological method mainly uses the adsorption and passivation of heavy metals by plants and microorganisms. Among them, microbial treatment of heavy metal pollution has the advantages of low cost, high efficiency, little damage to the environment, no secondary pollution, and is suitable for large-scale repairs. It has a good application prospect.

However, there are few reports on microorganisms that have been found to adsorb and remove low-concentration copper ions in heavy metal wastewater.

Contents of the invention

The technical problem to be solved by the present invention is to overcome the deficiencies and deficiencies of the lack of microorganisms that absorb and remove copper ions in heavy metal wastewater in the prior art, and provide a strain of rhizobia that efficiently adsorbs low-concentration copper ions. The bacterial strain is isolated and cultivated by bacteria The base is screened and separated from the activated sludge of Guangzhou Lijiao Sewage Plant. It can efficiently and quickly absorb low-concentration copper ions. It can be used for the treatment of low-concentration copper-containing wastewater to meet the wastewater discharge standard. It has great application prospect.

The object of the invention is to provide a strain of rhizobia capable of efficiently adsorbing low-concentration copper ions.

Another object of the present invention is to provide the application of the above rhizobia in the treatment of heavy metal elimination.

Above-mentioned purpose of the present invention is given to realize by following technical scheme:

A Rhizobium sp. S2 strain that efficiently adsorbs low-concentration copper ions, which belongs to the genus Rhizobium, was preserved in the Guangdong Microbial Culture Collection Center on September 24, 2017, and the strain preservation number is For GDMCC NO: 60247. The classification name is Rhizobium sp.; the preservation address is 5th Floor, Building 59, Compound, No. 100 Xianlie Middle Road, Guangzhou, China.

Specifically, the 16S rDNA of the rhizobia is shown in SEQ ID NO:1.

The rhizobium ( Rhizobium sp.) S2 strain of the present invention is obtained by screening and separating from the activated sludge of Guangzhou Lijiao Sewage Plant by using a bacterial isolation medium; specifically, the isolation and cultivation method of the strain comprises the following steps:

(1) Take the activated sludge and the enrichment medium that has been sterilized and cooled down to room temperature, mix evenly at a volume ratio of 1:3, put it into a wide-mouth bottle, and seal the bottle mouth with sterilized gauze. Replace half of the medium on 2d, and culture at 30°C in the dark for 30d.

(2) After 30 days of enrichment culture, water samples at the mud-water interface were taken and cultured for 3 days with separation medium. After 3 days, the enriched culture solution was taken for appropriate dilution, 1 mL was inoculated into the solid medium, placed in a constant temperature incubator at 30°C, and incubated in the dark for 3 days. After the colonies grow, pick out the colonies with different shapes, sizes, colors, etc. and inoculate them on the corresponding LB medium plates respectively, until there are no foreign colonies. Subsequently, the obtained strains were inoculated on the slant of LB medium, and stored in a refrigerator at 4°C for future use.

The formula of the enrichment medium is: 1.00 g of sodium succinate, 0.10 g of NaNO ₃ , 0.20 g of NaAc, 0.05 g of sodium thioglycolate, 2.0 ml of 0.01M ferric quinate solution, 10.0 mL of Wolfe’s vitamin mixture, and 10.0 ml of Wolfe’s vitamin mixture. Mineral mixture 5.0mL, deionized water 983mL.

The formula of the separation medium is: KH ₂ PO ₄ 0.68g, NaNO ₃ 0.12g, Wolfe's vitamin mixture 10.0mL, Wolfe's mineral mixture 5.0ml, 0.01M iron quinate solution 2.0mL, resazurin 2mg, NaAc 0.20 g, 983 mL of deionized water, adjust the pH to 7.0 with 10M NaOH, and add 1.5% agar to the solid medium.

Wherein, the formula of the mineral mixture is: 1.5g of ammonia (base) triacetic acid, 3.0g of MgSO ₄ 7H ₂ O, 0.5g of MnSO ₄ .H ₂ O, 1.0g of NaCl, 0.1g of FeSO ₄ .7H ₂ O , CoCl ₂ 6H ₂ O 0.1g, CaCl ₂ 0.1g, ZnSO ₄ 7H ₂ O 0.1g, CuSO ₄ 5H ₂ O 0.01g, AlK(SO ₄ ) ₂ 12H ₂ O 0.01g, H ₃ BO ₃ 0.01g, Na ₂ MoO ₄ ·2H2O 0.01g, deionized water 1.0L, adjust the pH to 7.0 with KOH.

The Rhizobium sp. S2 strain of the present invention can efficiently and rapidly adsorb low-concentration copper ions, and can be used for the treatment of low-concentration copper-containing wastewater to meet wastewater discharge standards, and has great application prospects.

Therefore, the application of the rhizobia of the present invention in the treatment of low-concentration copper-containing wastewater or in the preparation of microbial preparations that absorb low-concentration copper ions is within the protection scope of the present invention.

Preferably, the concentration of copper ions in the low-concentration copper-containing wastewater is 2-30 mg/L.

Preferably, the pH of the low-concentration copper-containing wastewater is 4-7 (preferably 5-6).

Specifically, the application is to first adjust the pH of the low-concentration copper-containing wastewater to 4-7, and then add the rhizobia or its fermented liquid of the present invention to the wastewater for treatment.

At the same time, the present invention also provides a bacterial strain suspension capable of efficiently adsorbing low-concentration copper ions, and the bacterial suspension contains the above-mentioned rhizobia.

A microbial preparation for efficiently adsorbing low-concentration copper ions, said microbial preparation comprising the above-mentioned fermented rhizobia or strains thereof.

In addition, the application of the above bacterial strain suspension or microbial preparation in the treatment of low-concentration copper-containing wastewater is also within the protection scope of the present invention.

Specifically, the application is to add the above-mentioned bacterial strain suspension or microbial preparation to low-concentration copper-containing wastewater.

Compared with the prior art, the present invention has the following beneficial effects:

The present invention discovered a Rhizobium sp. S2 strain that efficiently adsorbs low-concentration copper ions. The strain was preserved in the Guangdong Microbial Culture Collection Center (GDMCC) on September 24, 2017, and the strain preservation number is It is GDMCC NO: 60247; the strain is screened and separated from the activated sludge of Guangzhou Lijiao Sewage Plant by means of bacterial isolation medium, and can efficiently and quickly absorb low-concentration copper ions, with a specific concentration of 2mg/L The adsorption and removal rate of copper ions can reach 90% in 15 minutes, and the maximum adsorption capacity of bacteria to copper ions is 7.6mg/g wet cells. It can be used for the treatment of low-concentration copper-containing wastewater to meet the wastewater discharge standard, and has a large application prospects.

Description of drawings

Fig. 1 is the dendrogram of the development of Rhizobium sp. S2 bacterial strain of the present invention;

Fig. 2 is the growth curve figure of Rhizobium sp. S2 bacterial strain of the present invention;

Fig. 3 is the graph of the adsorption of copper by Rhizobium sp. S2 strain of the present invention as a function of time;

Fig. 4 is the effect of the dosage of the rhizobium ( Rhizobium sp.) S2 strain of the present invention on the adsorption effect of copper;

Fig. 5 is a diagram showing the removal effect of Rhizobium sp. S2 strain of the present invention in the MBR of actual wastewater operation;

Fig. 6 is a contrast electron micrograph of the Rhizobium sp. S2 strain of the present invention before and after copper adsorption.

Detailed ways

The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the technical field.

Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

Example 1 Isolation and Identification of Bacterial Strains

1. Source of the strain

Activated sludge from Lijiao sewage plant in Guangzhou.

2. Medium

(1) Enrichment medium: sodium succinate 1.00g, NaNO ₃ 0.10g, NaAc 0.20g, sodium thioglycolate 0.05g, 0.01M ferric quinate solution 2.0ml, Wolfe's vitamin mixture 10.0mL, Wolfe's minerals Mixed liquid 5.0mL, deionized water 983mL.

(2) Separation medium: KH ₂ PO ₄ 0.68g, NaNO ₃ 0.12g, Wolfe's vitamin mixture 10.0mL, Wolfe's mineral mixture 5.0ml, 0.01M iron quinate solution 2.0mL, resazurin 2mg, NaAc 0.20 g, 983 mL of deionized water, adjust the pH to 7.0 with 10M NaOH, and add 1.5% agar to the solid medium.

(3) Wolfe's vitamin mixture: biotin 2.0mg, folic acid 2.0mg, pyridoxine hydrochloride 10.0mg, thiamine hydrochloride 5.0mg, riboflavin 5.0mg, niacin 5.0mg, calcium pantothenate 5.0mg, vitamin B ₁₂ 0.1mg, p-aminobenzoic acid 5.0mg, lipoic acid 5.0mg, deionized water 1.0L.

(4) Mineral mixture: ammonia (base) triacetic acid 1.5g, MgSO ₄ ·7H ₂ O 3.0g, MnSO ₄ ·H ₂ O 0.5g, NaCl 1.0g, FeSO ₄ ·7H ₂ O 0.1g, CoCl ₂ 6H ₂ O 0.1g, CaCl ₂ 0.1g, ZnSO ₄ 7H ₂ O 0.1g, CuSO ₄ 5H ₂ O 0.01g, AlK(SO ₄ ) ₂ 12H ₂ O 0.01g, H ₃ BO ₃ 0.01g, Na ₂ MoO ₄ ·2H2O 0.01g, deionized water 1.0L, adjust the pH to 7.0 with KOH.

(5) Fermentation medium: 10g of bacto-peptone, 5g of yeast extract powder, 5g of NaCl, 1g of glucose and 1000mL of pure water, and adjust the pH to 7.0±0.2.

3. Isolation, screening and identification of strains

(1) Take the activated sludge and the enrichment medium that has been sterilized and cooled down to room temperature, mix evenly at a volume ratio of 1:3, put it into a wide-mouth bottle, and seal the bottle mouth with sterilized gauze. Replace half of the medium on 2d, and culture at 30°C in the dark for 30d. After 30 days of enrichment culture, the water samples at the mud-water interface were taken and cultured for 3 days with the separation medium. After 3 days, the enriched culture solution was taken for appropriate dilution, 1 mL was inoculated into the solid medium, placed in a constant temperature incubator at 30°C, and incubated in the dark for 3 days. After the colonies grow, pick out the colonies with different shapes, sizes, colors, etc. and inoculate them on the corresponding LB medium plates respectively, until there are no foreign colonies. Subsequently, the obtained strains were inoculated on the slant of LB medium, and stored in a 4°C refrigerator for later use.

(2) The strains identified after the screening culture were cultured at 30°C for 1-2 days, and then identified by 16S rDNA sequencing. The 16S rDNA sequence of the strain was obtained by PCR (as shown in SEQ ID NO: 1). After sequence determination, BLAST homology comparison and phylogenetic evolution analysis (as shown in Figure 1), the results showed that the taxonomic significance of the isolated strain was The above is the genus Rhizobium ( Rhizobium sp.), named Rhizobium sp.S2 strain, and its growth curve was measured by plate counting method, the results are shown in Figure 2: 4-12h is the logarithmic growth phase, 12h-36h For the slow growth period, after 36h for the decline period. The strain was preserved in the Guangdong Provincial Microbial Culture Collection Center on September 24, 2017. The strain preservation number is GDMCC NO: 60247; the classification name is Rhizobium sp.; the preservation address is No. 100 Xianlie Middle Road, Guangzhou City, China. 5th Floor, Building 59, Yard.

Example 2 Research on the Adsorption Performance of Bacterial Strains to Copper

1. The influence of adsorption time on the adsorption effect

(1) The strain of Example 1 was inoculated in the liquid fermentation medium, and cultured on a shaker at 30°C and 150 rpm for 2 days. Wet bacteria were collected by centrifugation and washed three times with deionized water. Weigh 0.06g of the collected and washed wet bacteria, put it into a solution (pH=6.0±0.2) containing 100mL of copper (2mg/L), put it in a shaker and shake it evenly for adsorption (30°C, 150rpm). At the same time, the aqueous solution containing the same concentration of copper ions without adding bacteria was used as a control. After the adsorption time was 0, 15, 30, 60, 90, 120, and 180 min, the samples were taken in sequence, and centrifuged at 5000 rpm for 10 min. Membrane filtration, the filtrate was taken to measure the change of copper ion concentration in the solution before and after adsorption.

Calculation method of removal rate (R):

R(%)=(C ₀ -Ct)/C0*100%

Wherein, C ₀ is the initial concentration of copper ions in the solution, and Ct is the concentration of copper ions in the solution after adding bacterium to absorb for t minutes.

(2) The results are shown in Figure 3. The adsorption of copper ions by the rhizobia strains of Example 1 is a fast adsorption stage between 0 and 15 minutes, and the adsorption efficiency mainly depends on this stage. After 15 minutes, it enters a slow adsorption stage. Equilibrium was reached at 90 min.

2. The influence of the initial concentration of copper ions on the adsorption effect

(1) The strain of Example 1 was inoculated in the liquid fermentation medium, and cultured on a shaker at 30°C and 150 rpm for 2 days. Wet bacteria were collected by centrifugation and washed three times with deionized water. Weigh 0.06g of the collected and washed wet bacteria, put them into the solution (pH=6.0±0.2) containing 100mL of copper (2, 5, 10, 15, 20, 30mg/L) respectively, and put them on the shaker Shake well in medium for adsorption for 2h (30°C, 150rpm). At the same time, the aqueous solution containing the same concentration of copper ions without adding thallus was used as a contrast, and samples were taken in sequence after adsorption for 2 hours, centrifuged at 5000rpm for 10 minutes, filtered with a 0.22 micron filter membrane, and the filtrate was taken to measure the concentration of copper ions in the solution before and after adsorption. Variety.

Calculation method of adsorption capacity:

Q=(C ₀ -Ct)/C _b *100%

Wherein, C ₀ is the initial concentration of copper ions in the solution, Ct is the concentration of copper ions in the solution after adding bacterial cells for t minutes of adsorption, _and C is the concentration of bacteria in the solution.

(2) The results showed that the adsorption efficiency decreased with the increase of copper ion concentration. Because the ability of a single bacterium to adsorb copper ions is limited, the amount of bacterial input is fixed, and the removal rate decreases as the concentration of copper ions increases. The unit adsorption capacity of bacteria increases, the maximum value is 7.6mg/g wet cells, reaching an equilibrium state.

3. The effect of the amount of bacteria on the adsorption effect

(1) The strain of Example 1 was inoculated into a liquid fermentation medium, and cultured on a shaker at 30° C. and 150 rpm for 2 days. The cells were collected by centrifugation and washed three times with deionized water. Weigh 0.02, 0.04, 0.06, 0.08, 1.0, and 1.2 g of the collected and washed bacteria, respectively, and put them into 100 mL of copper-containing (2 mg/L) solution (pH=6.0±0.2), Put it in a shaker and shake it well for adsorption for 2h (30°C, 150rpm). At the same time, the aqueous solution containing the same concentration of copper ions without adding thallus was used as a contrast, and samples were taken in sequence after adsorption for 2 hours, centrifuged at 5000rpm for 10 minutes, filtered with a 0.22 micron filter membrane, and the filtrate was taken to measure the concentration of copper ions in the solution before and after adsorption. Variety.

(2) The results are shown in Figure 4. With the increase of the bacterial dosage, the removal rate of copper ions gradually increased, and when the bacterial dosage was 0.6g/L, the removal rate gradually decreased.

4. The influence of pH on the adsorption effect

(1) The strain of Example 1 was inoculated in the liquid fermentation medium, and cultured on a shaker at 30°C and 150 rpm for 2 days. The cells were collected by centrifugation and washed three times with deionized water. Weigh 0.06g of wet bacteria collected and washed, put them into 100mL copper ion solution (2mg/L) with pH 1, 2, 3, 4, 5, 6, 7 respectively, and put them in a shaker Shake well to absorb for 2h (30°C, 150rpm). At the same time, the aqueous solution containing the same concentration of copper ions without adding thallus was used as a contrast, and samples were taken in sequence after adsorption for 2 hours, centrifuged at 5000rpm for 10 minutes, filtered with a 0.22 micron filter membrane, and the filtrate was taken to measure the concentration of copper ions in the solution before and after adsorption. Variety.

(2) The results show that the unit adsorption capacity of the strain on copper ions is the largest at pH=6, and there is almost no adsorption effect when the pH is 1-3. When the pH is 3-6, the adsorption effect increases with the pH value. High and enhanced, and then the adsorption effect decreased with the increase of pH value.

To sum up, the optimal condition for the removal of copper by bacteria is that the removal rate of copper ions with an initial concentration of 2 mg/L after adsorption for 15 minutes reaches more than 90% when the pH is 6 and the bacterial dosage is 1.2 g/L.

Example 3 Application of bacterial strains on actual industrial tail water

1. Application in actual industrial tail water

An electroplating treatment sewage plant in Zhongshan City, Guangdong Province was built in 2011, mainly to treat some heavy metal sewage. Due to the improvement of environmental protection requirements in recent years, the original treatment facilities of the water plant can no longer meet the requirements of wastewater treatment. The company plans to Carry out technological transformation. The tail water treated by the plant has been tested, and its copper ion concentration is about 2-5 mg/L, pH is between 4-5, and COD is between 20-30 mg/L. The tail water is taken in the laboratory for rhizobia adsorption experiments.

An MBR system was built in the laboratory, with a system volume of 9L, and two groups of systems, namely the blank group and the experimental group. The system runs continuously (hydraulic retention time is 8 hours). The tail water of the sewage plant is used as the influent, and rhizobia is added. After observing its good film formation, the concentration of copper ions in the water is detected.

The treatment effect is shown in Figure 5. The influent concentration is about 2.2mg/L, and the effluent effect is significantly enhanced and decreased after 2 hours, and tends to be stable after 6 hours. The removal rate of copper in the actual wastewater by the MBR system is about 76%.

2. Changes in the morphology of microorganisms before and after adsorption of copper ions under the microscope

The biofilm on the experimental group and the blank group was observed under the scanning electron microscope, and the results are shown in Figure 6. The shape of the bacteria in the blank group can be clearly and completely seen to be rod-shaped, and the shape of the bacteria is plump and round; the cell surface of the experimental group Adhesive, covered by substance, cell shape is incomplete. The cell surface of the experimental group will be brighter than that of the blank group. According to the principle of the scanning electron microscope, the heavy metal substances appear as bright white spots on the instrument. After the bacteria absorb zinc ions, they accumulate on a part of the surface, so there is bright light around the bacteria, indicating that the cells The surface adhesion substance is metal copper substance.

According to the application experiment, the rhizobia isolated by the present invention has a good adsorption effect on the low-concentration heavy metal wastewater.

sequence listing

<110> Sun Yat-sen University

<120> A Rhizobia Strain Efficiently Adsorbing Low Concentration Copper Ions and Its Application

<130> YG18100886AA042

<141> 2018-03-14

<160> 1

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<213> Rhizobium sp.S2

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ccctcgccct tagttgcagc atttagtttg gcacttttaa ggggactgcc gggtgaataa 1080

g 1081

Claims (9)

1. a plant height imitates the rhizobium of adsorption of Low Concentration copper ion（Rhizobiumsp.）S2 bacterial strains, which is characterized in that the bacterium Strain was preserved in Guangdong Province's Culture Collection on the 24th in September in 2017, and culture presevation number is GDMCC NO：60247.

2. rhizobium according to claim 1, which is characterized in that the 16S rDNA of the rhizobium such as SEQ ID NO：1 It is shown.

3. rhizobium described in claim 1 administer in low concentration copper-containing wastewater or in the microorganisms for preparing adsorption of Low Concentration copper ion Application in preparation.

4. application according to claim 3, which is characterized in that a concentration of the 2 of copper ion in the low concentration copper-containing wastewater ~30mg/L.

5. application according to claim 3, which is characterized in that first adjust the pH to 4~7 of low concentration copper-containing wastewater, then will Bacterial strain described in claim 1 or its zymotic fluid are added in waste water.

6. a kind of bacterial strain suspension of efficient absorption low concentration copper ion, which is characterized in that include root nodule described in claim 1 Bacterium.

7. a kind of microorganism formulation of efficient absorption low concentration copper ion, which is characterized in that include rhizobium described in claim 1 Or its zymotic fluid.

8. the application of bacterial strain suspension described in claim 6 or 7 microorganism formulations in administering low concentration copper-containing wastewater.

9. application according to claim 8, which is characterized in that the application is to add power into copper-containing wastewater containing low concentration Profit requires the 6 bacterial strain suspension or 7 microorganism formulations.