CN110241111B - A method for enhancing the chromium reduction ability of bacteria by immobilizing cells with modified activated carbon - Google Patents

Abstract

The invention discloses a method for enhancing the chromium reduction ability of micrococci by immobilizing cells with modified activated carbon, and belongs to the technical field of environmental restoration. Main parameters of immobilized pellet reagent: sodium alginate (3%, w/v), calcium chloride (4%, w/v), micrococcus bacteria liquid, modified activated carbon 0.1-0.5g, sodium alginate solution, The micrococcus bacteria liquid and the modified activated carbon are evenly mixed, and the calcium chloride solution is dropped into the cross-linking reaction. After standing at room temperature for four hours, it is added into the hexavalent chromium solution to carry out the reduction reaction. Compared with the reduction effect of adding 0.1-0.5g modified activated carbon to hexavalent chromium, the reduction rate increased by 8.4%-13.4%. The invention utilizes the adsorption characteristics of activated carbon to promote the efficient reduction of hexavalent chromium ions in water by immobilized bacteria, and significantly improves the reduction efficiency of micrococcus to hexavalent chromium in water.

Description

A method for enhancing bacterial chromium reduction ability by immobilizing cells with modified activated carbon

technical field

The invention belongs to the technical field of environmental restoration, and in particular relates to a method for enhancing the chromium reduction ability of bacteria by immobilizing cells with modified activated carbon.

Background technique

Chromium is an element with relatively high content and wide distribution in natural resources. Chromium mainly enters into various environments through two ways of natural existence and human activities, such as surface water, groundwater, seawater, soil, rock and air, and exists in the crust in different concentrations and in different combinations. Chromium is one of the most commonly used metal pollutants. It is one of the 129 key pollution hazards recognized by the US EPA and one of the three internationally recognized carcinogens. In recent years, chromium pollution incidents have occurred frequently in Hunan, Yunnan and other places in my country, causing serious social impact. Therefore, it has been widely concerned by scientists and has become a hot and difficult research topic in the field of environmental research in recent years. There are various shortcomings in the physical and chemical treatment methods of chromium pollution. Therefore, researchers at home and abroad began to shift their research goals to a new method - bioremediation. The bioremediation method converts highly toxic and highly mobile Cr(VI) into low toxic and weakly mobile Cr(III) through the adsorption and enrichment of Cr(VI) by microorganisms or the metabolism of microorganisms, and finally achieves the reduction of Cr(Ⅵ). The purpose of Cr(VI) ions contained in the environment. The use of bioremediation to control heavy metal pollution was not applied in practice until the 1980s. The advantages of bioremediation are easy control, small investment, high efficiency, low cost and no secondary pollution, so it has a good application prospect in environmental remediation. However, microbial remediation has disadvantages such as poor microbial adaptability, susceptibility to external environment, and low efficiency, which limit its wide application. In order to improve the tolerance of microorganisms and increase the metal reduction efficiency of strains, a large number of researchers have explored various methods of strengthening microorganisms to repair chromium pollution from multiple directions such as physics, chemistry, and biology.

Microbial immobilization technology has been widely used in the treatment of refractory toxic metal and organic pollution and has achieved good results. Compared with the traditional biological treatment technology, the use of microbial immobilization technology combined with the adsorption characteristics of activated carbon to reduce hexavalent chromium pollution in water has obvious advantages. The present invention can significantly increase the reduction efficiency of hexavalent chromium by adding modified activated carbon, so the achievement of the present invention has good practicability.

Contents of the invention

The purpose of the present invention is to improve the efficiency of microbes reducing hexavalent chromium, and invent a method for increasing the chromium reducing ability of bacteria by immobilizing cells with modified activated carbon. The present invention can significantly enhance the reducing ability of microbes to hexavalent chromium.

Technical scheme of the present invention is summarized as follows:

A method for improving the chromium reduction ability of bacteria by immobilizing cells with modified activated carbon, comprising the following steps:

1. Break the activated carbon into a particle size of more than 200 mesh;

2. Add 5g of activated carbon into a flask containing 50ml of 70% _HNO3 , and stir at room temperature at 170rpm for 1h to remove impurities in the activated carbon, wash with deionized water until neutral, and finally fully dry; add the dried activated carbon 100ml 10% (w/v) PEI (polyetherimide) methanol solution, stirred at 170rpm at room temperature for 24 hours, then immediately transferred the activated carbon to 200ml 1% (w/v) glutaraldehyde solution for cross-linking , and finally washed with deionized water to obtain modified activated carbon;

3. The strains were isolated from laboratory screening, and the selection medium was improved liquid LB medium, formulated with: peptone 10g·L ^-1 , sodium chloride 5g·L ^-1 , yeast extract 5g·L ^-1 , hydrogen phosphate Dipotassium 0.05g·L ^-1 , magnesium sulfate heptahydrate 0.2g·L ^-1 , if it is a solid LB medium, add 1.5-2.0% agar, pH 8.5-9.0, and sterilize;

4. The micrococci obtained by screening were cultured in shake flasks containing LB medium, the culture conditions were initial pH 8.0-9.0, temperature 30°C, shaker speed 170rpm;

5. The conditions for immobilizing the pellets are: 3g sodium alginate, 4g calcium chloride, 0.1-0.5g modified activated carbon, dissolve sodium alginate and modified activated carbon in 90ml distilled water, and sterilize;

6. Filter and centrifuge the cultured micrococcus in step (4), and make 10ml of sterile water into 10ml of the inoculum according to the _OD600 value of 1.5. The mass concentration is 4% w/v calcium chloride solution, after cross-linking for four hours, it is added to LB liquid medium containing 120 mg/L Cr(Ⅵ) and cultured on a shaking table at room temperature for 84 hours.

7. Prepare diphenylcarbazide solution, dissolve 0.2g of diphenylcarbazide in 50ml of acetone, transfer to a 100ml volumetric flask, add water to dilute to the mark, shake well, and gradually add 12.5ml into the cooling solution Concentrated H ₂ SO ₄ and 12.5ml concentrated phosphoric acid, stored in the dark.

In the step (3), take 10 g of the sample and add it to 90 ml of improved sterile liquid LB medium, dilute it with a 10-fold gradient, draw 0.2 ml each, and apply it on a solid containing 200 mg/L Cr(VI). On the improved LB medium, spread the bacterial solution evenly with a coating stick, and cultivate it in a constant temperature incubator at 30°C for 24 hours until the colony grows, and identify the isolated strain as Micrococcus.

To step (7) gained diphenylcarbazide solution, adopt the diphenylcarbazide spectrophotometric method to measure the concentration of hexavalent chromium in the sample, the calculation formula of Cr (Ⅵ) concentration is y=0.6031x+0.0065, standard The correlation coefficient of the curve is R ² =0.0997.

Modified activated carbon materials have their own advantages in the removal of pollutants. After modification, their own structure (specific surface area, porosity, etc.) or performance is optimized, and the repair effect on heavy metal pollution is better. The method of the invention can significantly improve the micrococcus reducing efficiency of hexavalent chromium in the water body by improving the active carbon and controlling the dosage (0.3g) of the active carbon, and the reduction rate is increased by 13.4%.

Description of drawings

Fig. 1 is the trend chart of hexavalent chromium concentration over time of embodiment 1;

Fig. 2 is the trend chart of hexavalent chromium concentration over time of embodiment 2;

Fig. 3 is the trend graph of hexavalent chromium concentration over time of embodiment 3;

Fig. 4 is the trend chart of hexavalent chromium concentration over time of embodiment 4;

Fig. 5 is the hexavalent chromium concentration over time chart of embodiment 5;

Fig. 6 is the display diagram without adding modified activated carbon immobilized pellets;

Fig. 7 is a display diagram of adding modified activated carbon to immobilize pellets.

Detailed ways

The purpose of the following specific examples or embodiments is to further illustrate the present invention, rather than limit the present invention.

Example 1

The method described in this embodiment mainly proceeds according to the following steps:

(1) The activated carbon is crushed into a particle size of more than 200 mesh;

(2) 5g of activated carbon is added into the flask containing 50ml 70% _HNO3 , and stirred at room temperature with 170rpm for 1h to remove impurities in the activated carbon, washed with deionized water to neutrality, and finally fully dried; the dried activated carbon Add 100ml 10% (w/v) PEI (polyetherimide)/methanol solution, stir at 170rpm at room temperature for 24 hours, then immediately transfer the activated carbon to 200ml 1% (w/v) glutaraldehyde solution for Cross-linking, and finally washing with deionized water to obtain modified activated carbon;

(3) Configure LB liquid medium, culture micrococci in shake flasks to the logarithmic growth phase, take the bacterial liquid with an _OD600 value of about 1.5, collect it by centrifugation, and prepare 10 ml bacterial suspension with sterile water;

(4) Weigh 3g of sodium alginate and 0.1g of modified activated carbon, add 90ml of distilled water system, sterilize, mix 10ml of bacterial suspension in step (2) with it, and evenly drop into 4% w/v calcium chloride solution In the medium, stand at room temperature for four hours, pour the washed pellets into LB liquid medium containing 120 mg/L Cr(Ⅵ), the culture conditions are initial pH 8.0, temperature 30°C, shaker speed 170 rpm, and culture for 84 hours;

Conclusion: As shown in Figure 1, the reduction rate of hexavalent chromium with 0.1 g of modified activated carbon was 72.5%, and the reduction rate of hexavalent chromium without modified activated carbon was 64.1%, and the reduction rate of hexavalent chromium increased by 8.4%.

Example 2

The method described in this embodiment mainly proceeds according to the following steps:

(1) The activated carbon is crushed into a particle size of more than 200 mesh;

(2) 5g of activated carbon is added into the flask containing 50ml 70% _HNO3 , and stirred at room temperature with 170rpm for 1h to remove impurities in the activated carbon, washed with deionized water to neutrality, and finally fully dried; the dried activated carbon Add 100ml 10% (w/v) PEI (polyetherimide)/methanol solution, stir at 170rpm at room temperature for 24 hours, then immediately transfer the activated carbon to 200ml 1% (w/v) glutaraldehyde solution for Cross-linking, and finally washing with deionized water to obtain modified activated carbon;

(3) Configure LB liquid medium, culture micrococci in shake flasks to the logarithmic growth phase, take the bacterial liquid with an _OD600 value of about 1.5, collect it by centrifugation, and prepare 10 ml bacterial suspension with sterile water;

(4) Weigh 3g of sodium alginate and 0.2g of modified activated carbon, add 90ml of distilled water system, sterilize, mix 10ml of bacterial suspension in step (2) with it, and evenly drop into 4% w/v calcium chloride solution In the medium, stand at room temperature for four hours, pour the washed pellets into LB liquid medium containing 120 mg/L Cr(Ⅵ), the culture conditions are initial pH 8.0, temperature 30°C, shaker speed 170 rpm, and culture for 84 hours;

Conclusion: As shown in Figure 2, the reduction rate of hexavalent chromium with 0.2g of modified activated carbon was 73.3%, and the reduction rate of hexavalent chromium without modified activated carbon was 64.1%, and the reduction rate of hexavalent chromium increased by 9.2%.

Example 3

The method described in this embodiment mainly proceeds according to the following steps:

(1) The activated carbon is crushed into a particle size of more than 200 mesh;

(2) 5g of activated carbon is added into the flask containing 50ml 70% _HNO3 , and stirred at room temperature with 170rpm for 1h to remove impurities in the activated carbon, washed with deionized water to neutrality, and finally fully dried; the dried activated carbon Add 100ml 10% (w/v) PEI (polyetherimide)/methanol solution, stir at 170rpm at room temperature for 24 hours, then immediately transfer the activated carbon to 200ml 1% (w/v) glutaraldehyde solution for Cross-linking, and finally washing with deionized water to obtain modified activated carbon;

(3) Configure LB liquid medium, culture micrococci in shake flasks to the logarithmic growth phase, take the bacterial liquid with an _OD600 value of about 1.5, collect it by centrifugation, and prepare 10 ml bacterial suspension with sterile water;

(4) Weigh 3g of sodium alginate and 0.3g of modified activated carbon, add 90ml of distilled water system, sterilize, mix 10ml of bacterial suspension in step (2) with it, evenly drop into 4% w/v calcium chloride solution In the medium, stand at room temperature for four hours, pour the washed pellets into LB liquid medium containing 120 mg/L Cr(Ⅵ), the culture conditions are initial pH 8.0, temperature 30°C, shaker speed 170 rpm, and culture for 84 hours;

Conclusion: As shown in Figure 3, the reduction rate of hexavalent chromium with 0.3g of modified activated carbon was 77.5%, and the reduction rate of hexavalent chromium without modified activated carbon was 64.1%, and the reduction rate of hexavalent chromium increased by 13.4%.

Example 4

The method described in this embodiment mainly proceeds according to the following steps:

(1) The activated carbon is crushed into a particle size of more than 200 mesh;

(2) 5g of activated carbon is added into the flask containing 50ml 70% _HNO3 , and stirred at room temperature with 170rpm for 1h to remove impurities in the activated carbon, washed with deionized water to neutrality, and finally fully dried; the dried activated carbon Add 100ml 10% (w/v) PEI (polyetherimide)/methanol solution, stir at 170rpm at room temperature for 24 hours, then immediately transfer the activated carbon to 200ml 1% (w/v) glutaraldehyde solution for Cross-linking, and finally washing with deionized water to obtain modified activated carbon;

(3) Configure LB liquid culture medium, culture micrococci in shake flasks to the logarithmic growth phase, take the bacterial liquid with an OD600 value of about 1.5, collect it by centrifugation, and prepare 10 ml bacterial suspension with sterile water;

(4) Weigh 3g of sodium alginate and 0.4g of modified activated carbon, add 90ml of distilled water system, sterilize, mix 10ml of bacterial suspension in step (2) with it, and evenly drop into 4% w/v calcium chloride solution In the medium, stand at room temperature for four hours, pour the washed pellets into LB liquid medium containing 120 mg/L Cr(Ⅵ), the culture conditions are initial pH 8.0, temperature 30°C, shaker speed 170 rpm, and culture for 84 hours;

Conclusion: As shown in Figure 4, the reduction rate of hexavalent chromium with 0.4g of modified activated carbon was 75.83%, and the reduction rate of hexavalent chromium without modified activated carbon was 64.1%, and the reduction rate of hexavalent chromium increased by 11.73%.

Example 5

The method described in this embodiment mainly proceeds according to the following steps:

(1) The activated carbon is crushed into a particle size of more than 200 mesh;

(2) 5g of activated carbon is added into the flask containing 50ml 70% _HNO3 , and stirred at room temperature with 170rpm for 1h to remove impurities in the activated carbon, washed with deionized water to neutrality, and finally fully dried; the dried activated carbon Add 100ml 10% (w/v) PEI (polyetherimide)/methanol solution, stir at 170rpm at room temperature for 24 hours, then immediately transfer the activated carbon to 200ml 1% (w/v) glutaraldehyde solution for Cross-linking, and finally washing with deionized water to obtain modified activated carbon;

(3) Configure LB liquid medium, culture micrococci in shake flasks to the logarithmic growth phase, take the bacterial liquid with an _OD600 value of about 1.5, collect it by centrifugation, and prepare 10 ml bacterial suspension with sterile water;

(4) Weigh 3g of sodium alginate and 0.5g of modified activated carbon, add 90ml of distilled water system, sterilize, mix 10ml of bacterial suspension in step (2) with it, and evenly drop into 4% w/v calcium chloride solution In the medium, stand at room temperature for four hours, pour the washed pellets into LB liquid medium containing 120 mg/L Cr(Ⅵ), the culture conditions are initial pH 8.0, temperature 30°C, shaker speed 170 rpm, and culture for 84 hours;

Conclusion: As shown in Figure 5, the reduction rate of hexavalent chromium with 0.5 g of modified activated carbon is 75%, and the reduction rate of hexavalent chromium without modified activated carbon is 64.1%, and the reduction rate of hexavalent chromium is increased by 10.9%.

Claims (2)

1. a kind of method that modified active carbon immobilized cell strengthens bacterial chromium reduction ability, it is characterized in that comprising the following steps: (1) Crush the activated carbon into a particle size of 200 mesh or more; (2) Add 5 g of activated carbon into a flask containing 50 ml of 70% HNO ₃ , and stir at 170 rpm at room temperature for 1 h to remove impurities in the activated carbon, wash with deionized water until neutral, and finally fully dry; the dried Activated carbon was added to 100ml of 10% w/v PEI/methanol solution, stirred at 170rpm at room temperature for 24 hours, then immediately transferred to 200ml of 1% w/v glutaraldehyde solution for cross-linking, and finally washed with deionized water to obtain the improved Activated carbon; (3) Configure LB liquid medium, culture micrococci in shake flasks to the logarithmic growth phase, take the bacterial liquid with an _OD600 value of 1.5, collect it by centrifugation, and prepare 10 ml bacterial suspension with sterile water; (4) Weigh 3g of sodium alginate, and 0.1, 0.2, 0.3, 0.4 or 0.5g of modified activated carbon, add 90 ml of distilled water system, sterilize, mix 10 ml of bacterial suspension with it evenly, drop evenly into 4% w /v calcium chloride solution, stand at room temperature for four hours, pour the washed pellets into LB liquid medium containing 120 mg/L Cr(Ⅵ), the culture conditions are initial pH 8.0, temperature 30 °C, shaking The bed rotates at 170 rpm and cultivates for 84 h; prepare diphenylcarbazide solution, 0.2 g of diphenylcarbazide, dissolve in 50 ml of acetone, transfer to a 100 ml volumetric flask, add water to dilute to the mark, shake well, and Slowly add 12.5 ml of concentrated H ₂ SO ₄ and 12.5 ml of concentrated phosphoric acid to the refrigerated solution, and store in the dark. 2. The method for enhancing bacterial chromium reduction ability of modified activated carbon immobilized cells according to claim 1, characterized in that the diphenylcarbazide solution of step (4) is obtained by using diphenylcarbazide spectrophotometry To measure the concentration of hexavalent chromium in the sample, the formula for calculating the concentration of Cr(VI) is y=0.6031x+0.0065, and the correlation coefficient R ² of the standard curve is 0.0997.

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