CN111647539A - Method for strengthening film forming capability of aerobic denitrifying bacteria - Google Patents
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Abstract
The invention belongs to the technical field of biological treatment of wastewater, and particularly discloses a method for enhancing the film forming capability of aerobic denitrifying bacteria. A method for strengthening the film forming capability of aerobic denitrifying bacteria comprises the following steps: step 1, preparing an HM culture medium and an LB culture medium; step 2, adding ZnO NPs solution with the concentration of 1.0-1.5mM into the HM culture medium in the step 1; step 3, putting the aerobic denitrifying bacteria strain into the HM culture medium in the step 2 for culture; step 4, selecting and finishing the HM culture medium in the step 3, and adding the HM culture medium into an LB culture medium for rejuvenation; and 5, adding the aerobic denitrifying bacteria strain obtained in the step 4 into the HM culture medium obtained in the step 1 for continuous culture. The invention aims to provide a method for strengthening the film forming capability of aerobic denitrifying bacteria, which aims to solve the problems of weak film forming capability and poor ZnO NPs resisting effect of the aerobic denitrifying bacteria.
Description
Technical Field
The invention belongs to the technical field of biological treatment of wastewater, and particularly discloses a method for enhancing the film forming capability of aerobic denitrifying bacteria.
Background
Currently, with rapid development of national economy and further progress of urbanization, the total amount of discharge of urban domestic and industrial wastewater is increasing year by year. The wide use of pesticides, fertilizers, synthetic detergents and the like causes the concentration of nutrient substances in water to be continuously increased, wherein nitrogen is one of the main reasons for eutrophication of water. Conventional denitrification processes typically separate the nitrification and denitrification processes in two reaction units, since most denitrifying bacteria require anaerobic conditions, while nitrifying bacteria require aerobic conditions. The discovery of the aerobic denitrifying bacteria enables the nitrification and denitrification processes to be carried out in the same reaction unit, thereby saving the construction and the operation and maintenance cost.
Zinc oxide nanoparticles (ZnO NPs) are one of the representatives of metal oxide nanomaterials and widely used in the industries of semiconductors, additives, coatings, cosmetics, electronic devices, catalysts and the like. Its widespread use makes it inevitable to enter sewage treatment systems by way of sewage and wastewater discharge. Research indicates that the content of nano ZnO in sludge of European and American sewage treatment plants in 2008 is about 17.1mg/kg and 23.2mg/kg respectively. The ZnO NPs can inhibit the denitrification effect of denitrifying bacteria on wastewater, and can effectively relieve the inhibition effect of the ZnO NPs when the denitrifying bacteria exist in a form of a biological film.
Disclosure of Invention
The invention aims to provide a method for strengthening the film forming capability of aerobic denitrifying bacteria, which aims to solve the problems of weak film forming capability and poor ZnO NPs resisting effect of the aerobic denitrifying bacteria.
In order to achieve the purpose, the basic scheme of the invention is as follows: a method for strengthening the film forming capability of aerobic denitrifying bacteria comprises the following steps: step 1, preparing an HM culture medium and an LB culture medium;
step 2, adding ZnO NPs solution with the concentration of 1.0-1.5mM into the HM culture medium in the step 1;
step 3, putting the aerobic denitrifying bacteria strain into the HM culture medium in the step 2 for culture;
step 4, adding the aerobic denitrifying bacteria in the HM culture medium after the step 3 into an LB culture medium for rejuvenation;
and 5, adding the aerobic denitrifying bacteria strain obtained in the step 4 into the HM culture medium obtained in the step 1 for culture to obtain the enhanced aerobic denitrifying bacteria.
The working principle and the beneficial effects of the basic scheme are as follows: because ZnO NPs inhibit aerobic denitrifying bacteria, the technical scheme is that the aerobic denitrifying bacteria are cultured in a high-concentration ZnO NPs environment. The aerobic denitrifying bacteria cultured in the high-concentration ZnO NPs environment have resistance and tolerance to the ZnO NPs, and the mode of the finally cultured aerobic denitrifying bacteria for resisting the ZnO NPs is reflected in that the film forming capability is obviously increased.
1. Compared with the prior art, the technical scheme has the advantage that the film forming capability is remarkably improved.
2. Compared with the prior art, the ZnO NPs resistance of the technical scheme is obviously enhanced.
Further, the LB medium comprises tryptone, yeast extract and NaCl in a ratio of 2:1: 2.
Further, the pH of the LB medium is 7.2-7.8, and the pH value of the LB medium is limited to meet the growth environment of the bacteria.
Further, the method also comprises an LB culture medium sterilization step, wherein the LB culture medium is sterilized for more than 25min by high-pressure steam at the temperature of not less than 120 ℃ so as to meet the aim of sterilizing the LB culture medium.
Further, the HM culture medium comprises potassium nitrate, glucose, disodium hydrogen phosphate dodecahydrate, sodium dihydrogen phosphate dihydrate, sodium chloride and a trace element solution in a ratio of 0.72:3:4.78:1.24: 4; the trace element solution comprises MgSO4·7H2O、MnSO4·H2O、H3BO3、ZnSO4·7H2O、FeSO4·7H2O and CaCl2。
Further, the method also comprises a glucose sterilization step, wherein the glucose is prepared into a solution and is subjected to high-pressure steam sterilization at the temperature of not less than 110 ℃ for more than 15min so as to meet the purpose of sterilizing the glucose.
Further, the method also comprises a sterilization step of the HM culture medium except the glucose, wherein the HM culture medium is sterilized by high-pressure steam at the temperature of not less than 120 ℃ for more than 25min so as to meet the purpose of sterilization.
Further comprising a step 6 of measuring the film forming capability of the enhanced aerobic denitrifying bacteria by adopting a crystal violet staining method after the step 5 is finished.
Drawings
FIG. 1 is a bar graph showing the film forming ability of wild-type bacteria according to the present invention as a reference.
Detailed Description
The following is further detailed by way of specific embodiments:
the aerobic denitrifying strain is Enterobacter cloacae strain HNR (GenBank accession number KP308150), the activated sludge is screened from an A2/O process secondary sedimentation tank of a gravity feed crowned rock sewage treatment plant, the activated sludge and the Enterobacter cloacae strain ATCC 39978 are in the same development branch, and the 16SrRNA gene sequence comparison homology similarity rate is up to 99 percent.
The following describes a method for enhancing the film forming capability of aerobic denitrifying bacteria, which specifically comprises the following steps:
step 1, preparing an aerobic denitrification culture medium (hereinafter referred to as HM culture medium) per liter, wherein the aerobic denitrification culture medium comprises the following specific components: 0.72g of potassium nitrate, 3g of glucose, 4.78g of disodium hydrogenphosphate dodecahydrate, 1.24g of sodium dihydrogenphosphate dihydrate, 4.0g of sodium chloride and 3mL of a trace element solution were weighed and mixed. Wherein the trace element solution is added per liter: 3.01g MgSO4·7H2O，3.36g MnSO4·H2O，1.12g H3BO3，3.00g ZnSO4·7H2O，0.30g FeSO4·7H2O，0.60g CaCl2. Glucose was sterilized separately from the other component solutions of the HM media. A high-concentration glucose solution (300g/L) was prepared and then autoclaved at 110 ℃ for 15 min. After adjusting the other component solutions (except glucose) to pH 7.5 with 0.5M NaOH solution, autoclaving at 121 ℃ for 25min, and finally mixing all the sterilized solutions in a clean bench.
Preparing an LB culture medium: wherein the preparation is carried out in the presence of 10g tryptone, 5g yeast extract, 10g NaCl per liter. The pH of the 0.5M NaOH solution was adjusted to about 7.5. Sterilizing with high pressure steam at 121 deg.C for 25 min.
And 2, the generation inhibition of the ZnO NPs on the aerobic denitrifying bacteria by releasing zinc ions in the solution is one of toxicity mechanisms of the ZnO NPs, so that the aerobic denitrifying bacteria are cultured by using a 0.15mM zinc ion HM culture medium, and the strain with the growth condition obviously better than that of a wild type strain under the stress of the ZnO NPs is selected as a resistant strain, and is named as an enhanced type.
And 3, using a 250mL triangular flask containing 100mL liquid HM culture medium when the aerobic denitrifying bacteria are cultured, adding a proper amount of bacterial liquid, and culturing at the rotating speed of 140rpm and the temperature of 28 ℃ by using a constant-temperature shaking table.
Step 4, rejuvenating the aerobic denitrifying bacteria strain HNR in an LB culture medium, and adjusting the bacterium liquid OD by using 1 × PBS600About 1.0 (6000rpm, 10min, 4 ℃).
Step 5, to verify whether the bacteria after continuous culture have resistance to ZnO NPs, the bacteria were cultured continuously in HM medium containing ZnO NPs of different concentrations using enriched HNR bacteria liquid (hereinafter referred to as wild type) and HNR bacteria liquid without treatment in step 2, as described in examples 1-4 below
Example one
Respectively adding 1mL of wild type and enhanced bacterial liquid into HM culture medium with ZnO NPs concentration of 0mM for culture, continuously culturing, and recording the film forming capability of HNR strain.
Example two
Respectively adding 1mL of wild type and enhanced bacterial liquid into HM culture medium with ZnO NPs concentration of 0.25mM for culture, continuously culturing, and recording the film forming capability of HNR strain.
EXAMPLE III
Respectively adding 1mL of wild type bacteria liquid and 1mL of enhanced bacteria liquid into HM culture medium with ZnO NPs concentration of 0.50mM for culture, continuously culturing, and recording the film forming capability of the HNR strain.
Example four
Respectively adding 1mL of wild type bacteria liquid and 1mL of enhanced bacteria liquid into HM culture media with ZnO NPs concentration of 0.75mM for culture, continuously culturing, and recording the film forming capability of the HNR strain.
The detection method is that ZnO inhibits aerobic denitrifying bacteria, the reinforced type is cultured in a high-concentration ZnO environment, the ZnO has resistance and tolerance, and the increase of the film forming capability is a mode of resisting ZnO, so that the film forming capability of the aerobic denitrifying bacteria is measured by adopting a crystal violet staining method in examples 1-4, the strain rejuvenated by an LB culture medium is adjusted to OD by using 1 × PBS to adjust the concentration of the strain liquid to OD600mu.L of the bacterial suspension was pipetted by a sterile pipette and inoculated into a 96-well plate containing 180. mu.L of a medium (HM liquid medium or HM medium containing ZnO NPs at various concentrations), and after static culture at 28 ℃ for 21 hours under aseptic conditions, the culture was gently aspirated, and the surface of the biofilm was carefully washed 2 times with sterile purified water. Then, the 96-well plate is placed in a 45 ℃ oven for drying, taken out and added with 200 mu L of 0.1% (w/v) crystal violet solution, dyed for 5min at room temperature, sucked with dyeing solution, washed with distilled water for 2 times, and then placed in the 45 ℃ oven for drying. Finally, 200 μ L of 95% ethanol solution was added to the dried 96-well plate to dissolve the crystal violet dye bound to the biofilm, which was continued for 30 min. Finally, measuring the absorbance at 570nm on a microplate reader, and taking the absorbance as the absorbanceAnd (4) a biofilm amount evaluation standard.
The values measured after continuous culture in examples 1 to 4 of the present invention are shown in Table 1, Table 2 and FIG. 1.
TABLE 1
Table 2 (reference to the wild type film Forming ability value in example I)
And (4) test conclusion: the enhanced film forming ability is far larger than that of the wild type, and is about 3.7 times of that of the wild type in a normal culture state. Under the stress of ZnO NPs with different concentrations, the film forming capacities of the wild type and the enhanced type in the second example, the third example and the fourth example are inhibited, wherein the film forming capacities are 91.96%, 93.41% and 97.30% and 58.1%, 70.0% and 75.8% of the film forming capacity in the normal state of the first example. Although the film formation is inhibited, the film formation effect of the wild type is not obvious under different ZnO concentrations with the increase of the concentration of the ZnO NPs, the film formation amount of the wild type is slightly increased, the film formation amount of the reinforced type still shows an obvious rising trend, and the film formation amount is always far larger than that of the wild type. Therefore, the reinforced type has proved that the reinforced type not only has better film forming capability, but also has obviously enhanced tolerance to ZnO NPs.
The foregoing is merely an example of the present invention and common general knowledge of known specific structures and features of the embodiments is not described herein in any greater detail. It should be noted that, for those skilled in the art, without departing from the structure of the present invention, several changes and modifications can be made, which should also be regarded as the protection scope of the present invention, and these will not affect the effect of the implementation of the present invention and the practicability of the present invention.
Claims (8)
1. A method for strengthening the film forming capability of aerobic denitrifying bacteria is characterized by comprising the following steps:
step 1, preparing an HM culture medium and an LB culture medium;
step 2, adding a ZnO NPs solution with the concentration of 1.0-1.5mM into the HM culture medium in the step 1;
step 3, putting the aerobic denitrifying bacteria strain into the HM culture medium in the step 2 for culture;
step 4, adding the aerobic denitrifying bacteria in the HM culture medium after the step 3 into an LB culture medium for rejuvenation;
and 5, adding the aerobic denitrifying bacteria strain obtained in the step 4 into the HM culture medium obtained in the step 1 for culture to obtain the enhanced aerobic denitrifying bacteria.
2. The method for enhancing the film forming capability of aerobic denitrifying bacteria as claimed in claim 1, wherein: the LB culture medium comprises tryptone, yeast extract and NaCl in a ratio of 2:1: 2.
3. The method for enhancing the film forming capability of aerobic denitrifying bacteria as claimed in claim 2, wherein: the pH value of the LB culture medium is 7.2-7.8.
4. The method for enhancing the film forming capability of aerobic denitrifying bacteria as claimed in claim 2, wherein: further comprises a step of sterilizing the LB culture medium, wherein the LB culture medium is sterilized for more than 25min by high-pressure steam at the temperature of not less than 120 ℃.
5. The method for enhancing the film forming capability of aerobic denitrifying bacteria as claimed in claim 1, wherein: the HM culture medium comprises potassium nitrate, glucose, disodium hydrogen phosphate dodecahydrate, sodium dihydrogen phosphate dihydrate, sodium chloride and a trace element solution in a ratio of 0.72:3:4.78:1.24: 4; the trace element solution comprises MgSO4·7H2O、MnSO4·H2O、H3BO3、ZnSO4·7H2O、FeSO4·7H2O and CaCl2。
6. The method for enhancing the film forming capability of aerobic denitrifying bacteria as claimed in claim 5, wherein: and a glucose sterilization step, wherein the glucose is prepared into a solution and is subjected to high-pressure steam sterilization at the temperature of not less than 110 ℃ for more than 15 min.
7. The method for enhancing the film forming capability of aerobic denitrifying bacteria as claimed in claim 6, wherein: further comprises a step of sterilizing the HM culture medium except glucose, wherein the HM culture medium is sterilized by high-pressure steam at a temperature of not less than 120 ℃ for more than 25 min.
8. The method for enhancing the film forming capability of aerobic denitrifying bacteria as claimed in claim 1, wherein: and 6, measuring the film forming capability of the enhanced aerobic denitrifying bacteria by adopting a crystal violet dyeing method after the step 5 is finished.
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