Screening of Denitrifying Bacteria and Study on Culture Conditions
2024-06-14 06:07:34
Microorganisms play an important role in the natural nitrogen cycle, such as nitrogen fixation, ammoniation, nitrification, denitrification. Among them, nitrification and denitrification maintain the balance of ammonia in nature and the normal circulation of nitrogen. Ammoniaization is the decomposition of organic nitrogen into ammonia and ammonia compounds by the action of ammoniated bacteria or fungi. Nitrification oxidizes ammonia compounds to nitrites and nitrates by nitrite bacteria and nitrate bacteria; denitrification is also called Nitrogen acts to reduce nitrate to nitrous oxide or nitrogen by microorganisms under anaerobic or microanaerobic conditions. Denitrification has great value in controlling eutrophication of water bodies, treating sewage and purifying waters. Certain plants, fungi and bacteria have been found to have denitrification. More than 50 microorganisms are known to be capable of denitrification, the vast majority of which are bacteria.
Denitrifying bacteria reduce nitrate and nitrite by inducing nitrate reductase and nitrite reductase. The denitrification ability of different denitrifying bacteria is different, and the growth conditions are also very different. The authors selected 45 strains of denitrifying bacteria from the physiological and biochemical laboratory of Yuxi Hongta Group Technology Center to further screen the strains with strong denitrification ability, and optimized the medium and growth temperature of the target strain. Aerobic measurements are made to provide a basis for the use of denitrifying bacteria.
l Materials and methods
1.1 Materials
1.1.2 medium and preparation
1.1.2.1 slant medium
Beef extract peptone medium.
1.1.2.2 Giltay medium
Solution A: KNO 3 1.0g, asparagine 1.0 g, 1% BTB alcohol solution 5 mL, distilled water 500 mL.
Solution B: Sodium citrate 8.5 g, MgSO 4 · 7 H 2 O 1.0 g , F e Cl 3 ·6 H 2 O 0.05 g , KH 2 PO 4 1.0 g , CaC1 2 · 2 H 2 O 0.2 g , distilled water 500 mL.
Mix the A and B solutions to adjust the p H value to 7.0-7.2, and fill them into 250 mL flasks, 150 mL each, and sterilize for use.
1.1.2.3 Medium to be sieved
Nitrate medium: beef extract 3g, peptone 5 g, KNO 3 (NaNO 3 ) 1 g , distilled water 1000m L
Denitrifying bacteria culture medium: KNO 3 2 g, MgSO 4 ·7 H 2 O 0.2 g, K 2 HPO 4 0.5 g, potassium sodium tartrate 20 g, distilled water 1000 mL.
YB medium.
Adjust the pH of the medium to be sieved and the YB medium to 7.2, and respectively fill the 250 mL flasks, 150 mL per bottle, and sterilize for use.
1.1.3 Instruments and equipment
Automatic chemical analyzer, CO 2 incubator, blood cell counting plate, shaker.
1.2 method
1.2.1 Determination of the ability of the strain to denitrify
1.2.1.1 Determination of gas production characteristics
Determination of gas production characteristics of denitrifying strains by reference to the Giltay method:
The prepared Giltay liquid medium was added to a large test tube (20 mm × 200 mm), and a small tube (12 mm × 75 mm) wrapped with a thin wire was placed upside down in a large test tube, and the gas in the small test tube was drained and stoppered. Live a large test tube, leaving some thin lines outside the large test tube, easy to lift and drop the small test tube, and sterilize for use.
The activated strain was washed with 2 mL of Giltay liquid medium, transferred to a large test tube containing a small test tube and Gi ltay liquid medium, and the small tube was lifted a distance to receive the gas, so be careful not to make the tube inside the tube. Enter the gas. Each strain was connected to 4 tubes, and cultured at 30 ° C for 10 to 14 days, and the results were observed. Repeat 3 times.
1.2.1.2 Determination of nitrate degradation ability
The medium filter paper in the gas-generating test tube was filtered, and the filtrate was taken, diluted 100 times with distilled water, and the nitrogen content in the medium was measured by an automatic chemical analyzer to determine the denitrification ability of the strain, which was repeated 3 times. The sterilized medium was used as a control.
1.2.2 suitable medium for the strain and determination of aerobicity
The selected strains were cultured on a slope at 30 ° C for 1 d, washed with 10 ml of sterile water, and shaken and mixed. Transfer 1 mL of the washed bacterial solution to the sterilized nitrate medium, denitrifying bacteria selection medium, and YB medium, respectively. Each treatment was shaken for 1 d at 30 ° C in a shaker and CO 2 incubator (20% CO 2 concentration). The optimum medium and aerobic properties of the selected strains were determined by dilution plate method. Repeat 3 times.
1.2.3 Determination of growth temperature
Five kinds of temperature gradients were set at 20 °C, 25 °C, 30 °C, 35 °C, 40 °C, and cultured in 30 ° CYB medium for 1 d. The optimum temperature for strain growth was determined by dilution plate method.
1.2.4 Determination of bacterial concentration
Seven time gradients, such as 18, 21, 24, 27, 30, and 33 h, were cultured in 30° CYB medium, and the highest concentration of the bacteria in the optimal growth conditions was determined by blood cell counting method.
2 Results and analysis
Screening of 2.1 Denitrification Strength of Strain
2.1.1 Determination of gas production
In the test tube inoculated with B88 and B237 strains, the medium began to turn blue after 2 days, and a small amount of gas was produced, which was retained in the small test tube. After 3 days, the medium changed from green to blue, and the gas in the small test tube increased significantly. After 3 to 4 days, the gas production rate is the fastest, and the gas volume is the highest. In the next few days, the gas volume increase is not observed. In the test tube inoculated with other strains, the color of the medium gradually turned blue from top to bottom, but did not produce gas.
The strain was screened by Giltay medium method. The medium in the test tube was reduced to nitrite by the action of denitrifying bacteria nitrate reductase, and the medium was made alkaline, and the medium color changed from green to blue. The strain with strong denitrification is further reduced to nitrous oxide or nitrogen by the action of nitrite reductase, and is retained in a small test tube. Among the 45 tested strains, B88 and B237 strains were able to degrade the nitrate and nitrite in the medium well, producing gas early and fast, and the gas production reached a peak at 3 to 4 days, indicating that the denitrification was higher. Strong. Although other strains can also reduce nitrate to nitrite, but can not further reduce nitrite, denitrification is weak.
2.1.2 Determination of nitrate degradation characteristics In the medium inoculated with B88 and B237, the nitrogen content decreased significantly, indicating that B88 and B237 strains have strong denitrification and can effectively degrade the nitrate in the medium. Table 1 .
Table 1 Determination of the effect of strain denitrification (r=0.99921, 100 mg·L -1 )
project | B88 | B237 | CK | ||||||
1 | 2 | 3 | 1 | 2 | 3 | 1 | 2 | 3 | |
Nitrogen content% | 0.0015 | 0.001 | 0.001 | 0.001 | 0.001 | 0.0015 | 0.065 | 0.068 | 0.070 |
average value% | 0.0013 | 0.0013 | 0.0677 | ||||||
2.2 medium and aerobic determination
Since denitrifying bacteria are denitrifying by nitrate-induced nitrate reductase and nitrite reductase under anaerobic or microanaerobic conditions, most researchers invest in denitrifying bacteria in the laboratory. It is carried out under microanaerobic conditions. However, the results of this experiment show that the rapid growth of B88 and B237 strains is aerobic, with or without the induction of nitrate. The growth is limited by the oxygen supply, and the CO 2 content (20%) is increased. Strong inhibition; the growth rate of the medium is different on different media; the growth rate is slower on the denitrifying bacteria medium, and the growth rate is faster in the YB medium, Table 2 and Table 3.
Table 2 Effect of different media on the growth of B 8 8 strain in incubator and CO 2 incubator
Training environment | Measurement item | Nitrate medium | Denitrifying bacterial culture medium | YB medium |
temperate box | Dilution factor | 10 5 | 10 3 | 10 5 |
Bacterial concentration / one mL -1 | 3.2315×10 7 | 3.6×10 5 | 10.96×10 8 | |
CO 2 incubator | Dilution factor | 10 5 | 10 3 | 10 3 |
Bacterial concentration / one mL -1 | 1.6×10 7 | 1.0×10 4 | 9.485×10 5 |
Table 2 Effect of different media on the growth of B237 strain in incubator and CO 2 incubator
Training environment | Measurement item | Nitrate medium | Denitrifying bacterial culture medium | YB medium |
temperate box | Dilution factor | 10 5 | 10 4 | 10 5 |
Bacterial concentration / one mL -1 | 3.2×10 7 | 4.98×10 6 | 9.2×10 7 | |
CO 2 incubator | Dilution factor | 10 5 | 10 5 | 10 4 |
Bacterial concentration / one mL -1 | 8.4×10 6 | 7.6×10 6 | 4.4532×10 6 |
2 . 3 growth temperature determination
The temperature has a significant effect on the growth rate of B88 and B237 strains. Under aerobic conditions, the strain is cultured in YB medium, and the strain can grow well between 20 °C and 40 °C. The optimal growth temperature is about 30 °C. .
2.4 Determination of growth concentration
After the denitrifying bacteria have undergone a slow growth period and an exponential growth period, the bacterial concentration is gradually reduced due to the consumption of nutrients and the inhibition between bacteria, the aging and digestion of the bacteria. B88 strains under optimal culture conditions for growth of the highest concentration of 6.1 × 1 0 th · mL -1, B237 strains under optimal culture conditions for growth of the highest concentration of 1.54 × 10 8 th · mL -1.
3 Conclusions and discussion
Denitrifying bacteria are mainly used in industrial wastewater or water treatment of polluted rivers and ponds to reduce harmful nitrogenous substances in water and improve water quality. For the culture of denitrifying bacteria, a dedicated denitrifying bacteria culture medium is generally used. However, different denitrifying bacteria have different aerobic properties, and different carbon sources, pH values, temperatures, and medium components have a great influence on the growth of denitrifying bacteria. In the commonly used denitrifying bacteria culture medium, screening the optimal growth medium is of great significance for the enrichment culture and industrial application of bacteria.
The conventional Giltay medium method was used to screen denitrifying bacteria using a test tube of 15 mm × 150 mm and a Duham tube, so that the amount of gas retained in the Duham tube was small and it was not easy to collect. This test uses a large test tube of 20 mm × 200 mm, replaces the Duham tube with a small test tube of 12 mm × 75 mm, and suspends the small test tube with a thin wire, which is beneficial to retain more gas and is easy to collect. It is more conducive to the detection of gas components. Two strains of B88 and B237 strains with strong denitrification were screened out from 45 denitrifying bacteria by conventional methods. YB medium suitable for strain growth was screened from several commonly used denitrifying bacteria culture media, and determined. The optimum growth temperature of the strain was 30 °C, and the highest concentration of the strain in YB medium: B 8 8 was 6.1 × 10 8 · mL -1 ; B237 was 1.54 × 10 8 · mL -1 .
The identification of B88 and B237 strains and their industrial application are for further study.
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