Method for selectively and efficiently inhibiting reducing activity of sulfate reducing bacteria

Abstract

The invention discloses a method for selectively and efficiently inhibiting the reducing activity of sulfate reducing bacteria, and relates to a method for inhibiting the sulfate reducing activity of the sulfate reducing bacteria. The problems that an existing sulfate reduction inhibitor is poor in effect and large in influence on the environment are solved. According to the invention, a selective sulfate reduction inhibitor is added into a system containing sulfate reducing bacteria, and the selective sulfate reduction inhibitor is used for inhibiting the growth of the sulfate reducing bacteria, so that the sulfate reduction activity of the sulfate reducing bacteria is inhibited; the selective sulfate reduction inhibitor is neutral red. According to the invention, the neutral red with a micromole-level addition amount is added as a selective efficient sulfate reduction inhibitor, so that the growth of sulfate reducing bacteria is efficiently inhibited, the growth of the sulfate reducing bacteria and the sulfate reduction activity are remarkably inhibited, and no negative effect is generated on non-sulfate reducing bacteria in a system. And the half inhibition concentration of the neutral red to common sulfate reducing bacteria in the environment is only micromole level, so that the influence on the environment is small.

Description

Technical Field

[0001] The present invention relates to a method for inhibiting the sulfate reducing activity of sulfate reducing bacteria. Background Art

[0002] Sulfate-reducing bacteria are widely present in anoxic environments. They use sulfate as the terminal electron acceptor to oxidize organic compounds or hydrogen, and reduce sulfate to sulfide. In industrial production, such as oil extraction and biogas fermentation, sulfate reduction will produce highly active, corrosive, toxic and strongly pungent sulfides, which will have a serious impact on equipment, environment, economy, health of employees and methane production.

[0003] Adding non-selective broad-spectrum sulfate reduction inhibitors such as formaldehyde and glutaraldehyde to the system will affect the growth and activity of various microorganisms, and after stopping the addition, the sulfate-reducing bacteria in the system quickly recover their growth ability and carry out sulfate reduction activities; when adding selective sulfate reduction inhibitors including inorganic oxygen-containing anions, they show weak sulfate reduction inhibition ability. Therefore, it is of great significance to develop a sulfate-reducing bacteria inhibitor that is efficient, selective, and has little impact on the environment. Summary of the invention

[0004] In order to solve the problems of poor effect and great environmental impact of existing sulfate reduction inhibitors, the present invention provides a method for selectively and efficiently inhibiting the reduction activity of sulfate-reducing bacteria.

[0005] The method for selectively and efficiently inhibiting sulfate reduction of the present invention is carried out according to the following steps: adding a selective sulfate reduction inhibitor to a system containing sulfate-reducing bacteria, using the selective sulfate reduction inhibitor to inhibit the growth of sulfate-reducing bacteria, and then inhibiting the sulfate reduction activity of the sulfate-reducing bacteria; the selective sulfate reduction inhibitor is neutral red.

[0006] The beneficial effects of the present invention are:

[0007] The present invention is directed to a system containing sulfate-reducing bacteria, and by adding a micromolar amount of neutral red as a selective and efficient sulfate-reducing inhibitor, the growth of sulfate-reducing bacteria is efficiently inhibited, and the growth and sulfate-reducing activity of sulfate-reducing bacteria are significantly inhibited, while having no negative effect on non-sulfate-reducing bacteria in the system. Moreover, the half-inhibitory concentration of neutral red on common sulfate-reducing bacteria in the environment is only at the micromolar level, and the impact on the environment is small.

[0008] After sulfate-reducing bacteria were exposed to neutral red, the transcription levels of genes encoding sulfate reductase in sulfate-reducing bacteria were significantly downregulated, so the central pathway of inhibiting sulfate reduction is one of the mechanisms by which neutral red inhibits the growth of sulfate-reducing bacteria. In addition, the transcription levels of electron transfer-related genes, ATP synthase, and cytoplasmic / periplasmic hydrogenase-related genes in the bacteria were downregulated, indicating the collapse of the intracellular proton gradient of sulfate-reducing bacteria. These findings are of great significance for the environmental control of sulfate-reducing bacteria and may improve the economic benefits of industrial operations. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 This is a curve diagram showing the effect of neutral red on the growth of sulfate-reducing bacteria enrichment and the sulfate reduction process;

[0010] Figure 2 This is a curve diagram showing the effect of neutral red on the growth of pure sulfate-reducing bacteria;

[0011] Figure 3 This is a curve diagram showing the effect of neutral red on the growth of non-sulfate-reducing bacteria (pure nitrate-reducing bacteria);

[0012] Figure 4 The transcriptomic analysis diagram of Desulfotomaculumacetoxidans DSM771 and Desulfovibriodesulfuricans DSM642; DETAILED DESCRIPTION

[0013] The technical solution of the present invention is not limited to the specific implementation modes listed below, but also includes any reasonable combination of the specific implementation modes.

[0014] Specific implementation method 1: The method for selectively and efficiently inhibiting the reduction activity of sulfate-reducing bacteria in this implementation method is carried out according to the following steps: adding a selective sulfate reduction inhibitor to a system containing sulfate-reducing bacteria, using the selective sulfate reduction inhibitor to inhibit the growth of sulfate-reducing bacteria, and then inhibiting the sulfate reduction activity of the sulfate-reducing bacteria; the selective sulfate reduction inhibitor is neutral red.

[0015] This implementation has the following beneficial effects:

[0016] Specific embodiment 2: This embodiment is different from specific embodiment 1 in that the concentration of the selective sulfate reduction inhibitor added to the system containing sulfate-reducing bacteria is 0.5-3.0 μmol / L.

[0017] Specific embodiment three: This embodiment is different from specific embodiment one or two in that the sulfate-reducing bacteria in the system containing sulfate-reducing bacteria are Desulfotomaculumacetoxidans DSM 771, Desulfovibriodesulfuricans DSM 642, Desulfomicrobium baculatum DSM 4028, or Desulfovibriovulgaris DP4CGMCC 1.5190.

[0018] Specific implementation method 4: This implementation method is different from any one of specific implementation methods 1 to 3 in that the system containing sulfate-reducing bacteria is a solution containing sulfate-reducing bacteria, sewage containing sulfate-reducing bacteria, or sludge containing sulfate-reducing bacteria.

[0019] Specific embodiment 5: This embodiment is different from specific embodiments 1 to 4 in that the temperature of the system containing sulfate-reducing bacteria is 30-37°C.

[0020] Specific embodiment 6: This embodiment is different from any one of specific embodiments 1 to 5 in that: the concentration of the selective sulfate reduction inhibitor added to the system containing sulfate-reducing bacteria is 3.0 μmol / L.

[0021] Specific embodiment 7: This embodiment is different from any one of specific embodiments 1 to 6 in that: the concentration of the selective sulfate reduction inhibitor added to the system containing sulfate-reducing bacteria is 0.5 μmol / L.

[0022] Specific embodiment eight: This embodiment differs from any one of specific embodiments one to seven in that: the concentration of the selective sulfate reduction inhibitor added to the system containing sulfate-reducing bacteria is 1.0 μmol / L.

[0023] Specific embodiment 9: This embodiment is different from any one of specific embodiments 1 to 8 in that: the concentration of the selective sulfate reduction inhibitor added to the system containing sulfate-reducing bacteria is 1.5 μmol / L.

[0024] Specific embodiment ten: This embodiment differs from any one of specific embodiments one to nine in that: the concentration of the selective sulfate reduction inhibitor added to the system containing sulfate-reducing bacteria is 2.0 μmol / L.

[0025] Verification test:

[0026] 1. Four representative pure sulfate-reducing bacteria and a sulfate-reducing bacteria enrichment with sulfate-reducing function were inoculated into sulfate-reducing medium to obtain five bacterial liquids;

[0027] The representative sulfate-reducing pure bacteria are DesulfotomaculumacetoxidansDSM 771, Desulfovibrio desulfuricansDSM642, Desulfomicrobium baculatumDSM4028, and Desulfovibrio vulgarisDP4CGMCC1.5190;

[0028] The sulfate-reducing bacteria concentrate is taken from the expanded granular sludge bed and can stably reduce sulfate to sulfide;

[0029] The sulfate reduction medium includes Na2SO4 and yeast extract, the concentration of Na2SO4 is 14.1-20mmol / L, and the concentration of yeast extract is 1-3g / L; the sulfate reduction medium is suitable for the growth and culture of various pure bacteria and activated sludge enrichment;

[0030] 2. Centrifuge the five bacterial solutions obtained in step 1 respectively, remove the supernatant, wash the solid products obtained by centrifugation with PBS buffer solution, remove the yeast extract remaining in the bacterial solution, and obtain bacterial cells without culture medium components;

[0031] 3. Add neutral red, a highly efficient and selective sulfate reduction inhibitor, to the new sulfate reduction medium, and then inoculate the five bacteria obtained in step 2 respectively. The initial inoculation OD 600 The OD values ​​were all 0.02. The culture was carried out in a shaker at 30-37°C and 160 rpm. After 48 h, the OD values ​​were measured by UV spectrophotometer. 600 value and sulfide production; the new sulfate-reducing medium has a Na2SO4 concentration of 14.1-20mmol / L and does not contain yeast extract;

[0032] The concentration of neutral red in the culture medium was 0.0 μmol / L, 0.5 μmol / L, 1.0 μmol / L, 1.5 μmol / L, 2.0 μmol / L, 2.5 μmol / L, and 3.0 μmol / L;

[0033] 4. Inoculate the nitrate-reducing pure bacteria into LB medium to obtain a bacterial solution; the nitrate-reducing pure bacteria is Paracoccus denitrificans ATCC 19367;

[0034] 5. Add neutral red inhibitor to the bacterial solution in step 4. The neutral red is added at the midpoint of the logarithmic growth phase of the inoculated strain in the culture medium. Cultivate in a shaker at 30°C and 160 rpm. After 48 hours, measure the OD using a UV spectrophotometer. 600 value;

[0035] The concentration of neutral red in the culture medium was 0 μmol / L, 20 μmol / L, 40 μmol / L, 60 μmol / L, 80 μmol / L, and 100 μmol / L;

[0036] The inhibition of neutral red on bacterial growth and sulfate reduction activity was recorded. The inhibitory efficacy was evaluated by comparing the half-inhibitory concentration (IC50) of neutral red on bacterial growth and sulfate reduction. 50 ).

[0037] Figure 1 The figure is a curve diagram of the effect of neutral red on the growth of sulfate-reducing bacteria enrichment and sulfate reduction process. In the enrichment, the half-inhibitory concentration of the sulfate reduction process is 14.2μM, and the half-inhibitory concentration of microbial growth exceeds 0.75mM. After calculation, the selectivity index of neutral red in inhibiting the sulfate reduction process is 52.8. Figure 2 This is a curve chart showing the effect of neutral red on the growth of pure sulfate-reducing bacteria; at 48 hours, the half-inhibitory concentration of neutral red on the growth of each sulfate-reducing bacteria is: 8.2 μmol / L for D. acetoxidans, 10.3 μmol / L for D. desulfuricans, 4.0 μmol / L for D. baculatum, and 38.0 μmol / L for D. vulgaris DP4. Figure 3 The figure is a graph showing the effect of neutral red on the growth of non-sulfate-reducing bacteria (nitrate-reducing pure bacteria). It was found that the growth of nitrate-reducing pure bacteria was not affected by neutral red. Further, in order to explore the principle of neutral red's selective inhibition of sulfate-reducing bacteria, transcriptomic analysis was performed on Desulfotomaculum acetoxidans DSM771 and Desulfovibriodesulfuricans DSM642. As shown in the figure, when the two strains of sulfate-reducing bacteria were exposed to the neutral red environment, the transcription levels of the genes encoding sulfate reductase (Sat, AprAB and DsrAB) in the bacteria were significantly downregulated. Therefore, the central pathway of inhibiting sulfate reduction is one of the mechanisms by which neutral red inhibits the growth of sulfate-reducing bacteria. In addition, when the two strains of sulfate-reducing bacteria were exposed to the neutral red environment, the electron transfer-related genes (QmoABC and DsrMKJOP), ATP synthase and cytoplasmic / periplasmic hydrogenase-related genes in the bacteria were downregulated, indicating the collapse of the intracellular proton gradient of sulfate-reducing bacteria.

Claims

1. A method for selectively and efficiently inhibiting the reducing activity of sulfate-reducing bacteria, characterized in that: The method for selectively and efficiently inhibiting the reduction activity of sulfate-reducing bacteria is carried out according to the following steps: adding a selective sulfate reduction inhibitor to a system containing sulfate-reducing bacteria, using the selective sulfate reduction inhibitor to inhibit the growth of sulfate-reducing bacteria and inhibit the sulfate reduction activity of the sulfate-reducing bacteria; the selective sulfate reduction inhibitor is neutral red.

2. The method for selectively and efficiently inhibiting the reducing activity of sulfate-reducing bacteria according to claim 1, characterized in that: The concentration of the selective sulfate reduction inhibitor added to the system containing sulfate reducing bacteria is 0.5-3.0 μmol / L.

3. The method for selectively and efficiently inhibiting the reducing activity of sulfate-reducing bacteria according to claim 1, characterized in that: The sulfate-reducing bacteria in the system containing sulfate-reducing bacteria are Desulfotomaculumacetoxidans DSM771, Desulfovibriodesulfuricans DSM642, Desulfomicrobium baculatum DSM 4028 or Desulfovibrio vulgaris DP4 CGMCC 1.5190.

4. The method for selectively and efficiently inhibiting the reducing activity of sulfate-reducing bacteria according to claim 1, characterized in that: The system containing sulfate-reducing bacteria is a solution containing sulfate-reducing bacteria, sewage containing sulfate-reducing bacteria, or sludge containing sulfate-reducing bacteria.

5. The method for selectively and efficiently inhibiting the reducing activity of sulfate-reducing bacteria according to claim 1, characterized in that: The temperature of the system containing sulfate-reducing bacteria is 30-37°C.

6. The method for selectively and efficiently inhibiting the reducing activity of sulfate-reducing bacteria according to claim 2, characterized in that: The concentration of the selective sulfate reduction inhibitor added to the system containing sulfate-reducing bacteria is 3.0 μmol / L.

7. The method for selectively and efficiently inhibiting the reducing activity of sulfate-reducing bacteria according to claim 2, characterized in that: The concentration of the selective sulfate reduction inhibitor added to the system containing sulfate-reducing bacteria is 0.5 μmol / L.

8. The method for selectively and efficiently inhibiting the reducing activity of sulfate-reducing bacteria according to claim 2, characterized in that: The concentration of the selective sulfate reduction inhibitor added to the system containing sulfate-reducing bacteria is 1.0 μmol / L.

9. The method for selectively and efficiently inhibiting the reducing activity of sulfate-reducing bacteria according to claim 2, characterized in that: The concentration of the selective sulfate reduction inhibitor added to the system containing sulfate-reducing bacteria is 1.5 μmol / L.

10. The method for selectively and efficiently inhibiting the reducing activity of sulfate-reducing bacteria according to claim 2, characterized in that: The concentration of the selective sulfate reduction inhibitor added to the system containing sulfate-reducing bacteria is 2.0 μmol / L.

Images