Determination method of Thiobacillus ferrooxidans density in sewage sludge and its application

Abstract

The present invention belongs to the field of environmental detection technology, and relates to a method for determining the density of Thiobacillus ferrooxidans in sludge and its application. It is aimed at the technical problem that the operation of detecting the density of Thiobacillus ferrooxidans in the prior art is cumbersome and time-consuming. The present application provides a method for determining the density of Thiobacillus ferrooxidans in sludge and its application, comprising the following steps: using iron salts and Thiobacillus ferrooxidans model bacteria to determine the linear relationship between the ferrous oxidation rate and the bacterial density of the Thiobacillus ferrooxidans model bacteria; determining the oxidation rate of ferrous iron in the sample to be tested to obtain the ferrous oxidation rate of the sample to be tested; substituting the ferrous oxidation rate of the sample to be tested into the linear relationship to obtain the density of the Thiobacillus ferrooxidans. The present application uses the quantitative relationship between the ferrous oxidation rate and its bacterial density to quickly determine the density of Thiobacillus ferrooxidans in the sample, thereby shortening the detection time. The present application also provides the application of the method for determining the density of Thiobacillus ferrooxidans in sludge.

Description

Technical Field

[0001] The invention belongs to the technical field of environmental detection, and in particular relates to a method for determining the density of Thiobacillus ferrooxidans in sludge and an application thereof. Background Art

[0002] Bioleaching is an effective sludge conditioning method that improves the dewatering performance of sludge by utilizing the metabolic activities of microorganisms such as Acidithiobacillus ferrooxidans. Acidithiobacillus ferrooxidans is a Gram-negative bacterium that can oxidize ferrous iron (Fe) at low pH (2.0-3.0) and an optimal growth temperature of 30°C. 2+ ) to obtain energy and synthesize necessary organic matter. The ferrous iron oxidation ability of this bacterium is a necessary condition for microbial leaching. In-depth research on its ferrous iron oxidation-related genes and their functions will help improve its ferrous iron oxidation ability and make it better applied in actual production. The bacterial density of Acidithiobacillus ferrooxidans has a significant effect on the bioleaching effect. During the bioleaching process, the bacteria completely replaced the activated sludge cells in the original sludge, and the final density reached about 10 8 cells·mL -1 The replacement effect of bacteria includes releasing interstitial water, reducing capillary water and surface bound water, thereby improving the dewatering performance of sludge. Studies have shown that after bioleaching, the centrifugal dewatering rate of sludge can be increased from 60% of the original sludge to 74.4%, and the sludge specific resistance can be increased from 1.83×10 9 s 2 ·g -1 Down to 0.39×10 9 s 2 ·g -1 , decreased by 79%, and the compression coefficient also decreased from 0.98 of the original sludge to 0.58. The bacterial density of Acidithiobacillus ferrooxidans is an important parameter in the bioleaching process because it directly affects the effect of bioleaching. In addition, changes in sludge pH, Fe 2+ and Fe 3+ The concentration, Zeta potential of sludge particles and changes in the particle size distribution of sludge particles are also important factors affecting the bioleaching effect.

[0003] The traditional method for detecting the density of acidophilic ferrooxidans is the double-layer plate counting method, such as the Chinese invention patent publication number CN1746315A, the application date is September 19, 2005, and the invention name is "A method for improving the detection rate of chemoautotrophic thiobacillus plate", which discloses the following operation method: 2% water agar is used as the bottom plate, yeast Rhodotorula sp. R30 bacterial solution is applied on it, and then the solid culture medium for separating acidophilic ferrooxidans is poured into it as the upper plate, and a suspension of thiobacillus containing a certain dilution is applied, and the plate is inverted and cultured at 30°C until the number of colonies no longer increases. Finally, the bacterial density of the bacteria in the sample is calculated by the number of colonies and the dilution multiple. However, this method is not only cumbersome to operate but also usually requires 11 days of culture time, which makes it unable to meet the needs of bioleaching process commissioning and monitoring. Therefore, it is urgent to invent a method for rapidly detecting the density of acidophilic ferrooxidans. Summary of the invention

[0004] 1. Problems to be solved

[0005] Aiming at the technical problem that the detection of the density of Thiobacillus ferrooxidans in the prior art is complicated and time-consuming, the present application provides a method for determining the density of Thiobacillus ferrooxidans in sludge. By determining the linear relationship between the ferrous oxidation rate and the density of the model bacteria of Thiobacillus ferrooxidans, the density of Acidithiobacillus ferrooxidans in the bioleaching sludge is quickly determined, thereby shortening the detection time. The present application also provides an application of the method for determining the density of Thiobacillus ferrooxidans in sludge.

[0006] 2. Technical solution

[0007] In order to achieve the above purpose, the technical solution provided is:

[0008] The method for determining the density of Thiobacillus ferrooxidans in sludge comprises the following steps:

[0009] S1. The linear relationship between the ferrous oxidation rate and the density of the model bacteria of Thiobacillus ferrooxidans was determined using iron salts and the model bacteria of Thiobacillus ferrooxidans.

[0010] S2. Determine the oxidation rate of ferrous iron in the sample to be tested to obtain the oxidation rate of ferrous iron in the sample to be tested;

[0011] S3. Substitute the ferrous oxidation rate of the sample to be tested into the linear relationship to obtain the density of the Thiobacillus ferrooxidans.

[0012] Furthermore, the iron salt is one or more of anhydrous ferrous sulfate, ferrous sulfate monohydrate, ferrous sulfate tetrahydrate, ferrous sulfate pentahydrate or ferrous sulfate heptahydrate.

[0013] Furthermore, the linear relationship is determined as follows:

[0014] The model bacteria of Thiobacillus ferrooxidans with different gradient bacterial densities are cultured in a basic culture medium, and the ferrous oxidation rates of the model bacteria of Thiobacillus ferrooxidans with different gradient bacterial densities are measured respectively to obtain the ferrous oxidation rates;

[0015] The linear relationship is obtained by taking the ferrous oxidation rate as the abscissa and the different gradient bacteria densities as the ordinate.

[0016] Preferably, the different gradient bacteria density is 5 gradients, which are 2.6×10 6 CFU / mL, 1.3×10 7 CFU / mL, 5.2×10 7 CFU / mL, 2.1×10 8 CFU / mL, 8.2×10 8 CFU / mL.

[0017] Furthermore, the basal salt culture medium contains: (NH4)2SO4 3.5g / L, KCl 0.119g / L, K2HPO4·3H2O0.058g / L, MgSO4·7H2O 0.583g / L, Ca(NO3)2·4H2O 0.017g / L, FeSO4.7H2O 2.5-3.0g / L; and the pH value is 2.5-3.0.

[0018] Preferably, the pH value is adjusted using 50% (v / v) dilute sulfuric acid.

[0019] Preferably, the culture conditions are: culture in a shaking table, 28° C., shaking at 180 rpm for 2 h, and measuring the water-soluble ferrous iron concentration every 0.5 h.

[0020] Furthermore, in step S2, a modified culture medium is used: (NH4)2SO4 35 g / L, KCl 1.19 g / L, K2HPO4·3H2O 0.58 g / L, MgSO4·7H2O 5.83 g / L, and Ca(NO3)2·4H2O 0.17 g / L.

[0021] Preferably, the culture conditions are: culture in a shaking table, 28° C., shaking at 180 rpm for 2 h, and measuring the water-soluble ferrous iron concentration every 0.5 h.

[0022] Further, in step S2: the volume ratio of the improved culture medium, dilute sulfuric acid with a pH value of 2.5 to 3.0 and 25 to 30 g / LFeSO4.7H2O is 1:3:1.

[0023] Furthermore, the ferrous iron oxidation rate is the slope of the linear relationship equation between the ferrous iron oxidation amount and the culture time; the ferrous iron oxidation amount is the difference between the initial ferrous iron concentration and the ferrous iron concentration at the detection time point.

[0024] Furthermore, it is characterized in that: the ferrous iron concentration is the water-soluble ferrous iron concentration; and the determination method adopts the o-phenanthroline colorimetric method.

[0025] Furthermore, the Thiobacillus ferrooxidans model bacteria is Thiobacillus ferrooxidans LX5 with a preservation number of CGMCCNO.0727, or the Thiobacillus ferrooxidans model bacteria is separated from the sample to be tested.

[0026] The invention discloses an application of a method for determining the density of Thiobacillus ferrooxidans in sewage sludge, and the application of the method in determining the density of Thiobacillus ferrooxidans in biological leaching sludge.

[0027] Thiobacillus ferrooxidans is Thiobacillus ferrooxidans LX5 (A.ferrooxidansLX5, deposit number is CGMCCNO.0727, deposit time: March 13, 2002, classification name: Thiobacillus ferrooxidans, deposit unit: General Microbiology Center of China Microbiological Culture Collection Administration, deposit address: Institute of Microbiology, Chinese Academy of Sciences, Datun Road, Chaoyang District, Beijing), see Chinese invention patent publication number CN1375553A for details.

[0028] 3. Beneficial effects

[0029] Compared with the existing known technology, the technical solution provided by the present invention has the following beneficial effects:

[0030] (1) The method for determining the density of Acidithiobacillus ferrooxidans in sludge of the present invention uses iron salts and Acidithiobacillus ferrooxidans model bacteria to determine the linear relationship between the ferrous oxidation rate and the density of the Acidithiobacillus ferrooxidans model bacteria; determines the oxidation rate of ferrous iron in the sample to be tested to obtain the ferrous oxidation rate of the sample to be tested; substitutes the ferrous oxidation rate of the sample to be tested into the linear relationship to obtain the density of Acidithiobacillus ferrooxidans. The method uses the quantitative relationship between the ferrous oxidation rate and its bacterial density to quickly determine the density of Acidithiobacillus ferrooxidans in bioleaching sludge, and the bacterial density can be measured within 3 hours. Compared with the traditional detection method (double-layer plate counting method), the method shortens the detection time from 11 days to 3 hours. The present invention is simple to operate, and the instruments and reagents involved are generally available, and it has the advantages of being simple, convenient and easy to implement.

[0031] (2) The method for determining the density of Thiobacillus ferrooxidans in sludge of the present invention is applied to determining the density of Thiobacillus ferrooxidans in bioleaching sludge. Since the determination time is short, it meets the needs of bioleaching process debugging and monitoring. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1The linear fitting diagram of the ferrous oxidation amount and the oscillation time in the 2h ferrous oxidation rate test of the model bacteria of Thiobacillus ferrooxidans (a) and the linear fitting diagram of the bacterial density of the model bacteria of Thiobacillus ferrooxidans and the ferrous oxidation rate (b);

[0033] Figure 2 This is the linear fitting diagram of ferrous oxidation amount and oscillation time in the 2h ferrous oxidation rate test of bioleaching sludge sample. DETAILED DESCRIPTION

[0034] In order to further understand the content of the present invention, the present invention is described in detail in conjunction with embodiments.

[0035] Example 1

[0036] The method for determining the density of Thiobacillus ferrooxidans in sludge of this embodiment and its application include the following steps:

[0037] S1. The linear relationship between ferrous iron oxidation rate and the density of T. ferrooxidans model bacteria was determined using iron salts and T. ferrooxidans model bacteria.

[0038] 1. Cultivate the model bacteria of Thiobacillus ferrooxidans with different gradient bacterial densities in the basic culture medium.

[0039] Take 5 250mL conical flasks and prepare 50mL 9K basal salt medium, each medium contains 2.5g / LFeSO4.7H2O, add the suspension of Thiobacillus ferrooxidans cells to dilute the bacterial density in each medium to 2.6×10 6 CFU / mL, 1.3×10 7 CFU / mL, 5.2×10 7 CFU / mL, 2.1×10 8 CFU / mL, 8.2×10 8 CFU / mL;

[0040] 9K basic salt culture medium contains: (NH4)2SO4, 3.5g / L; KCl, 0.119g / L; K2HPO4·3H2O, 0.058g / L; MgSO4·7H2O, 0.583g / L; Ca(NO3)2·4H2O, 0.017g / L; the pH of 9K basic salt culture is 3, adjusted with 50% (v / v) dilute sulfuric acid, and stored at 4°C for future use after preparation.

[0041] 2. Determine the ferrous oxidation rate of the model bacteria of Thiobacillus ferrooxidans at different gradient bacterial densities, and obtain the ferrous oxidation rate of each type.

[0042] The five conical flasks were placed in a shaker at 28°C and 180 rpm for 2 h, and 2 mL of sample was taken every 0.5 h to determine the concentration of water-soluble ferrous iron;

[0043] Calculate the biological oxidation rate of ferrous iron at different gradient bacterial densities. Subtract the water-soluble ferrous iron concentration of the sample from the initial ferrous iron concentration to obtain the ferrous iron oxidation amount (mg / L). The ferrous iron oxidation rate is the slope of the linear fitting equation of the ferrous iron oxidation amount and the oscillation time, as shown in Figure 1 As shown in a, the biological oxidation rate of ferrous iron at different gradient bacterial densities was obtained;

[0044] 3. With the ferrous oxidation rate (mg / L / h) as the abscissa and the different gradient bacterial density (CFU / mL) as the ordinate, a linear relationship was obtained, and the fitting linear equation was y=0.2972x, such as Figure 1 Therefore, we only need to measure the biological oxidation rate of ferrous iron in the future to infer the bacterial density based on this relationship.

[0045] S2. Determine the oxidation rate of ferrous iron in the sample to be tested to obtain the oxidation rate of ferrous iron of the sample to be tested.

[0046] 1. Take 5 mL of 10-fold modified 9K medium (pH 3.0), 15 mL of dilute sulfuric acid with pH 3.0 and 5 mL of 25 g / LFeSO4.7H2O (pH 3.0) solution and add them to a 250 mL conical flask (No. 1 conical flask), and place the conical flask in a water bath to keep the solution temperature at 28°C. The pH is adjusted with 50% (v / v) dilute sulfuric acid.

[0047] The 10-fold modified 9K culture medium contains: (NH4)2SO4, 35g / L; KCl, 1.19g / L; K2HPO4·3H2O, 0.58g / L; MgSO4·7H2O, 5.83g / L; Ca(NO3)2·4H2O, 0.17g / L. The pH of the 10-fold modified 9K culture medium is 3.0, and the pH is adjusted with 50% (v / v) dilute sulfuric acid.

[0048] 2. Take out 25 mL of the bioleaching sludge to be tested and add it to another 250 mL Erlenmeyer flask (No. 2 Erlenmeyer flask). Adjust its pH value to 3.0 with 50% (v / v) dilute sulfuric acid. Then place the Erlenmeyer flask in a water bath to stabilize the sludge temperature at 28°C.

[0049] 3. Add the liquid in the No. 1 flask to the No. 2 flask, mix quickly and take out 2 mL of sample for determination of water-soluble ferrous iron concentration (0h sample ferrous iron concentration), then place the No. 2 flask in a shaker and shake at 28°C and 180 rpm for 2h. Take 2 mL of sample every 0.5h for determination of water-soluble ferrous iron concentration.

[0050] Method for determining the concentration of water-soluble ferrous iron: centrifuge the collected 2 mL sample at 10,000 rpm for 1 minute, filter the supernatant through a 0.45 μm PES filter membrane, add 2 drops of 1:1 (v / v) HCl to the filtrate, and determine the concentration of ferrous iron in the sample using the o-phenanthroline colorimetric method.

[0051] 4. Calculate the ferrous iron oxidation rate (mg / L / h). Subtract the initial ferrous iron concentration (i.e., the ferrous iron concentration of the sample at 0h) from the sample ferrous iron concentration to calculate the ferrous iron oxidation amount (mg / L). Perform a linear fit between the ferrous iron oxidation amount and the oscillation time (e.g., Figure 2 The ferrous oxidation rate (mg / L / h) was obtained by the slope of the linear fitting equation, and its value was 77.79 mg / L / h.

[0052] S3. Substitute the ferrous oxidation rate of the sample to be tested into the linear relationship to obtain the density of the Thiobacillus ferrooxidans.

[0053] The ferrous iron biological oxidation rate (mg / L / h) obtained by S2 was substituted into the linear equation of S1, and the dilution factor was converted to obtain the density of Acidithiobacillus ferrooxidans in the bioleaching sludge sample (CFU / mL).

[0054] The biological oxidation rate of ferrous iron is 77.79 mg / L / h multiplied by 2 (dilution factor) and then multiplied by 0.2972×10 7 The bacterial density (CFU / mL) is: (77.79×2)×0.2972×10 7 =4.62×10 8 CFU / mL.

[0055] In this example, the density of Thiobacillus ferrooxidans in the bioleaching sludge sample was rapidly detected within 3 hours, and the density was 4.62×10 8 CFU / mL.

[0056] Comparison column 1

[0057] The bacterial density of Acidithiobacillus ferrooxidans in bioleached sludge was determined by microscopy and was 4.56×10 8 CFU / mL.

[0058] References for this method:

[0059] Comparative Example 2

[0060] The bacterial density of Acidithiobacillus ferrooxidans in bioleached sludge was determined by plate count method and was 4.58×10 8 CFU / mL.

[0061] Reference for this method: Annika Meyers, Christoph Furtmann, Joachim Jose, Direct optical density determination of bacterial cultures in microplates for high-throughput screening applications, Enzyme Microb Technol. 2018 Nov: 118: 1-5. DOI: 10.1016 / j.enzmictec.2018.06.016. Epub 2018 Jul 3.

[0062] Comparative Examples 1 and 2 respectively measured the bacterial density of Thiobacillus ferrooxidans in bioleaching sludge by microscopy and plate counting methods, which were basically consistent with the bacterial density of Thiobacillus ferrooxidans measured by the method of the present application, indicating that the present application is highly accurate.

[0063] In summary, the present application quickly determines the density of acidophilic ferrooxidans in biological leaching sludge by determining the linear relationship between ferrous oxidation rate and density of model bacteria of Acidithiobacillus ferrooxidans, thereby shortening the detection time and achieving high accuracy, and can be applied to the determination of density of Acidithiobacillus ferrooxidans in biological leaching sludge.

[0064] The above-mentioned embodiments only express the preferred implementation modes of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the present invention. It should be pointed out that, for a person skilled in the art, several modifications, improvements and substitutions can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention patent shall be subject to the attached claims.

Claims

1. A method for determining the density of Thiobacillus ferrooxidans in sludge, characterized in that: The following steps are involved: S1. The linear relationship between the ferrous oxidation rate and the density of the model bacteria of Thiobacillus ferrooxidans was determined using iron salts and the model bacteria of Thiobacillus ferrooxidans. S2. Determine the oxidation rate of ferrous iron in the sample to be tested to obtain the oxidation rate of ferrous iron in the sample to be tested; S3. Substitute the ferrous oxidation rate of the sample to be tested into the linear relationship to obtain the density of the Thiobacillus ferrooxidans.

2. The method for determining the density of Thiobacillus ferrooxidans in sludge according to claim 1, wherein: The iron salt is one or more of anhydrous ferrous sulfate, ferrous sulfate monohydrate, ferrous sulfate tetrahydrate, ferrous sulfate pentahydrate or ferrous sulfate heptahydrate.

3. The method for determining the density of Thiobacillus ferrooxidans in sludge according to claim 1, characterized in that: The linear relationship is determined as follows: The model bacteria of Thiobacillus ferrooxidans with different gradient bacterial densities are cultured in a basic culture medium, and the ferrous oxidation rates of the model bacteria of Thiobacillus ferrooxidans with different gradient bacterial densities are measured respectively to obtain the ferrous oxidation rates; The linear relationship is obtained by taking the ferrous oxidation rate as the abscissa and the different gradient bacteria densities as the ordinate.

4. The method for determining the density of Thiobacillus ferrooxidans in sludge according to claim 3, characterized in that: The basic salt culture medium contains: (NH4)2SO4 3.5g / L, KCl 0.119g / L, K2HPO4·3H2O 0.058g / L, MgSO4·7H2O 0.583g / L, Ca(NO3)2·4H2O 0.017g / L, FeSO4.7H2O 2.5-3.0g / L; and the pH value is 2.5-3.

0.

5. The method for determining the density of Thiobacillus ferrooxidans in sludge according to claim 1, characterized in that: In step S2, a modified culture medium is used: (NH4)2SO4 35 g / L, KCl 1.19 g / L, K2HPO4·3H2O 0.58 g / L, MgSO4·7H2O 5.83 g / L, and Ca(NO3)2·4H2O 0.17 g / L.

6. The method for determining the density of Thiobacillus ferrooxidans in sludge according to claim 5, characterized in that: In step S2: the volume ratio of the modified culture medium, dilute sulfuric acid with a pH value of 2.5 to 3.0 and 25 to 30 g / L FeSO4.7H2O is 1:3:

1.

7. The method for determining the density of Thiobacillus ferrooxidans in sludge according to claim 1, characterized in that: The ferrous iron oxidation rate is the slope of the linear relationship equation between the ferrous iron oxidation amount and the culture time; the ferrous iron oxidation amount is the difference between the initial ferrous iron concentration and the ferrous iron concentration at the detection time point.

8. The method for determining the density of Thiobacillus ferrooxidans in sludge according to any one of claims 1 to 7, characterized in that: The ferrous iron concentration is the water-soluble ferrous iron concentration; the determination method adopts the o-phenanthroline colorimetric method.

9. The method for determining the density of Thiobacillus ferrooxidans in sludge according to claim 8, characterized in that: The model bacterium of Thiobacillus ferrooxidans is Thiobacillus ferrooxidans LX5 with a preservation number of CGMCCNO.0727, or the model bacterium of Thiobacillus ferrooxidans is separated from the sample to be tested.

10. Application of the method for determining the density of Thiobacillus ferrooxidans in sludge, characterized in that: Application of the method according to any one of claims 1 to 9 in determining the density of Thiobacillus ferrooxidans in bioleaching sludge.

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