Method for determining protein content of sewage sludge

Abstract

The invention provides a method for determining the protein content of sewage sludge, which comprises the following steps: (1) determining the concentration of the sewage sludge, (2) separating mud and water, and storing separated supernate, (3) extracting the protein in the separated sludge, (4) separating after extracting, (5) collecting and storing the separated supernate, (6) extracting the protein in the sludge for the second time, and repeating the steps (3), (4), (5) and (7) preparing a color developing agent. (8) preparing a standard curve sample; (9) purifying, developing and standing the standard curve sample and a to-be-detected sample; (10) determining the sample; and (11) calculating the content of the sludge protein. The protein concentration determination method provided by the invention is more accurate, higher in protein extraction efficiency and short in time consumption, so that the accuracy of determining the protein content of the sewage sludge is higher.

Description

Technical Field

[0001] The invention provides a method for determining the protein content of sewage sludge, and belongs to the field of sludge protein measurement. Background Art

[0002] With the development of biological processes in wastewater treatment systems, it is becoming increasingly important to understand the composition of soluble microbial products (SMPS) and extracellular polymeric substances (EPS). SMPS and EPS are mixtures of various compounds, such as proteins, carbohydrates, lipids, DNA, humus, and small molecules. Proteins, as major compounds in various wastewater treatment systems, are important for aggregation processes and flocculation capacity. Therefore, accurate quantification of proteins in biological wastewater treatment sludge is required.

[0003] At present, there is no standard method for sludge protein extraction and quantitative measurement. For protein measurement, the commonly used colorimetric methods are Lowry method, modified Lowry (M-Lowry) method, BCA (Bicinchonic Acid) method and Bradford method. For Lowry method, first, under alkaline conditions, Cu 2+ Reduced to Cu by protein + ; Secondly, the amplification stage will be carried out by reducing the Folin-Ciocalteu reagent (phosphomolybdate and phosphotungstate), showing a characteristic blue color, and then the determination is carried out. However, the humus in the sludge will interfere with the accuracy of the Lowry method, so it was modified to reduce the interference of humic acid and became the M-Lowry method. In addition, Smith et al. developed the BCA method by replacing the Folin-Cioalteu reagent with Bicinchonic Acid, which improved the sensitivity of protein determination. However, there is a contradiction between the three methods: Ras et al. showed that BCA is more suitable than the M-Lowry method. Jimenez et al. showed that the Lowry method is superior to the BCA method based on the statistical criteria of sensitivity, linearity, accuracy, correctness and specificity. Some studies have shown the advantages of the M-Lowry method compared with the Lowry method. As for the Bradford method, it is based on the binding of Coomassie Brilliant Blue G-250 to histidine arginine, phenylalanine, tryptophan and tyrosine residues at acidic pH, as well as hydrophobic interactions. Studies have shown that compared with the Lowry method, the Bradford method underestimates the protein content by 2-4 times when measuring sewage samples and extracted EPS. However, the exact mechanism is still not fully understood, but it is certain that the protein is not fully dissolved.

[0004] Another method is the Kjedahl-N method, which measures the organic nitrogen content and calculates the protein content using a protein / nitrogen conversion factor of 6.25 (or 6.25 g protein g N -1 ). However, it is generally acknowledged that the Kjedahl-N method is not accurate due to the different protein / nitrogen conversion factors of different sludge samples. To avoid the variation of the protein / nitrogen conversion factor in the Kjedahl-N method, it has been proposed to express the protein content by the sum of amino acids. However, this method ignores the fact that the molecular weight of free amino acids is 18 mass units larger than when bound to proteins. In addition, amino acids may be destroyed during sample preparation for amino acid analysis. For example, tryptophan loses about 5-10% during alkalinization. When hydrochloric acid is used for hydrolysis, almost all tryptophan and some others such as tyrosine, serine and threonine are destroyed. In addition, it requires more complex equipment than the Kjeldahl method and may therefore be beyond the capabilities of many laboratories, especially those that only perform intermittent analysis and cannot meet the requirements for timely protein determination. Therefore, new methods need to be found for the determination of protein concentration.

[0005] On the other hand, in order to accurately determine the protein content of sewage sludge, the extraction efficiency of protein is equally important. A variety of physical and chemical methods are used to increase the extraction efficiency of protein in sewage sludge, such as physical methods such as cation exchange resin, ultrasound, ultracentrifugation, heating and chemical methods such as alkaline reagents, ethylenediaminetetraacetic acid (EDTA), aldehyde solution, etc. Studies have shown that Triton X-100 can be extracted more effectively. However, the extraction efficiency of this method needs to be further improved, and new methods need to be found. Summary of the invention

[0006] Purpose of the invention: The technical problem to be solved by the present invention is to overcome the above-mentioned existing defects and provide a method for extracting sewage sludge protein with higher efficiency and more accurately measuring the concentration of the extracted protein, thereby more accurately determining the protein content in sewage sludge.

[0007] Technical solution: In order to solve the above technical problems, such as Figure 4 As shown, the present invention provides a method for determining the protein content of sewage sludge, the method comprising the following steps:

[0008] (1) The concentration of sludge is measured by weight method and recorded as ρ1;

[0009] (2) 1-2 mL of sludge is measured and its volume is recorded as V1, and placed in a 2 mL lysis tube for centrifugation at a speed of 10000-15000 g, a time of 10-20 min, and a temperature of 4°C. After centrifugation, the supernatant is separated and collected, and the sludge sediment is retained. The supernatant is marked as sample ①;

[0010] (3) 1-2 mL of protein extraction reagent is measured, and its volume is recorded as V2, and placed in the lysis tube in step (2), and the lysis tube is placed in a grinder for oscillation and grinding at a speed of 3000-5000 rpm for 30-60 seconds to evenly mix the sludge sediment and the protein extraction agent;

[0011] (4) After shaking and grinding, place the lysis tube in a centrifuge for centrifugation at a speed of 10,000-15,000 g for 10-20 min at a temperature of 4° C. Transfer the supernatant to a centrifuge tube, mark it as sample ②, and store it at 4° C.

[0012] (5) After the supernatant is transferred in step (4), the lysis tube is extracted again according to the operation steps of steps (3) and (4) to obtain the supernatant, which is marked as sample ③; wherein, when repeating step (3), the volume of 1-2 mL of protein extraction reagent added is marked as V3;

[0013] (6) 100 parts of developer A and 1 part of developer B are mixed evenly to prepare a mixed developer;

[0014] (7) Take 0, 5, 10, 15, 20 and 25 μL of 2 mg / mL bovine serum albumin in 6 2 mL centrifuge tubes, respectively. The contents of bovine serum albumin in the 6 centrifuge tubes are 0, 10, 20, 30, 40 and 50 μg, respectively;

[0015] (8) Take 1-50 μL of samples ①, ② and ③ respectively, and record their volumes as V 11 , V 22 , V 33 , placed in three 2 mL centrifuge tubes;

[0016] (9) Steps (7) and (8) are performed in a total of 9 2 mL centrifuge tubes. For each centrifuge tube, 0.5 mL of precipitant C is added and mixed evenly using a vortex mixer. After standing at room temperature for 2-3 min, 0.5 mL of precipitant D is added and mixed evenly using a vortex mixer. The centrifuge tube is placed in a centrifuge for centrifugation at a speed of 12000 g for 5 min. After removing the supernatant, 100 μL of copper solution and 400 μL of ultrapure water are added and mixed evenly using a vortex mixer. At this time, the precipitate in the centrifuge tube is in a dissolved state. 1 mL of the mixed developer in step (6) is added to the centrifuge tube, mixed evenly, and then stood at room temperature for 15-20 min. At this time, the liquid volume in each 2 mL centrifuge tube is 1.5 mL.

[0017] The 6 2 mL centrifuge tubes corresponding to step (7) have the corresponding bovine serum protein concentrations of 0 μg / 1.5 mL, 10 μg / 1.5 mL, 20 μg / 1.5 mL, 30 μg / 1.5 mL, 40 μg / 1.5 mL, and 50 μg / 1.5 mL, and the corresponding concentrations are denoted as ρ X1 , ρ X2 , ρ X3 , ρ X4 , ρ X5 , ρ X6 ;

[0018] (10) After the standing in step (9), each centrifuge tube was inverted and mixed three times, and then transferred to a cuvette, and the cuvette was placed in a spectrophotometer to read the absorbance, and 9 absorbance values ​​were obtained, corresponding to the 9 centrifuge tubes in step (9);

[0019] (11) Six of the nine absorbance values ​​in step (10) correspond to the bovine serum albumin concentrations ρ contained in the six centrifuge tubes in step (9) X1 , ρ X2 , ρ X3 , ρ X4 , ρ X5 , ρ X6 , the absorbance and bovine serum albumin concentration corresponding to the 6 groups are plotted into a standard curve, and a two-dimensional standard curve with the abscissa being the bovine serum albumin concentration and the ordinate being the absorbance is obtained. After linear fitting of the standard curve, a linear equation y=ax+b is obtained, where y represents absorbance, x represents bovine serum albumin concentration, and a and b are fitting coefficients;

[0020] The other three of the nine absorbance values ​​in step (10) correspond to the three centrifuge tubes of samples ①, ② and ③ in step (9), respectively. The three absorbance values ​​obtained are used to obtain the equivalent bovine serum albumin concentrations X1, X2, and X3 in the centrifuge tubes corresponding to samples ①, ② and ③ in step (9) through y=ax+b;

[0021] (12) Obtaining sludge mass M = ρ1 × V1 by the sludge concentration ρ1 in step (1) and the sludge volume V1 in step (2);

[0022] The volume of step (8) is obtained by using the bovine serum albumin concentrations X1, X2, and X3 in step (10) and the corresponding volume 1.5 mL in step (9), and is recorded as V 11 , V 22 , V 33 The amount of protein contained is recorded as m1, m2, and m3, that is, m1 = X1 × 1.5 mL, m2 = X2 × 1.5 mL, and m3 = X3 × 1.5 mL. The protein content in sample ① is obtained by the volume V1 in step (2), recorded as M1 = m1 × V1 / V 11, the protein content in sample ② is obtained by the volume V2 in step (3), which is recorded as M2 = m2 × V2 / V 22 , through step (5), the protein content in sample ③ is obtained, denoted as M3 = m3 × V3 / V 33 ;

[0023] The total mass of sludge protein was obtained by adding the protein amounts in samples ①, ② and ③, which was recorded as M0 = M1 + M2 + M3;

[0024] The sludge protein content obtained is M0 / M=(M1+M2+M3) / (ρ1×V1).

[0025] Furthermore, in step (2), the lysis tube used is a Lysing Matrix E tube.

[0026] Furthermore, in step (3), the protein extraction reagent is B-PER reagent.

[0027] Furthermore, in step (6), the color developer A is GE Healthcare 28955980, and the color developer B is GE Healthcare 28955984.

[0028] Furthermore, in step (9), the precipitant C is GE Healthcare 28955971, the precipitant D is GE Healthcare 28955977, and the resistivity of ultrapure water is 18-18.3 MΩ*cm.

[0029] Furthermore, in step (10), the analysis wavelength of the spectrophotometer is 480 nm, and ultrapure water is used as a reference solution.

[0030] Beneficial effects: Compared with the prior art, the technical solution of the present invention has the following beneficial technical effects:

[0031] (1) The protein concentration determination method of the present invention is more accurate;

[0032] (2) The protein extraction efficiency of the present invention is higher and the time consumption is shorter;

[0033] (3) The present invention has higher accuracy in determining the protein content of sewage sludge. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 These are the standard curve fitting of different protein concentration determination methods in Experiment 1.1 of the present invention, (a) the linear fitting curve of the BCA method, (b) the method of the present invention, (c) the M-Lowry method, and (d) the binary fitting curve of the M-Lowry method.

[0035] Figure 2This is a graph of the extraction efficiency of different protein extraction methods in Experiment 2.1 of the present invention.

[0036] Figure 3 This is the diagram of repeated extraction of sludge by the extractant and vibration grinding method of the present invention in Experiment 2.2 of the present invention.

[0037] Figure 4 Flow chart of the method of the present invention. DETAILED DESCRIPTION

[0038] Experiment 1, this experiment includes the following contents:

[0039] Experiment 1.1: Comparison of Linearity and Sensitivity of Different Protein Concentration Determination Methods

[0040] The protein concentration determination methods selected in Experiment 1.1 include: the protein determination method of the present invention, the BCA method, and the M-Lowry method. For each method, a standard curve was drawn and a fitting analysis was performed (e.g. Figure 1 ).

[0041] Depend on Figure 1 It can be seen that the regression coefficient (R 2 =0.9983) and slope (-0.0086)( Figure 1 (b)) is superior to the BCA method (R 2 =0.9945, slope 0.0010( Figure 1 (a))) and M-Lowry method (R 2 =0.9515, slope 0.0014( Figure 1 (c)), therefore, the method of the present invention has better linearity and sensitivity.

[0042] Figure 1 (d) is the binary fitting curve of the M-Lowry method, and the fitting degree is better than the linear fitting. The M-Lowry 1 method and the M-Lowry 2 method are defined for the following content. The difference between them is that the method of calculating protein concentration is different.

[0043] Experiment 1.2: Comparison of the effects of interfering substances on different protein concentration determination methods

[0044] The interference substances selected in Experiment 1.2 were protein extractants (Triton X-100 and the extractant of the present invention). The selected interference substances were determined by the protein determination method of the present invention, the BCA method, the M-Lowry 1 method and the M-Lowry 2 method. The results are shown in Table 1.

[0045] Table 1 Effects of the extractant of the present invention and Triton X-100 on protein concentration determined by different methods

[0046]

[0047] The true protein concentration is 0 mg BSA / L;

[0048] Mean ± SD, n = 6;

[0049] As can be seen from Table 1, the protein determination method of the present invention is least interfered by the extractants (the extractants of the present invention and Triton X-100).

[0050] Experiment 1.3: Comparison of different protein concentration determination methods for measuring protein in sludge of different dilution multiples

[0051] Experiment 1.3 selected sludge sample 1 and sludge sample 2, and adjusted the concentration of each sample to 1g VSS / L and 4g VSS / L, respectively. The sludge samples after adjusting the concentration were subjected to protein extraction, and the protein determination method of the present invention, BCA method, M-Lowry 1 method and M-Lowry 2 method were used for determination. The results are shown in Table 2.

[0052] Table 2 Protein concentration of sludge at different dilution times determined by different protein concentration determination methods

[0053]

[0054] It can be seen from Table 2 that the dilution multiple of the sludge sample affects the final protein content determination result, and the protein determination method of the present invention is least affected.

[0055] Based on Experiments 1.1, 1.2 and 1.3, the protein concentration determination method of the present invention is the best, has the best linearity, the highest sensitivity, and is least affected by the extractant and sludge dilution. Therefore, the protein concentration determination method of the present invention is more accurate.

[0056] Experiment 2 This experiment includes the following contents:

[0057] Experiment 2.1: Efficiency of different sludge protein extraction methods

[0058] Experiment 2.1 Take sludge sample 1 and sludge sample 2, determine the concentration of sludge, take 1 ml of sludge sample in a 2 ml centrifuge tube, centrifuge (12000g, 15min, 4℃) to separate mud and water, collect the supernatant, retain the precipitate, and perform the following different extraction methods on the centrifuge tube retaining the precipitate:

[0059] Extraction method 1: Oscillating grinding: Add 1 ml of ultrapure water, mix well, transfer to a lysis tube, and oscillate for 40 seconds (4000 rpm).

[0060] Extraction method 2: Triton X-100 extraction: Add 1 ml of 0.5% Triton X-100 extraction solution, mix well, and shake for 1 hour at 500 rpm.

[0061] Extraction method 3: Extractant of the present invention: add 1 ml of the extractant of the present invention, mix well, and let stand for 30 minutes.

[0062] Extraction method 4: Triton X-100 and oscillating grinding: Add 1 ml of 0.5% Triton X-100 extraction solution, mix well, transfer to a lysis tube, and oscillate for 40 seconds (4000 rpm).

[0063] Extraction method 5: extractant of the present invention and vibration grinding: add 1 ml of the extractant of the present invention, mix well, transfer to a lysis tube, and vibration grind for 40 seconds (4000 rpm).

[0064] After the above treatment, centrifugal treatment (12000g, 15min, 4℃) was performed to separate mud and water, and the supernatant was collected and measured and calculated using the protein concentration measurement method of the present invention. The results are as follows: Figure 2 As shown. Figure 2 It can be seen that the above protein extraction method 5, i.e. the extraction of protein from sludge using the extractant of the present invention and vibration grinding, has the highest efficiency and takes a short time.

[0065] Experiment 2.2: Efficiency of different sludge protein extraction methods

[0066] Experiment 2.2 Take sludge sample 1 and sludge sample 2, determine the concentration of sludge, take 1 ml of sludge sample in a 2 ml centrifuge tube, centrifuge (12000g, 15min, 4°C) for mud and water separation, collect the supernatant, retain the precipitate, add 1 ml of the extractant of the present invention to the centrifuge tube retaining the precipitate, mix evenly, transfer to a lysis tube, shake and grind for 40s (4000rpm), centrifuge (12000g, 15min, 4°C) for mud and water separation, collect the supernatant, retain the precipitate, perform secondary and thrice repeated extraction on the retained precipitate, and use the protein concentration measurement method of the present invention to measure and calculate. The results are as follows: Figure 3 As shown. Figure 3 It can be seen that the amount of protein extracted in the second extraction cannot be ignored, so it is necessary to repeat the extraction for the second time. The necessity of the third extraction depends on the specific situation.

[0067] Based on Experiments 2.1 and 2.2, the present invention has the highest extraction efficiency and requires a short time, and it is necessary to perform repeated extraction to increase the extraction efficiency.

Claims

1. A method for determining the protein content of sewage sludge, characterized in that: The method comprises the following steps: (1) The concentration of sludge is measured by weight method and recorded as ρ1; (2) 1-2 mL of sludge is measured and its volume is recorded as V1, and placed in a 2 mL lysis tube for centrifugation at a speed of 10000-15000 g, a time of 10-20 min, and a temperature of 4°C. After centrifugation, the supernatant is separated and collected, and the sludge sediment is retained. The supernatant is marked as sample ①; (3) 1-2 mL of protein extraction reagent is measured, and its volume is recorded as V2, and placed in the lysis tube in step (2), and the lysis tube is placed in a grinder for oscillation and grinding at a speed of 3000-5000 rpm for 30-60 seconds to evenly mix the sludge sediment and the protein extraction agent; (4) After shaking and grinding, place the lysis tube in a centrifuge for centrifugation at a speed of 10,000-15,000 g for 10-20 min at a temperature of 4° C. Transfer the supernatant to a centrifuge tube, mark it as sample ②, and store it at 4° C. (5) After the supernatant is transferred in step (4), the lysis tube is extracted again according to the operation steps of steps (3) and (4) to obtain the supernatant, which is marked as sample ③; wherein, when repeating step (3), the volume of 1-2 mL of protein extraction reagent added is marked as V3; (6) 100 parts of developer A and 1 part of developer B are mixed evenly to prepare a mixed developer; (7) Take 0, 5, 10, 15, 20 and 25 μL of 2 mg / mL bovine serum albumin in 6 2 mL centrifuge tubes, respectively. The contents of bovine serum albumin in the 6 centrifuge tubes are 0, 10, 20, 30, 40 and 50 μg, respectively; (8) Take 1-50 μL of samples ①, ② and ③ respectively, and record their volumes as V 11 , V 22 , V 33 , placed in three 2 mL centrifuge tubes; (9) Steps (7) and (8) are performed in a total of 9 2 mL centrifuge tubes. To each centrifuge tube, 0.5 mL of precipitant C is added and mixed evenly using a vortex mixer. After standing at room temperature for 2-3 min, 0.5 mL of precipitant D is added and mixed evenly using a vortex mixer. The centrifuge tube is placed in a centrifuge for centrifugation at a speed of 12000 g for 5 min. After removing the supernatant, 100 μL of copper solution and 400 μL of ultrapure water are added and mixed evenly using a vortex mixer. At this time, the precipitant in the centrifuge tube is 1.5 μL. The precipitate is in a dissolved state, 1 mL of the mixed color developing agent in step (6) is added to the centrifuge tube, mixed evenly, and allowed to stand at room temperature for 15-20 min; at this time, the liquid volume in each 2 mL centrifuge tube is 1.5 mL; and the 6 2 mL centrifuge tubes corresponding to step (7) have corresponding bovine serum protein concentrations of 0 μg / 1.5 mL, 10 μg / 1.5 mL, 20 μg / 1.5 mL, 30 μg / 1.5 mL, 40 μg / 1.5 mL, and 50 μg / 1.5 mL, and the corresponding concentrations are recorded as ρ X1 , ρ X2 , ρ X3 , ρ X4 , ρ X5 , ρ X6 ; (10) After the standing in step (9), each centrifuge tube was inverted and mixed three times, and then transferred to a cuvette, and the cuvette was placed in a spectrophotometer to read the absorbance, and 9 absorbance values ​​were obtained, corresponding to the 9 centrifuge tubes in step (9); (11) Six of the nine absorbance values ​​in step (10) correspond to the bovine serum albumin concentrations ρ contained in the six centrifuge tubes in step (9) X1 , ρ X2 , ρ X3 , ρ X4 , ρ X5 , ρ X6 , the absorbances and bovine serum albumin concentrations corresponding to the 6 groups are plotted into a standard curve to obtain a two-dimensional standard curve with the abscissa being the bovine serum albumin concentration and the ordinate being the absorbance, and a linear equation y=ax+b is obtained after linear fitting of the standard curve, where y represents the absorbance, x represents the bovine serum albumin concentration, and a and b are fitting coefficients; the other 3 of the 9 absorbance values ​​in step (10) correspond to the 3 centrifuge tubes of samples ①, ② and ③ in step (9), respectively, and the 3 obtained absorbance values ​​are used to obtain the equivalent bovine serum albumin concentrations X1, X2, and X3 in the centrifuge tubes corresponding to samples ①, ② and ③ in step (9) through y=ax+b; (12) The sludge mass M = ρ1 × V1 is obtained through the sludge concentration ρ1 in step (1) and the sludge volume V1 in step (2), and the sludge protein content is calculated based on this.

2. The method for determining protein content in sewage sludge according to claim 1, characterized in that: In step (2), the lysis tube used is a Lysing Matrix E tube.

3. The method for determining protein content in sewage sludge according to claim 1, characterized in that: In step (3), the protein extraction reagent is B-PER reagent.

4. The method for determining protein content in sewage sludge according to claim 1, characterized in that: In step (6), the color developing agent A is GE Healthcare 28955980, and the color developing agent B is GE Healthcare 28955984.

5. The method for determining protein content in sewage sludge according to claim 1, characterized in that: In step (9), precipitant C is GE Healthcare 28955971, precipitant D is GE Healthcare 28955977, and the resistivity of ultrapure water is 18-18.3 MΩ*cm.

6. The method for determining protein content in sewage sludge according to claim 5, characterized in that: In step (10), the analysis wavelength of the spectrophotometer is 480 nm, and ultrapure water is used as a reference solution.

7. The method for determining protein content in sewage sludge according to claim 1, characterized in that: The method of step (12) is as follows: by using the bovine serum albumin concentrations X1, X2, X3 in step (10) and the corresponding volume 1.5 mL in step (9), the volume of step (8) is obtained and recorded as V 11 , V 22 , V 33 The amount of protein contained is recorded as m1, m2, and m3, that is, m1 = X1 × 1.5 mL, m2 = X2 × 1.5 mL, and m3 = X3 × 1.5 mL. The protein content in sample ① is obtained by the volume V1 in step (2), recorded as M1 = m1 × V1 / V 11 , the protein content in sample ② is obtained by the volume V2 in step (3), which is recorded as M2 = m2 × V2 / V 22 , through step (5), the protein content in sample ③ is obtained, denoted as M3 = m3 × V3 / V 33 ; Add the protein amounts in samples ①, ② and ③ to obtain the total mass of sludge protein, recorded as M0=M1+M2+M3; The sludge protein content obtained is M0 / M=(M1+M2+M3) / (ρ1×V1).

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