A method for selective extraction of microbial-occluded phosphorus in sediments based on surfactants

Abstract

This invention belongs to the field of environmental monitoring and sediment analysis technology, and discloses a method for selective extraction of microbial-possessed phosphorus from sediments based on surfactants. This invention removes weakly adsorbed phosphorus from the sample while retaining microbial-possessed phosphorus components. A mild extraction system using sodium dodecyl sulfate (DSS) is employed to simultaneously extract intracellular phosphorus and strongly adsorbed phosphorus from the cell surface of microorganisms. This system has extremely weak extraction capability for phosphorus in subsequent steps, enabling highly selective and accurate extraction and quantification of microbial solid phosphorus. This invention effectively eliminates interference from weakly adsorbed phosphorus, accurately extracts microbial-possessed phosphorus from sediments, achieves precise quantification of microbial solid phosphorus in sediments, and supports fine analysis and reclassification of phosphorus speciation, providing reliable technical support for revealing the role of microorganisms in phosphorus biogeochemical cycles.

Description

Technical Field

[0001] This invention relates to the fields of environmental analysis and sediment geochemical detection technology, and in particular to a method for selective extraction of microbial-immobilized phosphorus from sediments based on surfactants. Background Technology

[0002] In sediment phosphorus speciation and biogeochemical studies, microbial-associated phosphorus components are key elements regulating phosphorus biotransformation. Current traditional phosphorus speciation analysis methods primarily focus on classifying phosphorus into conventional components such as weakly adsorbed phosphorus, iron / aluminum bound phosphorus, calcium bound phosphorus, detrital phosphorus, and organic phosphorus, while the identification and definition of microbial-associated phosphorus components remain unsystematic.

[0003] While existing research mentions "microbial phosphorus," its definition is usually limited to intracellular phosphorus in microorganisms. Because its content is far lower than other forms of phosphorus, it is often broadly categorized as organic phosphorus by researchers using a subtraction method. Therefore, existing systems do not fully recognize or differentiate the contribution of strongly adsorbed phosphorus on the microbial cell surface, nor do they systematically define intracellular phosphorus and strongly adsorbed phosphorus on the cell surface as a unified, independent, and stable phosphorus pool.

[0004] In short, current extraction methods and their accompanying analytical systems have not yet established "microbial held phosphorus" as an independent primary morphological classification, failing to treat it as a key component alongside weakly adsorbed phosphorus, inorganic mineral-bound phosphorus, and organic phosphorus for targeted extraction and quantification. Furthermore, existing methods have failed to simultaneously extract intracellular phosphorus and strongly adsorbed phosphorus from microorganisms, resulting in a long-standing lack of precise and reliable methodological support for understanding the occurrence characteristics, content levels, and environmental behavior of microbial held phosphorus in sediments.

[0005] Therefore, establishing a selective extraction method that can simultaneously extract intracellular phosphorus and strongly adsorbed phosphorus on the surface of microorganisms, and treat microbial immobilized phosphorus as an independent primary form, can not only improve the phosphorus speciation system in sediments, but also accurately reveal the mechanism of microbial-mediated phosphorus cycling. At the same time, it provides key technical support for the scientific assessment of environmental phosphorus pollution and endogenous phosphorus load, which has important scientific significance and broad application prospects. Summary of the Invention

[0006] In order to overcome the problems of existing technologies such as inability to specifically extract microbial immobilized phosphorus, poor selectivity, low precision, and lack of standardized quality control, this invention provides a surfactant-based method for selective extraction of microbial immobilized phosphorus from sediments with a weak adsorption state removal and a rigorous quality control system.

[0007] This invention is achieved through the following technical solution: A surfactant-based method for selective extraction of microbially immobilized phosphorus from sediments, using sediments from lakes, estuaries, or oceans as samples, specifically includes the following steps: (1) The collected sample was dried and then ground to obtain a pretreated sample; (2) Place the pretreated sample in a centrifuge tube, add magnesium chloride solution, extract by constant temperature shaking, then centrifuge at low temperature and high speed, discard the supernatant, and retain the precipitate after washing; (3) Add sodium dodecyl sulfonate solution to the precipitate, extract by constant temperature shaking, then centrifuge at low temperature and high speed, retain the centrifuged liquid, and combine the washing liquid after washing into the centrifuged liquid to obtain a mixed liquid system; (4) The mixture system was brought to a constant volume, and the immobilized inorganic phosphorus index of microorganisms was measured by the molybdenum blue colorimetric method.

[0008] The microbial immobilized phosphorus described in this invention refers to phosphorus components that exist in the form of adsorption, precipitation, chelation, or intracellular accumulation, combined with microbial biomass, extracellular polymers, and biofilms. It mainly includes intracellular phosphorus and strongly adsorbed phosphorus on the surface of microbial cells, which is different from soluble phosphorus, weakly adsorbed phosphorus, inorganic mineral-bound phosphorus, detrital phosphorus, and residual phosphorus in the environment. This form of phosphorus is in the dynamic process of biogeochemical cycle and is a key phosphorus component with high biological activity and transformation potential in sediment phosphorus pools. It can also serve as an important indicator of environmental microbial activity.

[0009] This invention first removes weakly adsorbed phosphorus and soluble phosphorus using magnesium chloride solution to eliminate interference; then, it gently lyses microorganisms using sodium dodecyl sulfonate solution to release microbial-immobilized phosphorus. This invention was validated in a pure microbial system. By pre-removing weakly adsorbed phosphorus and using the total phosphorus residue from high-temperature pyrolysis as the true value for method validation, the results are reliable and demonstrate the ability to selectively extract microbial-immobilized phosphorus.

[0010] A more preferred technical solution of the present invention is as follows: In step (1), gravel, plant and animal remains and other impurities are removed from the collected samples. After air drying, the samples are ground through a 100-mesh sieve and then stored in a sealed container at a low temperature away from light for later use.

[0011] In step (2), 1 mol / L magnesium chloride (MgCl2) solution was added to the pretreated sample, and the sample was extracted by shaking on a shaker at 26℃ and 160 rpm / min for 1 h, and then centrifuged at 4℃ and 10000 rpm / min for 15 min.

[0012] In step (3), a 1% sodium dodecyl sulfonate solution was added to the precipitate, and the mixture was extracted by shaking on a shaker at 26°C and 160 rpm / min for 4 h. Then, it was centrifuged at 4°C and 10,000 rpm / min for 15 min.

[0013] In steps (2) and (3), the residue after centrifugation is rinsed with 1 mol / L magnesium chloride solution and then rinsed three times with Milli-Q water.

[0014] Further preferred, the ratio of sample to magnesium chloride solution and residue to sodium dodecyl sulfonate solution is 1g:25mL, i.e., 1g sample / residue + 25mL extract.

[0015] In step (4), the mixture system is filtered through a filter membrane with a pore size of 0.45 μm and then brought to a constant volume.

[0016] In a further preferred embodiment, a mixed solution system with an equal volume to that used for measuring the inorganic phosphorus index of microorganisms was taken, and 5% potassium persulfate was added. The mixture was then subjected to high-temperature digestion in a 121°C autoclave for 30 minutes to convert organic phosphorus into phosphate ions. The total phosphorus index of the extract was determined using the molybdenum blue colorimetric method, and the organic phosphorus index of microorganisms was calculated by the difference method.

[0017] The above subtraction method is: Microbial held organic phosphorus = Microbial held phosphorus - Microbial held inorganic phosphorus.

[0018] Based on the above method, microbial held phosphorus is further subdivided into microbial held inorganic phosphorus and microbial held organic phosphorus, ultimately achieving quantitative characterization of microbial held phosphorus components without distinguishing their location.

[0019] More preferably, the residue after centrifugation in step (3) is subjected to subsequent phosphorus extraction. Iron-bound phosphorus is extracted for 6 hours with 0.11 mol / L sodium bicarbonate solution + 0.11 mol / L sodium dithionite solution (NaHCO3-Na2S2O4 solution, i.e. BD solution, pH=7), calcium-bound phosphorus is extracted for 6 hours with 1 mol / L sodium acetate-acetic acid buffer solution (NaAc-HAc solution, pH=4), and detrital phosphorus is extracted for 16 hours with 1 mol / L hydrochloric acid solution. The organic phosphorus extracted from the above three extracts is medium-stable organic phosphorus. The phosphorus concentration in the extracts is determined by the molybdenum blue colorimetric method. The residue is calcined at 550℃ for 2 hours and extracted with 1 mol / L hydrochloric acid solution for 16 hours to obtain highly stable phosphorus (i.e., residual phosphorus or inert organic phosphorus).

[0020] The extraction was performed using a shaking incubator at a temperature of 26°C and a rotation speed of 160 rpm / min. Solid-liquid separation was then carried out using a high-speed refrigerated centrifuge at 4°C and 10,000 rpm / min for 15 minutes. The residue from the previous extraction was washed with a 1 mol / L magnesium chloride solution, followed by three washes with Milli-Q water before proceeding to the next extraction step. The supernatant was filtered and brought to a final volume, and the inorganic phosphorus content was directly determined. An equal volume of the supernatant was then added to a 5% potassium persulfate solution and autoclaved at 121°C for 30 minutes before determining the total phosphorus content.

[0021] The beneficial effects of this invention are as follows: (1) High selectivity. By pre-removing weakly adsorbed phosphorus interference components, microbial immobilized phosphorus can be specifically extracted, and there is almost no interference with the subsequent extraction of several different phosphorus forms, effectively distinguishing phosphorus of different binding forms; (2) High accuracy. The method adopts a self-control design using a pure microbial culture system and uses total phosphorus pyrolysis as the true value for quality control. No external standard is required to achieve method calibration, and the results are true and reliable. (3) The method is stable and has few interferences. The experimental system is simple, the pretreatment approach is clear, and after eliminating the interference of weakly adsorbed phosphorus, the parallelism and stability of the determination are good, and the results are highly reliable; (4) Outstanding scientific value. It can accurately characterize the content of microbial phosphorus in sediments, support the fine division and reclassification of phosphorus speciation in sediments, and provide reliable support for revealing the role of microorganisms in phosphorus biogeochemical cycles.

[0022] This invention can effectively eliminate the interference of weakly adsorbed phosphorus, accurately extract microbial phosphorus from sediments, achieve precise quantification of solid phosphorus in sediment microorganisms, and support fine analysis and reclassification of phosphorus speciation, providing reliable technical support for revealing the role of microorganisms in phosphorus biogeochemical cycles; it is applicable to the assessment of endogenous phosphorus and the study of phosphorus biogeochemical cycles in sediments of lakes, estuaries, and oceans. Attached Figure Description

[0023] The invention will now be further described with reference to the accompanying drawings.

[0024] Figure 1 This is a schematic diagram of the process for graded extraction of phosphorus speciation from sediments according to the present invention. Figure 2 This is a schematic diagram of the selective verification results of the method of the present invention; Figure 3 This is a schematic diagram of microbial-mediated phosphorus migration, transformation, and fate.

[0025] in, Figure 1 The dashed box indicates the extraction steps of microbial immobilized phosphorus according to the present invention; Figure 3 The abbreviations in Chinese and English are as follows: Weakly adsorbed phosphorus – Labial-P, Weakly adsorbed inorganic phosphorus – L-IP, Weakly adsorbed organic phosphorus – L-OP, Microbially held phosphorus – BaP, Microbially held organic phosphorus – Ba-OP, Iron-bound phosphorus – Fe-P, Calcium-bound phosphorus – Ca-P, Detrital phosphorus – Det-P, Medium-stable organic phosphorus – MS-OP, Residual phosphorus (highly stable phosphorus, inert phosphorus) – Res-P. Detailed Implementation

[0026] To make the above-mentioned objects, features, and advantages of the present invention more apparent and understandable, specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings. Many specific details are set forth in the following description to provide a thorough understanding of the present invention. However, the present invention can be practiced in many other ways different from those described herein, and those skilled in the art can make similar modifications without departing from the spirit of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0027] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0028] The present invention will now be described in detail with reference to the accompanying drawings.

[0029] Example 1: A method for selective extraction of microbially immobilized phosphorus from sediments The extraction method in this embodiment specifically includes the following steps: (1) Sample pretreatment Collect sediment samples, remove impurities such as gravel and plant and animal remains; air dry, grind and pass through a 100-mesh nylon sieve, and store in a sealed container at low temperature away from light for later use.

[0030] (2) Removal of weakly adsorbed phosphorus Weigh 1.0000g of pretreated sample, add 25mL of 1mol / L MgCl2 solution (pH=8), extract by constant temperature shaking (26℃, 160rpm / min, 1h), centrifuge at low temperature and high speed (4℃, 10000rpm / min, 15min) and discard the supernatant.

[0031] Wash the residue three times with 1 mol / L MgCl2 solution to remove residual weakly adsorbed phosphorus, and retain the precipitate for subsequent extraction.

[0032] (3) Extraction with sodium dodecyl sulfate solution Add 25 mL of 1% sodium dodecyl sulfate solution to the precipitate and extract by constant temperature shaking (26℃, 160 rpm / min, 6 h) to specifically lyse bacterial cells and extracellular biofilm structures and release microbial immobilized phosphorus.

[0033] (4) Solid-liquid separation and washing The mixture was centrifuged at low temperature and high speed (4℃, 10000 rpm / min, 15 min). The residue was rinsed with 1 mol / L MgCl2 solution and then rinsed three times with Milli-Q water. The washing liquid was then combined with the centrifuged liquid after centrifugation.

[0034] (5) Optional step after washing / separation: filtration step: filtering the obtained system through a microporous membrane to remove extremely small particulate impurities. This step is optional and can be implemented as needed. The microporous membrane has a pore size of 0.45 μm. Relevant phosphorus indicators will be measured after volume adjustment.

[0035] (6) Determination and calculation of phosphorus components The molybdenum blue colorimetric method was used to directly measure the supernatant after volume adjustment to obtain microbial immobilized inorganic phosphorus; Take an equal volume of the supernatant to be tested, add 2 mL of 5% potassium persulfate, and digest it at high temperature in an autoclave at 121℃ (30 min). The total phosphorus in the extract is determined by the molybdenum blue colorimetric method, thus obtaining the microbial immobilized phosphorus.

[0036] Calculation of microbially held organic phosphorus: Microbial held organic phosphorus = microbial held phosphorus - microbial held inorganic phosphorus; Through the above methods, the content of microbial held phosphorus, including microbial held inorganic phosphorus and microbial held organic phosphorus, was obtained.

[0037] (7) Quality control plan Method validation and quality control were performed using a pure microbial culture system. Sediment samples were inoculated into LB liquid medium and cultured for 48 hours to obtain an enriched mixed microbial community, which was used for subsequent method validation and quality control.

[0038] The resulting mixed microbial system was inoculated into a liquid culture medium containing inorganic phosphate as the sole phosphorus source (KH2PO4) at a volume of 100 μL, and the validation system was obtained after culturing.

[0039] Because the soluble phosphorus content in the pure culture system is much higher than that in natural sediment samples, the system needs to be pretreated: the culture system is centrifuged (4℃, 5000 rpm / min, 10 min), the supernatant is discarded to remove most of the soluble weakly adsorbed phosphorus; the residue is retained, and then weakly adsorbed phosphorus is removed using the aforementioned method (extraction-centrifugation-washing). The treated residue is then divided into two portions: one portion is extracted and determined using the method of this invention, and the other portion is ignited in a muffle furnace, extracted with 1 mol / L hydrochloric acid for 16 h, and then the total phosphorus is determined. The total phosphorus measured by pyrolysis is used as the true value for method verification and quality control. Three parallel replicates were set up for the experiment. Table 1 shows the results of microbial immobilized phosphorus determination obtained from the pure culture system at 28℃, 160 rpm / min, and a culture time of 36 h.

[0040] Table 1 Comparison of Microbial Immobilized Phosphorus Measurement Results with True Values

[0041] As can be seen from the above data, the results obtained by the method of the present invention are in good agreement with the true values, with a recovery rate of 96.74%. The method has high accuracy, strict quality control, and reliable results. Example

[0042] Columnar samples of sediments from the mouth of the Yellow River Delta island were collected at a depth of 100 cm. The samples were cut evenly at 1 cm intervals, dried, ground through a 100-mesh nylon sieve, and stored at low temperature in the dark. Weigh 1.0000 g of the pretreated sediment sample into a 50 mL centrifuge tube. Extract the weakly adsorbed phosphorus and microbially immobilized phosphorus from the sample sequentially using 1 mol / L MgCl2 solution (26℃, pH=8, 1 h) and 1% sodium dodecyl sulfate solution (26℃, 6 h).

[0043] Further extraction of phosphorus in various forms was carried out on the residue: extraction was performed using 0.11 mol / L NaHCO3 + 0.11 mol / L Na2S2O4 (BD solution, 26℃, pH=7, 6h), 1 mol / L NaAc-HAc buffer solution (26℃, pH=4, 6h), and 1 mol / L HCl solution (26℃, 16h).

[0044] Each extraction step was rinsed three times with 1 mol / L MgCl2 solution and Milli-Q water. The supernatant from each extraction step was centrifuged, filtered through a 0.45 μm filter membrane, and brought to a final volume. The inorganic phosphorus content was then directly determined. Separately, an equal volume of the final volume solution was taken, 2 mL of 5% potassium persulfate solution was added, and the mixture was digested at 121 °C for 30 min before determining the total phosphorus content. The difference between the two determinations represents the organic phosphorus content extracted in each step.

[0045] As attached Figure 1 As shown, sediment phosphorus is classified into weakly adsorbed phosphorus (weakly adsorbed inorganic phosphorus and weakly adsorbed organic phosphorus), microbially held phosphorus (microbially held inorganic phosphorus and microbially held organic phosphorus), iron-bound phosphorus, calcium-bound phosphorus, detrital phosphorus, and moderately stable organic phosphorus (i.e., co-extractable organic phosphorus, including the sum of organic phosphorus extracted from iron-bound phosphorus, calcium-bound phosphorus and detrital phosphorus).

[0046] The residue was calcined at 550℃ for 2 hours and then leached with 1 mol / L HCl solution for 16 hours to obtain highly stable phosphorus (i.e., residual phosphorus or inert phosphorus).

[0047] The phosphorus concentration in the extract was determined using the molybdenum blue colorimetric method.

[0048] In each of the above extraction steps, 25 mL of extractant was added, and extraction was performed by shaking on a shaker at a temperature of 25°C and a rotation speed of 160 rpm / min. Solid-liquid separation was performed using a high-speed refrigerated centrifuge at 4°C, 10,000 rpm / min, for 15 min.

[0049] The extract obtained from a 1% sodium dodecyl sulfonate solution (26°C, 4h) is microbial immobilized phosphorus.

[0050] This invention uses a pure microbial culture system to verify the method: First, weakly adsorbed phosphorus in the sample is removed, and the remaining components are divided into two equal parts: one part is extracted and determined using the method of this invention, and the other part is pyrolyzed in a muffle furnace to determine the total phosphorus. The value is used as the true value for quality control to ensure that the determination results are accurate and reliable.

[0051] Each sample group was set up with 3 parallel groups. The relative standard deviation (RSD) of the parallel samples was ≤3%, indicating good accuracy. (See attached image) Figure 2 As shown, selective verification indicates that the method of the present invention can specifically extract microbially held phosphorus (refined in the figure as microbially held organic phosphorus and microbially held inorganic phosphorus) after removing weakly adsorbed phosphorus. The interference of different forms of phosphorus to be extracted subsequently is negligible, and it can achieve systematic and accurate hierarchical analysis of different phosphorus forms in sediments.

[0052] As attached Figure 3 As shown, the results of the determination of phosphorus based on microbial immobilized phosphorus reveal the role of microorganisms in the biogeochemical cycle of phosphorus in sediments, as well as the migration, transformation and fate relationships among different forms of phosphorus based on microbial metabolism.

[0053] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention, and they should all be covered within the scope of the claims and specification of the present invention.

Claims

1. A method for selective extraction of microbially immobilized phosphorus from sediments based on surfactants, characterized in that, The samples were collected from sediments in lakes, estuaries or oceans, and the specific steps included: (1) drying and grinding the collected samples to obtain pretreated samples; (2) placing the pretreated samples in centrifuge tubes, adding magnesium chloride solution, extracting by constant temperature shaking, then centrifuging at low temperature and high speed, discarding the supernatant, and retaining the precipitate after washing. (3) Add sodium dodecyl sulfonate solution to the precipitate, extract by constant temperature shaking, then centrifuge at low temperature and high speed, retain the centrifuged liquid, and combine the washing liquid after washing into the centrifuged liquid to obtain a mixed liquid system; (4) make up the volume of the mixed liquid system, and use the molybdenum blue colorimetric method to determine the microbial immobilized inorganic phosphorus index.

2. The method for selective extraction of microbially immobilized phosphorus from sediments based on surfactants as described in claim 1, characterized in that: In step (1), gravel, plant and animal remains and other impurities are removed from the collected samples. After air drying, the samples are ground through a 100-mesh sieve and then stored in a sealed container at a low temperature away from light for later use.

3. The method for selective extraction of microbially immobilized phosphorus from sediments based on surfactants as described in claim 1, characterized in that: In step (2), 1 mol / L magnesium chloride solution was added to the pretreated sample, and the sample was extracted by shaking on a shaker at 26°C and 160 rpm / min for 1 h, and then centrifuged at 4°C and 10000 rpm / min for 15 min.

4. The method for selective extraction of microbially immobilized phosphorus from sediments based on surfactants as described in claim 1, characterized in that: In step (3), a 1% sodium dodecyl sulfonate solution was added to the precipitate, and the mixture was extracted by shaking on a shaker at 26°C and 160 rpm / min for 4 h. Then, it was centrifuged at 4°C and 10,000 rpm / min for 15 min.

5. The method for selective extraction of microbially immobilized phosphorus from sediments based on surfactants as described in claim 1, characterized in that: In steps (2) and (3), the residue after centrifugation is rinsed with 1 mol / L magnesium chloride solution and then rinsed three times with Milli-Q water.

6. The method for selective extraction of microbially immobilized phosphorus from sediments based on surfactants as described in claim 1, characterized in that: The ratio of the collected sample to magnesium chloride solution and the residue to sodium dodecyl sulfonate solution was 1 g: 25 mL.

7. The method for selective extraction of microbially immobilized phosphorus from sediments based on surfactants as described in claim 1, characterized in that: In step (4), the mixture system is filtered through a filter membrane with a pore size of 0.45 μm and then brought to a constant volume.

8. The method for selective extraction of microbially immobilized phosphorus from sediments based on surfactants as described in claim 1 or 7, characterized in that: In step (4), take a mixed solution system with an equal amount of the amount of inorganic phosphorus index of microorganisms to be measured, add 5% potassium persulfate, and digest it at high temperature in an autoclave at 121℃ for 30 min. The total phosphorus index of the extract is determined by the molybdenum blue colorimetric method, and the organic phosphorus index of microorganisms is calculated by the difference method.

9. The method for selective extraction of microbially immobilized phosphorus from sediments based on surfactants as described in claim 1, characterized in that: The residue after centrifugation in step (3) was subjected to subsequent phosphorus extraction. Iron-bound phosphorus was extracted with 0.11 mol / L sodium bicarbonate solution + 0.11 mol / L sodium dithionite solution for 6 h, calcium-bound phosphorus was extracted with 1 mol / L sodium acetate-acetic acid buffer solution for 6 h, and fragmented phosphorus was extracted with 1 mol / L hydrochloric acid solution for 16 h. The phosphorus concentration in the extract was determined by the molybdenum blue colorimetric method. The residue was calcined at 550℃ for 2 h and extracted with 1 mol / L hydrochloric acid solution for 16 h to obtain highly stable phosphorus.

10. The method for selective extraction of microbially immobilized phosphorus from sediments based on surfactants as described in claim 9, characterized in that: The extraction was performed using a shaking incubator at a temperature of 26°C and a rotation speed of 160 rpm / min. Solid-liquid separation was then carried out using a high-speed refrigerated centrifuge at 4°C and 10,000 rpm / min for 15 minutes. The residue from the previous extraction was washed with a 1 mol / L magnesium chloride solution, followed by three washes with Milli-Q water before proceeding to the next extraction step. The supernatant was filtered and brought to a final volume, and the inorganic phosphorus content was directly determined. An equal volume of the supernatant was then added to a 5% potassium persulfate solution and autoclaved at 121°C for 30 minutes before determining the total phosphorus content.

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