Synthetic Humic Acids Solubilize Otherwise Insoluble Phosphates to Improve Soil Fertility

Abstract Artificial humic acids (A‐HA) made from biomass in a hydrothermal process turn otherwise highly insoluble phosphates (e.g. iron phosphate as a model) into highly available phosphorus, which contributes to the fertility of soils and the coupled plant growth. A detailed electron microscopy study revealed etching of the primary iron phosphate crystals by the ‐COOH and phenolic groups of humic acids, but also illustrated the importance of the redox properties of humic matter on the nanoscale. The combined effects result in the formation of then bioavailable phosphate nanoparticles stabilized by humic matter. Typical agricultural chemical tests indicate that the content of total P and directly plant‐available P improved largely. Comparative pot planting experiments before and after treatment of phosphates with A‐HA demonstrate significantly enhanced plant growth, as quantified in higher aboveground and belowground plant biomass.


SI-1. Materials and Methods
Unless otherwise noted, all of the commercial reagents were used as received. Glucose (C6H12O6), iron phosphate (FePO4), phosphorites/apatite (Ca5(PO4)3(OH)) and potassium hydroxide (KOH) were purchased from Sigma-Aldrich Company. Various biomasses and sandy soils were collected in Brandenburg, Germany. Black soils for comparison were sampled in the campus of Northeast Agricultural University, Harbin, China. Before experiments, the crude biomasses are cut into a suitable size. Typically, 1.20 g rude biomass powders, 0.5 g insoluble P rock with various KOH qualities (ensuring the molar rations between degradable cellulose and alkali close to 1) in absence and presence of 5 g sandy soil and were put into the bottom of the glass tube in 50 ml autoclave in the oven to heat to 200 °C, and then keep for 24 h to insure the sufficient humification, then the products are collected after the temperature naturally cooled down to room temperature with subsequent drying procedure. In brief, the products are named as G-FePO4 (glucose), L-FePO4 (leaf), CS-FePO4 (corn straw), R-FePO4 (root) without the dirt addition and LS-FePO4 and RS-FePO4 with the dirt addition according.

SI-2: SOM examination
Typically, SOM examinations of sandy soil, a series of artificial high-fertility soil and natural soils are carried out following the procedures: firstly, those empty porcelain boats were placed in a muffle furnace, burned at 95 °C for 30 min, taken out and cooled in a desiccator for 20 min, and the mass (M1) was weighed; then, they were burned at the same temperature for 30 min, taken out, cooled, and weighed. Repeated the above steps, until their mass difference between the two times is less than 0.5 mg. Weigh out 0.50 g of dry sample in a known quality porcelain boat, put it in an oven at 105 °C for 12h, then take it out, put it into the desiccator, cooled it, weighted it, recorded the mass (M3); Then transfer these porcelain boats to a muffle furnace and heat up to 550 °C, burn for 5 h, cool in a desiccator, weigh, and record the mass (M2) and calculate.

Total Phosphorus
Weigh out 0.25g of dried soil sample passing through 100-mesh sieve into 50 mL flask, moisten with a small amount of water, add 8 mL of concentrated sulfuric acid, shake and place overnight, then add perchloric acid 10 drips, shake well, set a small funnel at the mouth of the bottle, put it in an electric furnace (or heating device), heat it and dilute it until the solution in the bottle begins to turn white, continue to cook for 20 min, and all the cooking time is 45-60 min. The cooled digestion liquid is carefully washed into a 100 mL volumetric flask with water, and the water should be washed a small amount several times during rinsing. The flask was gently shaken, and after it was completely cooled, it was made up to volume with water, and the solution was filtered through a dry funnel and a non-phosphorus filter paper into a dry 100 mL flask. At the same time, it was a reagent blank test.

Soil available phosphorus
0.25 g of the soil sample, placed in a dry 150 mL Erlenmeyer flask, adding a leaching agent (c (NaHCO3) = 0.5 mol L -1 , pH=8.5) 50.0 mL, it was stoppered with a rubber stopper and shaken on a reciprocating shaker at room temperature of (25 ± 1 °C) for 30 min. form an antimony-phosphomolybdate complex. This complex is reduced to an intensely bluecoloured complex by l(+)ascorbic acid. The complex is measured at 880 nm. The presence of P was analyzed with the molybdenum blue method, based on the reactions between P and MoO4, Sb, and ascorbic acid. Total P concentration was determined using a colorimetric assay. Acid molybdate and Fiske's SubbaRow reducer solution were added to the digest to form a phosphomolybdenum complex. The total phosphorus (TP) was measured by calcination in a muffle furnace (550 °C) and was then extracted using a solution of 1 mol L −1 HCl.

Available phosphorus
The P-solubility in neutral ammonium citrate (Pnac) is a widely accepted indicator for characterizing phosphate in hydrothermal products as a direct nutrient fertilizer for plant growth. The solubility of P was conducted to estimate the plant-availability of P in samples.

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For Pnac, 500 μL of sample solutions were placed in 50 mL flask and extracted in 25.0 mL neutral ammonium citrate solution (1%) for 2 h at room temperature and stirring. Fig. S1. Images and Corresponding elemental maps of insoluble P rock etched by humic substances prepared       Tables   Table S1. Surface acidic functional groups (mmol L -1 ) and molecular weight of artificial humic substances.