Synergy effect of peroxidase enzymes and Fenton reactions greatly increase the anaerobic oxidation of soil organic matter

Study sites and sampling

Three soil types were selected (Table 2). The first was an Andisol³¹ (Volcanic–allophanic) formed from recent Volcanic ash deposited in the Andes. This was collected from a native temperate rainforest dominated by old growth Nothofagus betuloides (Mirb.) located in Puyehue National Park, where mean annual precipitation is typically > 8,000 mm year⁻¹¹². The soils are derived from basaltic scoria with high levels of allophane, imogolite, and ferrihydrite material³². The second soil type was an Ultisol (Metamorphic) sampled in Alerce Costero National Park in the coastal range. It is derived from Metamorphic-schist with high levels of illite-kaolinite³³. Finally, an Inceptisol (Granitic) was selected from an ancient Araucaria araucana and Nothofagus pumilio forest in Nahuelbuta National Park. This soil type originates from intrusive Granitic rock parent materials. Mean annual precipitation in this ancient forest reaches > 1,491 mm and mean annual temperatures reach 13.3 °C³⁴ (Table 2). From each soil sample, four composite soil samples were taken from the Ah mineral horizon (5–10 cm) after removing litter and organic horizons. The samples were then transported to a laboratory under cold conditions. All soils were homogenized and sieved (< 2 mm). Hereafter, all derived soils are identified as Granitic, Volcanic–allophanic and Metamorphic.

Analytical procedure

pH and electrical conductivity were directly measured in an aliquot of soil in a 1:2.5 suspension of soil:water. Soil organic C was determined using TOC-VCSH (Shimadzu, Kyoto, Japan), and total N was determined by Kjeldahl distillation (VELP, Usmate, Italy). For the determination of Fe (Fe_o) and Mn (Mn_o), 0.2 M ammonium oxalate at pH 3 was used³⁵. Iron and Mn complexed with SOM (Fe_p and Mn_p) were obtained using a solution of 0.1 M sodium pyrophosphate³⁶. Dithionite-citrate-bicarbonate (Fe_d) was used to identify exchangeable, crystalline, and complexed-SOM metals in the samples (see³⁶ for further details). Fe and Mn concentrations were determined by atomic absorption spectroscopy (Perkin Elmer 3110, Waltham, Massachusetts, USA) conducted at 248.3 nm for Fe and at 279.5 nm for Mn using a nitrous oxide acetylene flame. Aluminium pyrophosphate (Al_p), Al oxalate (Al_o), and Si (Si_o) were also determined as described above. Cation exchange capacity (CEC) and nutrient characterization were conducted as indicated by Sadzawka et al.³⁵ (Table 3).

The total Fe concentration was determined from 100 mg of soil by adding 0.9 ml of 0.28 M hydroxylamine hydrochloride (2%) and 1 ml of 0.28 M HCl²⁶. Approximately 100 μl of the extract was added to 4 ml of colour reagent (1 g ferrozine in a 6.5 M ammonium acetate solution). The amount of reduced iron Fe(II) (or Fe²⁺) was quantified from 0.1 g of soil added to 1 ml of 0.5 M HCl (now on Fe(II)-HCl)³⁴ and shaken vigorously. Approximately 100 μl of this suspension was added to 4 ml of colour reagent (1 g ferrozine in 6.5 M ammonium acetate solution). The ferrozine complex standard was prepared with ferrous ethylene diammonium sulphate dissolved in 0.5 M HCl³⁷. Fe²⁺ was determined spectrophotometrically at 562 nm (absorbance ferrozine-Fe²⁺ complex) after the colour developed. The concentration of oxidized Fe(III)-(oxyhydr)oxide was determined by calculating the difference between total Fe and Fe²⁺ concentrations (Table 1).

Soil sterilization

Soils used for the incubation experiment (see below) were sterilized in an autoclave for 20 min at 121 °C for 3 consecutive days to remove the microbial population with resistant structures such as endospores and conidia. Furthermore, soils were fumigated with chloroform vapour in a vacuum chamber for 24 h³⁸. Autoclaving was used because it does not create significant changes in the SOM structure, mainly leading to changes in carbohydrate and N‐alkyl regions of the ¹³C‐CP/MAS spectra due to the lysis of microorganisms and the subsequent loss of microbial C in the aqueous phase³⁹. Gamma radiation was avoided because some reports indicate that it causes Fe reduction and oxidation^40,41,42. Gamma radiation increased the bioavailability of Fe(III)(oxyhydr)oxide minerals, which resulted in increased Fe(III)(oxyhydr)oxide reduction⁴³.

Microorganism culture and enzyme extraction

White rot fungi were isolated from wood logs in the same areas where soil sampling was conducted. The white rot fungi were cultured and maintained in Koroljova–Skorobogat’ko medium at pH 5⁴⁴. This is a typical medium used to grow fungi for enzymatic extraction when required^45,46. The medium (g l⁻¹) consisted of: 3.0 peptone, 10.0 glucose, 0.6 KH₂PO₄, 0.001 ZnSO₄, 0.4 KH₂PO₄, 0.0005 FeSO₄, 0,05 MnSO₄, and 0,5 MgSO₄ and the fungi exhibited strong growth^45,46. Sterile Koroljova–Skorobogat’ko medium was dispersed into sterile 250 ml Erlenmeyer flasks at a rate of 50 ml of medium per flask. The flasks were inoculated with homogenized mycelia suspension and incubated in an orbital shaker at 30 °C and at a rate of 200 rpm. The flasks with growing cultures of white root fungi were removed at different time intervals over the course of the experiment for processing. MnP and LiP were isolated from fungal culture slants in a Koroljova liquid broth medium (10 ml) after 24, 48, 72, 96 and 120 h to obtain mycelia and spores by centrifugation at 4 °C (10,000 rpm for 10 min). All purification and protein concentration steps were performed at 4 °C. Small pre-weighed quantities of ammonium sulphate were added to 250 ml of culture supernatant from 20 to 80% saturation. Each precipitated fraction was separated by centrifugation at 10,000 rpm for 15 min at 4 °C, dissolved in a minimum volume of 0.1 M Tris–HCl (pH 9.0) and dialyzed twice for 6–8 h against the same buffer. The dissolved fractions were stored at 4 °C. Ligninolytic enzyme activity (LiP and MnP) was estimated in the crude culture filtrate and ammonium sulphate precipitates using the standard protocols described below.

Enzyme assay

MnP activity was measured by the oxidation of MnSO₄ (1.0 mM) substrate. Reactions were conducted in a 3 ml cuvette containing 2.5 ml of buffer (20 mM sodium tartrate at pH 4.5), 1.0 ml of substrate, 1.0 ml of enzyme extract from the supernatant, and 0.5 ml of 2.0 mM H₂O₂. Manganese peroxidase was determined spectrophotometrically at 238 nm⁴⁷. Lignin peroxidase was evaluated using veratryl alcohol (2.0 mM) as a substrate. The reactions were carried out in 3 ml cuvettes containing 1.25 ml of 50 mM sodium tartrate buffer at pH 2.5, 0.5 ml of enzyme extract, 0.25 ml of substrate, and 0.5 ml of 500 μM H₂O₂. Lignin peroxidase was measured at 310 nm. All oxidative enzymatic activities were expressed as units (U) per millilitre (i.e., one millimole of substrate oxidized per minute)⁴⁸.

The standard Bradford method was used to estimate the concentration of proteins in the supernatant. Bovine serum albumin (BSA) was used as a standard. One millilitre of enzyme extract was mixed with 1 ml of Bradford reagent (Amresco, USA) and incubated for five days. Protein concentrations were measured at 595 nm. Approximately 3 ml of mixed enzyme extract was used for the identification of MnP and LiP. We used Type II horseradish peroxidase (Sigma Aldrich) dissolved in phosphate buffer as an enzyme standard. HPLC–MS/MS (GE Healthcare, USA) was used for protein separation. The outflow was monitored at 280 nm for protein detection. The fractions were assayed for MnP and LiP activity and total protein content.

Experiment 1: incubation under biotic and abiotic conditions

Four replicates of 13 g of moist (80% of field capacity) sterilized soil (abiotic) and another portion of non-sterilized soil (biotic) were incubated in serum bottles (120 mml) at 20 °C in anaerobic conditions. Another round of abiotic and biotic incubation was conducted under aerobic conditions. Serum bottles were equipped with a septum for gas sampling. Anaerobic incubations were previously tested by injecting oxygen-free gas (N₂) into the headspace of each serum bottle until < 2% O₂ was reached (PCE Instrument model PCE-228-R, Germany)⁴⁹. Anaerobic conditions were monitored and CO₂ was collected after 0.3, 4, 8, 12, 24 and 36 h of incubation. Preliminary studies indicate that after further incubation for > 3 days, CO₂ levels increased little. For gas sampling, 10 ml was extracted using a syringe, and this was then injected into a gas chromatograph coupled with thermal conductivity and a flame photometric detector.

Experiment 2: induced fenton reactions

Fenton reactions were induced by adding various hydrogen peroxide (H₂O₂) and one Fe(II) concentration to all sterilized soils in ratios of 5:1, 10:1, and 20:1^17,50,51 by adding 120–143 ml, 0.1 M H₂O₂ and 1.29 g Fe²⁺ kg⁻¹ soil as FeCl₂ (Sigma Aldrich, USA).

Experiment 3: LiP and MnP activity

Sterilized soils were inoculated with peroxidase enzymes and H₂O₂. Iron (II) was not added in this experiment because MnP and LiP do not require free Fe. Enzymatic activity in the soils was monitored for 36 h spectrophotometrically at 310 nm for LiP and at 238 nm for MnP using a Tecan Infinite 200 PRO spectrophotometer (Durham, NC). The Initial activity of both enzymes was determined to each soils previously to the sterilization. Detailed values of LiP (1.23 ± 0.9–2.31 ± 0.3 µg g⁻¹ soil) and MnP (2.69 ± 0.0–9.34 ± 1.1 µg g⁻¹ soil) are presented in Table 3. This experiment is referred to as Peroxidase.

Experiment 4: combined fenton reactions and ligninolitic enzymes LiP and MnP

One millilitre of inoculum from a combined extract of LiP and MnP was added to sterilized soils with the addition of H₂O₂ and Fe(II) to induce Fenton reactions as described above. The soils were incubated under anaerobic conditions (in Fenton + LiP + MnP).

Hydrogen peroxide and Fe(II)-HCl determination

Experiments 1–4 were performed in parallel for destructive sampling to monitor Fe(II)-HCl solubilization and hydrogen peroxide consumption. Hydrogen peroxide consumption was determined using the iodometric titration method⁵². This method measures the concentration (mg l⁻¹) of an oxidizing agent in solution. While it is somewhat less accurate than permanganate titration, it is less susceptible to interferences by SOM. The method has been applied to plant tissues^53,54 and soils⁵⁵. In brief, at each sampling time soil samples were frozen to − 18 °C to stop enzymatic activity. Then, 150 mg of frozen samples were homogenized with 1 ml of solution containing 0.25 ml of 0.1% trichloroacetic acid, 0.5 ml of 1 M KI, and 0.25 ml of 10 mM potassium phosphate buffer. The homogenized suspension was incubated at 4 °C for 10 min. H₂O₂ content were monitored spectrophotometrically at 390 nm and final values are expressed in μmol g⁻¹ of fresh weight soil⁴⁸. H₂O₂ consumption (%) was estimated as:

The initial H₂O₂ content from each soils were determined previously to sterilization and ranged between 25.6 ± 0.7 and 33.7 ± 0.5 µM g⁻¹ soil (Table 3).

Free radical detection

The presence of hydroxyl radicals in the soil was first tested using hexanol substrate. Approximately, 5 g of each sterilized soil (13 replicates) was incubated for 36 h in anaerobic conditions with 5 ml of hexanol (2 mM) (Sigma-Aldrich). A H₂O₂:Fe(II) ratio of 10:1 was used to induce Fenton reactions (see below). Hydroxyl radicals do not react strongly with the superoxide anion but with hexanol, as hydroxyl radicals oxidize preferentially short-range organic molecules^23,24. In total, 39 soil samples were analysed for 10 randomly selected regions of interest (ROI, 33,489 μ²). Hexanol oxidation was calculated as the difference between the initial amount and the amount of hexanol remaining. The hexanol was quantified using a Hewlett-Packard 5890A gas chromatograph (Thermo Fisher, Waltham, Massachusetts USA) with a flame ionization detector equipped with a 15 m × 0.53 mm DB-1 capillary column.

In the fluorescence experiment, the generation of ·OH radicals was examined in anaerobic and sterilized soils using a 2′,7′-dichlorodihydrofluorescein diacetate (DCFH₂)²⁵ fluorescent probe in an excitation/emission of 488/530 nm and via laser scanning confocal microscopy (CLSM) (Olympus Fluoview 1000, Florida, USA). Maximum fluorescence emissions were found for a ratio of 10:1 H₂O₂:Fe(II). Using a closed vacuum plate with 50 μl of the fluorescent DCFH₂ probe⁵⁶, the samples were analysed for free radicals after 36 h of incubation⁵⁷. The emissions observed using CLSM were attributed to hydroxyl radicals reacting in the soil. The images were processed using image processing software (software FV10-ASW v.0.2c), and fluorescence intensity was expressed as relative fluorescence (AU) as given by the software. A control soil without H₂O₂ and Fe(II) additions was also included.

Statistical analysis

Two-way ANOVAs were conducted to determine significant differences in CO₂ released, hydrogen peroxide consumed, and Fe(II)-HCl solubilized for experiment 1. To test differences for the same variables in the second, third, and fourth experiments, one-way ANOVAs were performed. Regressions between CO₂ and H₂O₂ or Fe(II)-HCl extractable and between H₂O₂ and Fe(II)-HCl extractable were performed. All analyses were conducted using XLSTAT software by Addinsoft (Premium) 2019, version 4.1. Significant differences were set at a p value of 0.05. Datasets were tested for normal distributions and homoscedasticity. Datasets abnormally distributed were log transformed when necessary.

Source: Ecology - nature.com