Apparent microbial carbon use efficiency in the fertosphere: short-term responses to nitrogen fertilizer granule dissolution in tropical soil Eduardo Mariano a,b,c,* , Ciro A. Rosolem d , J´essica P.Q. Barcelos a,b,d , Bruna Arruda a,e,f

,

Lílian A. Moreira c , David R. Chadwick a , Davey L. Jones a a Environment Centre Wales, School of Natural Sciences, Bangor University, Gwynedd, LL57 2UW, UK b College of Agricultural Sciences, S˜ao Paulo State University, Av. Universit´aria, 3780, Altos do Paraíso, Botucatu, SP, 18610-034, Brazil c Center for Nuclear Energy in Agriculture, University of S˜ao Paulo, Av. Centen´ario, 303, S˜ao Dimas, Piracicaba, SP, 13416-000, Brazil d University of Western S˜ao Paulo, Rod. Raposo Tavares, km 572, Presidente Prudente, SP, 19067-175, Brazil e Luiz de Queiroz College of Agriculture, University of S˜ao Paulo, Av. P´adua Dias, 11, S˜ao Dimas, Piracicaba, SP, 13418-900, Brazil f Agronomy Engineering Program, University of Applied and Environmental Sciences, Calle 222 # 55-37, Bogot´a, 5145554, Colombia A R T I C L E I N F O Keywords:

14C tracer Carbon mineralization Nitrogen fertilization Fertosphere Phosphorus limitation Soil microsite A B S T R A C T Understanding short-term microbial responses to N fertilizer granule dissolution is essential for elucidating how C substrates are transformed within localized fertilizer–soil microsites, hereafter the fertosphere. We conducted a 20-d laboratory incubation to evaluate the mineralization and apparent microbial C use efficiency (aCUE) of four 14C-labeled substrates — glucosamine, glucose, glycine, and malic acid — added to a tropical clayey soil treated with six granular N fertilizers: ammonium nitrate, ammonium sulfate, diammonium phosphate (DAP), potassium nitrate, struvite, and urea. Apparent microbial C use efficiency was operationally defined as the fraction of substrate label allocated to the slower-turnover pool of a double first-order exponential model, representing a kinetic proxy rather than a direct measure of biosynthetic efficiency. Nitrogen fertilizers stimulated 14C mineralization of labile substrates, particularly urea, which induced the highest 14CO2 emission and the lowest aCUE, concurrent with a pronounced alkalinization (pH up to 8.9) driven by urea hydrolysis. In contrast, DAP and struvite increased glycine half-life and aCUE relative to the unfertilized control, consistent with relief of P limitation in this low-P Ferralsol. Glucose and malic acid were predominantly allocated to the slower (anabolic) pool, whereas glucosamine and glycine were primarily respired. The aCUE reduction was greater for N-con­ taining than for C-only substrates. Overall, granular N fertilizers did not promote short-term retention of lowmolecular-weight C substrates in the fertosphere and may instead accelerate their mineralization. These find­ ings underscore that microsite-scale variations in aCUE can transiently modulate soil C turnover in fertilized tropical systems.

1. Introduction

In intensive cropping systems, synthetic N fertilizers are generally preferred over organic sources because they are cheaper, easier to transport, more straightforward to apply, and more readily available to plants (Robertson and Vitousek, 2009). However, their overall efficiency in crop N recovery remains low and variable, often leading to N losses that compromise ecosystem health and water quality (Conijn et al., 2018). While the negative effects of fertilizer N losses on air quality and freshwater ecosystems are well recognized, the short-term microbial response at the fertilizer–soil interface — hereafter referred to as the fertosphere — has received comparatively little attention. This interface exhibits nutrient concentrations orders of magnitude higher than those of the surrounding bulk soil and is the site of strong physicochemical gradients that can regulate microbial activity and C turnover. Laboratory incubations investigating fertilizer effects on soil func­ tion have typically added N in soluble forms at uniform rates (Ladd et al., 1992; Zagal and Persson, 1994; Saggar et al., 2000), usually in the range 0.01–0.075 mM, which falls well below the sub-lethal thresholds documented for microbial activity near dissolving granules (Ruiz et al., 2020). Although such approaches ensure homogeneity of nutrient dis­ tribution, they fail to capture the spatial heterogeneity that

* Corresponding author. Center for Nuclear Energy in Agriculture, University of S˜ao Paulo, Av. Centen´ario, 303, S˜ao Dimas, 13416-000, Piracicaba, SP, Brazil.

E-mail address: emariano@cena.usp.br (E. Mariano).

Contents lists available at ScienceDirect Soil Biology and Biochemistry journal homepage: www.elsevier.com/locate/soilbio https://doi.org/10.1016/j.soilbio.2026.110215 Received 25 October 2025; Received in revised form 1 June 2026; Accepted 3 June 2026 Soil Biology and Biochemistry 221 (2026) 110215 Available online 10 June 2026 0038-0717/© 2026 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license ( http://creativecommons.org/licenses/by/4.0/ ).

characterizes field conditions and can overlook the pronounced pH and ionic-strength fluctuations that accompany granule dissolution and that are known to shape microbial community composition and function (Malik et al., 2018; Jones et al., 2019). Granular N fertilizers generate localized, transiently harsh conditions in which mineral N (NH4 + + NO3 −) concentrations can exceed 4000 mg kg−1 (Yadvinder-Singh and Beauchamp, 1988; Geisseler and Scow, 2014). Ammonium-based fer­ tilizers can further inhibit microbial activity at high NH3 concentrations (Geisseler and Scow, 2014), whereas urea dissolution raises soil pH through urease-mediated proton consumption (Sommer et al., 2004), and nitrate-based fertilizers can induce osmotic stress without directly altering pH. The overall effect of N salts on soil pH and ionic strength is therefore fertilizer-specific, and depends on both the anionic partner and the rate of microbial transformation of the applied N. Quantifying how such transient conditions influence microbial C use efficiency (CUE) is central to understanding soil C cycling in fertilized agroecosystems. CUE describes the partitioning of assimilated C be­ tween biosynthesis and catabolism (Manzoni et al., 2012; Sinsabaugh et al., 2013) and, in global analyses, scales with soil C storage — higher CUE is associated with greater microbial necromass accumula­ tion and with larger soil C stocks (Tao et al., 2023). In short-term 14C tracer experiments, an apparent CUE (aCUE) is commonly used as a kinetic proxy for biomass partitioning, inferred from two-pool decay modeling of substrate-derived 14CO2 rather than measured directly as 14C incorporation into microbial biomass (Creamer et al., 2014, 2016; Glanville et al., 2016; Jones et al., 2018). While aCUE can be influenced by processes other than biomass synthesis — including substrate sorp­ tion–desorption, intracellular storage, and partial mineralization of structural components — it provides an internally consistent basis for comparing fertilizer and substrate treatments under identical incubation conditions. The response of CUE to N addition is, however, mechanis­ tically ambiguous. On the one hand, stoichiometric theory predicts that alleviation of microbial N limitation should increase CUE by enabling greater biomass synthesis per unit C assimilated (Sinsabaugh et al., 2013; Manzoni et al., 2017). On the other hand, N salts can impose osmotic and pH stresses that divert C to maintenance respiration and to the synthesis of compatible solutes such as glycine betaine, trehalose, and proline (Schimel et al., 2007; Warren, 2014), with empirical esti­ mates indicating that osmotic acclimation can consume part of the assimilated C and that salt-induced stress depresses CUE by up to 30–50% in soil microbial communities (Mavi et al., 2012; Rath et al., 2019; Brown et al., 2021, 2022; Dong et al., 2022). These opposing mechanisms are likely to be most pronounced at the fertilizer–soil interface, where solute concentrations and pH gradients are strongest and where CUE responses have rarely been resolved under realistic granular loading.

Glucose has been widely used to trace soil C turnover following N additions (Zagal and Persson, 1994; Saggar et al., 2000; Mariano et al., 2016) because it is rapidly assimilated by diverse microbial taxa. Yet amino acids, amino sugars, and organic acids are also abundant in soil solution and in fresh organic inputs (Jones, 1998; Jones et al., 2005; van Hees et al., 2005), and their fates differ from glucose because they carry N and bind differently to soil surfaces (Jones and Brassington, 1998; Kuzyakov and Jones, 2006; Roberts et al., 2007). Position-specific 13C and 14C labeling has shown that the C skeleton of amino acids and amino sugars can be incorporated directly into proteins, peptidoglycan, and chitin without passing through the tricarboxylic-acid cycle (Knowles et al., 2010; Dippold and Kuzyakov, 2013; Apostel et al., 2015; Gunina and Kuzyakov, 2015). Direct incorporation, however, coexists with mineralization–immobilization–turnover pathways, in which part of the substrate C is respired to generate energy while the substrate-derived N may be retained, mineralized, or recycled within the microbial pool (Barraclough, 1997; Geisseler et al., 2010). The relative contribution of these routes determines how much substrate-derived C is recovered as CO2 versus retained in slower-turnover pools. For N-containing substrates such as glycine and glucosamine, the available evidence indicates that a substantial fraction of the C skeleton is respired even when external mineral N is non-limiting, because the substrate itself supplies the N required by the microbial pool (Barraclough, 1997; Knowles et al., 2010; Roberts et al., 2007). In contrast, for C-only substrates such as glucose and low-molecular-weight (LMW) organic acids, biomass synthesis is contingent on concurrent acquisition of N from the soil mineral pool, so a larger fraction of substrate C tends to be incorporated into biomass and a smaller fraction respired when mineral N is available (Bremer and Kuikman, 1994; Manzoni et al., 2012). Malic acid follows a broadly similar pattern to glucose but is additionally subject to strong sorption on Fe-oxide surfaces in highly weathered soils such as Ferralsols (Jones and Brassington, 1998; van Hees et al., 2005; Strickland and Rousk, 2010). Few studies have examined how mineral N affects substrates other than glucose (Chen et al., 2007; de Sosa et al., 2018), and to our knowledge, no work has resolved fertosphere-scale aCUE responses across granular N sources and LMW substrate classes. To address this gap, we evaluated the short-term mineralization and aCUE of four 14C-labeled LMW substrates — glucose, malic acid, glycine, and glucosamine — applied directly beneath individual granules of six commonly used N fertilizers [ammonium nitrate, ammonium sulfate, diammonium phosphate (DAP), potassium nitrate, struvite, and urea] in addition to an unfertilized control. We tested two directional hypothe­ ses: (H1) The high local concentrations of N salts in the fertosphere will reduce aCUE relative to the unfertilized control across the four 14Clabeled substrates, because the C cost of acclimating to transient pH and ionic-strength stresses outweighs any stoichiometric benefit of N supply; and (H2) The magnitude of the fertosphere-induced reduction in aCUE will be substrate-dependent: greater for N-containing substrates (glycine and glucosamine) and smaller for C-only substrates (glucose and malic acid).

2. Materials and methods

2.1. Soil sampling and characterization

Four independent soil samples were collected from the top 15 cm within an approximately 50 m2 area of a non-saline (electrical conductivity = 93 ± 8 μS cm−1) field at Botucatu, S˜ao Paulo, south­ eastern Brazil (22◦49′S, 48◦26′W; 700 m a.s.l.) using a spade. At the time of sampling, the field was under fallow following a mixed tropical grass cover including signal grass (Urochloa decumbens, syn. Brachiaria decumbens), ruzigrass (U. ruziziensis, syn. B. ruziziensis), and Guinea grass (Megathyrsus maximus, syn. Panicum maximum). The soil is classified as a Rhodic Ferralsol (IUSS Working Group WRB, 2022) with a clay texture. The mean annual temperature and rainfall at the site are 20.3 ◦C and

1501 mm, respectively. Samples were sieved moist (<5 mm) to remove

visible roots, thoroughly homogenized, and pre-incubated at 20 ± 1 ◦C at 60% of their maximum water-holding capacity (WHC), following Mariano et al. (2017).

Particle-size distribution was determined by the hydrometer method (Gee and Bauder, 1986). Soil pH and electrical conductivity were measured in a 1:2.5 (w/v) soil:distilled water suspension using standard electrodes. Total C and N were quantified by dry combustion with a CHN-2000 analyzer (LECO, St. Joseph, MI, USA). Basal respiration was measured at 20 ◦C over 48 h with an automated EGM-5 CO2 infrared gas analyzer (PP Systems, Amesbury, MA, USA). Permanganate-oxidizable (active) C (Culman et al., 2012) was quantified by KMnO4 oxidation and colorimetry in a 96-well microplate spectrophotometer (Power­ Wave HT; BioTek Instruments, Winooski, VT, USA). Extractable micro­ bial biomass C and N were estimated by the chloroform-fumigation extraction method using 0.5 M K2SO4 (Brookes et al., 1985; Vance et al., 1987). Dissolved organic C (DOC) and total dissolved N (TDN) were measured in fumigated and unfumigated extracts on a multi N/C 2100S TOC-TN analyzer (Analytik Jena, Jena, Germany). Values are reported as extractable microbial biomass C and N (the difference between E. Mariano et al.

Soil Biology and Biochemistry 221 (2026) 110215

fumigated and unfumigated extracts) without applying a kEC/kEN correction factor, because site- and soil-specific extraction efficiencies have not been determined empirically for this Ferralsol. Extractable P was determined by ion-exchange resin followed by colorimetry, and exchangeable K, Ca, and Mg were determined by ionexchange resin followed by atomic-absorption spectroscopy (van Raij et al., 2001). Extractable S was determined as SO4 2−after Ca(H2PO4)2 extraction and turbidimetry (van Raij et al., 2001). Potential acidity (H + Al) was measured using the SMP buffer method (Shoemaker et al., 1961). Cation-exchange capacity (CEC) at pH 7.0 was calculated as the sum of exchangeable cations (K, Ca, and Mg) plus H + Al. Base saturation was calculated as the ratio of the sum of exchangeable bases to CEC × 100.

Soluble C and N were measured in 0.5 M K2SO4 extracts as described above. Samples were centrifuged at 8000 × g for 10 min, and the su­ pernatant was stored at −20 ◦C until analysis. Nitrate and NH4 + were determined colorimetrically following Miranda et al. (2001) and Mulvaney (1996), respectively, while DOC and TDN were analyzed as described above. Dissolved organic N was calculated as the difference between TDN and mineral N (NH4 + + NO3 −). Total free amino acids were measured by the fluorometric o-phthaldialdehyde–β-mercaptoethanol method (Jones et al., 2002), and total dissolved phenolics by reaction with the Folin–Ciocalteu reagent (Swain and Hillis, 1959). All physical, chemical, and biological soil properties are summarized in Table 1.

2.2. Experimental setup and treatments

The experiment followed a completely randomized factorial design, with seven N treatments (six granular N fertilizers plus an unfertilized control), four 14C-labeled LMW C substrates, and four independent replicates, giving a total of 112 experimental units (7 × 4 × 4). A con­ ceptual diagram of the experimental setup and sampling workflow is provided in Fig. 1.

For each experimental unit, 6.3 g of moist soil (equivalent to 5 g dry mass) was transferred into a sterile 50-mL polypropylene centrifuge tube. A single granule of ammonium nitrate (NH4NO3; 33% N), ammonium sulfate [(NH4)2SO4; 21% N and 24% S], DAP [(NH4)2HPO4; 18% N and 20% P], potassium nitrate (KNO3; 13% N and 37% K), or urea [(NH2)2CO; 46% N] was placed at the center of the soil surface. For struvite (NH4MgPO4⋅6H2O; 6% N, 13% P, 10% Mg), three granules were used to achieve an equivalent N input. All granules were individually selected for uniformity in shape, mass, and apparent density. Ammo­ nium sulfate and potassium nitrate granules were laboratory-prepared by slightly moistening reagent-grade salts and oven-drying them at

80 ◦C for 6 h. Commercial struvite granules recovered from wastewater

(Crystal Green®; Ostara Nutrient Recovery Technologies, Vancouver, BC, Canada) were used, and the remaining fertilizers were obtained from commercial suppliers.

Nitrogen was applied at a rate of 1600 mg N kg−1 dry soil, corre­ sponding to molar salt concentrations of 58–122 mmol kg−1 depending on the fertilizer. The rate was standardized on an N basis (rather than by ionic concentration) to enable meaningful comparison of soil mineral N transformations across sources. An unfertilized control (no granule added) was included. After fertilizer placement, tubes were hermetically sealed and incubated at 20 ◦C for 5 d to allow granule dissolution and fertosphere formation prior to substrate addition. By day 5, most gran­ ules had fully dissolved; DAP was nearly fully dissolved, and struvite remained only partially dissolved, consistent with its known slow dissolution kinetics (Degryse et al., 2017). On the same day, 250 μL of 14C-labeled substrate solution (100 mM; 6.7 kBq mL−1) was pipetted precisely onto the location where the granule had been placed. The four substrates were N-acetyl-D-[1-14C]-glucosamine (American Radio­ labeled Chemicals Inc.), D-[U–14C]-glucose (PerkinElmer Inc., Bea­ consfield, UK), [U–14C]-glycine (American Radiolabeled Chemicals Inc., St. Louis, MO, USA), and L-[U–14C]-malic acid (GE Healthcare, Buck­ inghamshire, UK). These substrates represent the four main classes of LMW compounds found in the soil solution — simple sugars, organic acids, amino acids, and amino sugars — and were selected because they differ in C:N stoichiometry and in their affinity for mineral surfaces. The

100 mM concentration matches that used by Creamer et al. (2016) and

approximates the concentration of these solutes in root cell sap that would be released upon lysis in the rhizosphere (Jones and Darrah, 1996; Ciereszko et al., 1999). The combined use of a locally high sub­ strate concentration and localized granular N was intentionally selected to generate a well-defined energy pulse that would resolve anabolic versus catabolic responses under the extreme physicochemical condi­ tions of the fertosphere. Results should therefore be interpreted as fundamental microbial physiological responses to concurrent nutrient and stress pulses rather than as absolute in situ turnover rates.

2.3. Microbial 14C uptake and mineralization

Respired 14CO2 was trapped in a 4-mL polypropylene vial containing

1 mL of 1 M NaOH that was suspended above the soil surface inside each

tube. Tubes were then sealed and re-incubated in the dark at 20 ◦C. Water loss during incubation was negligible. NaOH traps were replaced at 0.08, 0.17, 0.33, 1, 2, 3, 5, 8, 11, 15, and 20 d after 14C addition — i.e., 2, 4, and 8 h in the first day, and daily to every few days thereafter — a schedule designed to capture both the rapid first-order catabolic phase and the slower subsequent phase of 14CO2 evolution (Glanville et al., 2012, 2016). Trapped 14C–CO3 2- was quantified by liquid scintillation counting (Wallac 1404, PerkinElmer).

At the end of the incubation (20 d), 25 mL of distilled water was added to each tube, and soil pH was measured in a 1:2.5 soil:water ratio (w/v) using a standard electrode. Then, 1.86 g of K2SO4 was added to each tube to produce a 0.5 M K2SO4 solution, which displaces substratederived C sorbed to the solid phase. Soil suspensions were shaken on a reciprocating shaker at 200 rev min−1 for 30 min and centrifuged at 18,000 × g for 3 min 14C in the K2SO4 supernatant was measured by liquid scintillation counting, and NH4 + and NO3 − were determined colorimetrically. We acknowledge that 0.5 M K2SO4 efficiently extracts Table 1 Selected physical, chemical, and biological properties of the 0–15 cm layer of the tropical clayey Ferralsol, measured prior to experimentation. Values represent means ± SEM (n = 4).

Soil property Mean ± SEM Sand (g kg−1)

228 ± 6

Silt (g kg−1)

215 ± 12

Clay (g kg−1)

558 ± 14

pH (1:2.5, H2O)

5.5 ± 0.2

Electrical conductivity (μS cm−1)

93 ± 8

Total C (g kg−1)

21 ± 1

Total N (g kg−1)

2.4 ± 0.1

Extractable microbial biomass C (mg kg−1)a

146 ± 6

Extractable microbial biomass N (mg kg−1)a

17 ± 1

Dissolved organic C (mg kg−1)

142 ± 15

Dissolved organic N (mg kg−1)

0.75 ± 0.73

Exchangeable NH4 +-N (mg kg−1)

0.15 ± 0.08

Exchangeable NO3 −-N (mg kg−1)

29 ± 3

Free amino acids (mg N kg−1)

0.15 ± 0.02

Dissolved phenolics (mg kg−1)

1.1 ± 0.2

Permanganate-oxidizable C (mg kg−1)

429 ± 22

Basal respiration (mg CO2–C kg−1 d−1)

8.0 ± 0.6

Extractable P (mg kg−1)

11 ± 0

Extractable S (mg kg−1)

6 ± 0

Exchangeable K (mmolc kg−1)

3.1 ± 0.3

Exchangeable Ca (mmolc kg−1)

24 ± 2

Exchangeable Mg (mmolc kg−1)

12 ± 1

Potential acidity (H + Al; mmolc kg−1)

39 ± 3

Cation-exchange capacity at pH 7.0 (mmolc kg−1)

79 ± 1

Base saturation (%)

40 ± 4

Maximum water-holding capacity (g kg−1)

457 ± 9

a Extractable microbial biomass C and N correspond to fumigated minus unfumigated 0.5 M K2SO4 extracts; no kEC/kEN correction factor was applied. E. Mariano et al.

Soil Biology and Biochemistry 221 (2026) 110215

glucose, amino sugars, and amino acids, but may not fully desorb malic acid, which is known to strongly bind to mineral surfaces of Fe-oxiderich Ferralsols. All procedures involving 14C were performed in certi­ fied facilities under standard institutional radiation-safety protocols.

2.4. Calculations and statistical analysis

Low-molecular-weight compounds dominate the rapid component of total soil CO2 flux because of their fast microbial turnover (van Hees et al., 2005). Their mineralization typically follows a biphasic pattern of 14CO2 release (Chotte et al., 1998; Glanville et al., 2016), which can be described by a double first-order exponential decay model fitted to the cumulative 14C remaining in soil using the “nls” function in R v4.2.2 (R Core Team, 2022):

14Csoil = Ccatab × e(−kcatab × t) + Canab × e(−kanab × t) (1) where 14Csoil is the proportion of added 14C remaining in soil at time t (d); Ccatab and Canab are the model-derived fractions of applied 14C par­ titioned to the fast-turnover (catabolic), and slow-turnover (anabolic) pools, respectively; and kcatab and kanab are the corresponding first-order rate constants (d−1). Ccatab and Canab were obtained directly as the fitted coefficients of Eq. (1) and represent the fractions of applied 14C parti­ tioned to the fast- and slow-turnover pools, which by the initial condi­ tion at t = 0 sum to the 100% of 14C initially added. The half-life of the fast (catabolic) pool was calculated as: t½,catab = ln(2) / kcatab

(2)

A direct analytical half-life for the slow anabolic pool (Canab) cannot be obtained from Eq. (1) alone because connectivity and dilution be­ tween the two pools are not explicitly resolved (Glanville et al., 2016). The total substrate half-life (t½,total), accounting for both pools, was therefore estimated numerically. The amount of 14C remaining in soil at the combined half-life was defined following Glanville et al. (2012) and Creamer et al. (2014) as:

14Csoil,½ = (Ccatab + Canab) / 2 (3) This value was then substituted into Eq. (1) and t½,total was solved numerically by Newton–Raphson iteration. 14C not recovered in either the NaOH traps or the 0.5 M K2SO4 extracts was assumed to be incor­ porated into the extractable soil microbial community. Fig. 1. Schematic representation of the experimental workflow. (1) A single granule (three for struvite, to provide an equivalent N input) of one of six granular N fertilizers (ammonium nitrate, ammonium sulfate, DAP, potassium nitrate, struvite, or urea), or none in the unfertilized control, was placed in 6.3 g of moist soil in a 50 cm3 polypropylene tube and pre-incubated for 5 d to allow granule dissolution and fertosphere formation. (2) On day 5, 250 μL of a14C-labeled low-molecularweight substrate solution (100 mM; glucosamine, glucose, glycine, or malic acid) was pipetted onto the dissolution spot. (3) Respired 14CO2 was trapped in 1 mL of 1 M NaOH and quantified by liquid scintillation counting at 11 time points over 20 d (4) At day 20, soil pH, K2SO4-extractable mineral N (NH4 + and NO3 −), and K2SO4extractable 14C were measured. The seven N treatments and four substrates were combined factorially in four replicates (total n = 112). E. Mariano et al.

Soil Biology and Biochemistry 221 (2026) 110215

Apparent microbial C use efficiency (aCUE) was calculated from the fitted coefficients of Eq. (1) as:

aCUE = Canab / (Ccatab + Canab) (4) The term “apparent” reflects the model-inferred nature of Canab (Creamer et al., 2014, 2016; Glanville et al., 2016; Jones et al., 2018). We therefore base our conclusions on within-experiment contrasts among fertilizer × substrate treatments and interpret absolute aCUE values cautiously. The assumption of negligible loss of 14C to non-extractable pools implies that partial retention of strongly sorbed compounds (e.g., malic acid) may slightly underestimate aCUE. All statistical analyses were performed in R v4.2.2 and RStudio v2022.12.0 (Posit Software, Boston, MA, USA). Two-way ANOVA based on generalized linear models (GLMs) was used to assess the main effects of N fertilizer and C substrate, as well as their interaction, at α = 0.05. Model diagnostics included visual inspection of residuals versus fitted values and residual distributions using the “rcompanion” package (Mangiafico, 2022). Estimated marginal means and Tukey HSD post-hoc tests were computed using the “car”, “emmeans”, and “multcomp” pack­ ages (Hothorn et al., 2008; Fox and Weisberg, 2019; Lenth, 2022). Where interactions were not significant, only main effects were inter­ preted. All figures were produced with “ggplot2” (Wickham, 2016).

3. Results

3.1. Apparent microbial C use efficiency and substrate mineralization

All four LMW substrates were rapidly mineralized after addition, and the 14CO2 release patterns followed a clear biphasic trajectory (Fig. 2). The double first-order exponential decay model described the data well across all fertilizer × substrate combinations (r = 0.994 ± 0.001, mean ± SEM; n = 112). Nitrogen addition generally increased the pro­ portion of 14C partitioned to the fast catabolic pool (Ccatab) and reduced the proportion partitioned to the slow anabolic pool (Canab) (Supplementary Table S1). Exceptions were observed for DAP and struvite applied together with glycine, which showed the opposite pattern — a decrease in Ccatab and an increase in Canab. The rate constant of the fast pool (kcatab) was similar to or lower than that of the unfer­ tilized control for most treatments, except for malic acid combined with DAP, which yielded the highest kcatab. The rate constant of the slow pool (kanab) was reduced under urea, most markedly for glucose and glucosamine.

By the end of the incubation, the residual 14C in urea-treated soil was, on average, 27% and 51% lower than in the other N-fertilizer treatments for glucosamine and glucose, respectively (Supplementary Fig. S1ab). For glycine, both potassium nitrate and urea reduced resid­ ual 14C by about 51% relative to the other N treatments (Supplementary Fig. S1c). For malic acid, ammonium nitrate and urea reduced the re­ sidual 14C by approximately 21% compared with the other N treatments, except for potassium nitrate (Supplementary Fig. S1d). Across fertilizer treatments, glycine consistently showed the lowest 14C remaining in soil. Except for glycine, N addition did not increase aCUE (Fig. 3). For most substrates, aCUE under fertilizer treatments was equal to or lower than that of the unfertilized control. For glucosamine, aCUE under DAP was on average 32% higher than under ammonium nitrate, ammonium sulfate, and potassium nitrate (Fig. 3a). Urea decreased glucose aCUE by 20% on average compared with the other N treatments (Fig. 3b). For glycine, DAP and struvite produced the highest aCUE values (46% higher than in the unfertilized control), whereas potassium nitrate and urea produced the lowest (Fig. 3c). For malic acid, potassium nitrate increased aCUE by 22% on average relative to ammonium nitrate, struvite, and urea (Fig. 3d). The substrate-level means (n = 28) were Fig. 2. Amount of 14C remaining in soil after addition of (a) glucosamine, (b) glucose, (c) glycine, and (d) malic acid, in a tropical clayey Ferralsol as affected by granular N fertilizers. Symbols represent means (n = 4) and error bars the SEM; where not visible, error bars are smaller than the symbol. Solid lines are fits of the double first-order exponential decay model (Eq. (1)). E. Mariano et al.

Soil Biology and Biochemistry 221 (2026) 110215

0.48 ± 0.01 (glucosamine), 0.75 ± 0.01 (glucose), 0.37 ± 0.03 (glycine), and 0.59 ± 0.01 (malic acid), with an overall mean of 0.55 ± 0.02 across all substrates (n = 112).

Total substrate half-life (t½,total) was strongly affected by N fertil­ ization (Fig. 4). For glucosamine, N fertilizers reduced t½,total by an average of 77% compared with the unfertilized control (Fig. 4a). Simi­ larly, for glucose, all N fertilizers except struvite reduced t½,total by an average of 55% (Fig. 4b). For malic acid, ammonium nitrate and urea reduced t½,total by 75% on average relative to the unfertilized control (Fig. 4d). In contrast, DAP and struvite increased the glycine t½,total by an average of 1496% relative to the other N treatments (Fig. 4c). Among all substrates, glucose had the longest t½,total (44 d), whereas glucosamine, glycine, and malic acid had estimated t½,total values of 17, 11, and 15 d, respectively.

3.2. Substrate remaining in the K2SO4-extractable pool

At the end of the incubation, most of the substrate-derived 14C had been either respired or immobilized in the non-extractable pool, as indicated by the consistently low 14C recovery in the K2SO4 extract across all treatments (Supplementary Fig. S2). Urea, however, was an exception: it produced markedly higher residual 14C in the extract than the other N treatments — on average 156% higher for glucosamine and 580% higher for glucose (Supplementary Fig. S2ab). A similar pattern was observed for glycine and malic acid, for which urea increased 14C recovery by 202% and 156%, respectively, compared with the other N treatments (excluding ammonium nitrate for glycine and potassium nitrate for malic acid; Supplementary Fig. S2cd). Across substrates, glucosamine had the highest average K2SO4-extractable 14C (1.9 ± 0.2% of the total added; n = 28).

3.3. Soil pH and mineral N

Final soil pH and NO3 −-N content were strongly affected by N fertil­ izers but not by C-substrate type, and no significant fertilizer × substrate interaction was detected (Fig. 5). Soil pH followed the order urea >

DAP >

struvite ≈ unfertilized control > ammonium nitrate ≈ammonium sulfate ≈potassium nitrate (Fig. 5a). Urea raised soil pH to 8.9, 3.4 pH units above the unfertilized control. Nitrate-based fertilizers (ammonium nitrate and potassium nitrate) produced the highest NO3 −-N contents in soil (Fig. 5b).

In contrast, soil NH4 +-N content showed a significant interaction between C substrate and N fertilizer (Fig. 6). For glucosamine, glycine, and malic acid, ammonium sulfate, DAP, and urea produced the highest and statistically similar NH4 +-N contents (Fig. 6acd). For glucose, the order was ammonium sulfate ≈

DAP >

urea > ammonium nitrate > potassium nitrate ≈struvite (Fig. 6b). Apparent recovery of applied mineral N (NH4 +-N + NO3 −-N, minus background values of the unfertilized control) were 95%, 92%, 94%, 96%, 2%, and 86% for ammonium nitrate, ammonium sulfate, DAP, potassium nitrate, struvite, and urea, respectively. The very low apparent recovery of struvite-N reflected its poor solubility and partial granule dissolution over 20 d. Fig. 3. Apparent microbial C use efficiency (aCUE; Eq. (4)) of 14C-labeled (a) glucosamine, (b) glucose, (c) glycine, and (d) malic acid in a tropical clayey Ferralsol as affected by granular N fertilizers. Error bars represent the SEM (n = 4). Means sharing common lowercase letters within each panel do not differ at the 5% probability level (Tukey HSD).

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4. Discussion

4.1. Granular N fertilization accelerates microbial C turnover in the

fertosphere Consistent with the first hypothesis, all granular N fertilizers stimu­ lated short-term microbial C mineralization and generally decreased aCUE relative to the unfertilized control. The one clear exception was glycine under DAP or struvite. Urea caused the largest 14CO2 release and the lowest aCUE, in agreement with previous reports that soluble N inputs can accelerate C mineralization of labile substrates (Saggar et al., 2000; Creamer et al., 2014; Zheng et al., 2019). Importantly, microbial activity was not suppressed in the fertosphere: CO2 evolution increased rather than decreased, indicating that the microbial community remained active even under the strong physicochemical gradients imposed by dissolving granules.

Under N fertilization, a larger fraction of each 14C-substrate was released as 14CO2 rather than retained in the non-extractable pool, and aCUE declined. This pattern is consistent with a shift toward mainte­ nance respiration, which is typically increased under salinity or pH stress because microbial cells allocate C to ion regulation and osmo­ protectant synthesis (Brown et al., 2022; Dong et al., 2022). Our data cannot resolve the relative contributions of cell lysis, maintenance respiration, or altered community composition, but the consistency of the aCUE decline across five of the six fertilizer treatments supports a general stress-driven mechanism. Over longer timescales, chronic N enrichment has been linked to slower decomposition and greater soil C retention (Tao et al., 2023); our observations therefore describe the immediate, microsite-scale response, which may differ from the cumulative field-scale outcome.

4.2. Substrate-specific responses to granular N fertilization

In agreement with the second hypothesis, the magnitude of the fertilizer-induced aCUE reduction was substrate-dependent: under the majority of N fertilizers, the N-containing substrates (glycine and glucosamine) showed the largest relative reductions, while the C-only substrates (glucose and malic acid) showed smaller reductions, except under urea (Fig. 3). The selective increase in aCUE of glycine and glucosamine under DAP and struvite departs from this pattern and is discussed next.

This asymmetric sensitivity is consistent with the contrasting meta­ bolic routes of these substrate classes. Glucose and malic acid enter central catabolism rapidly and are efficiently assimilated into biomass when external mineral N is available, because their biosynthetic use is stoichiometrically constrained by concurrent N acquisition from the soil pool (Bremer and Kuikman, 1994; Gunina and Kuzyakov, 2015). The additional mineral N supplied by the fertilizers therefore partially offsets the C cost of acclimating to the physicochemical stresses generated in the fertosphere. Glycine and glucosamine, in contrast, carry their own N and are at least partially channeled through the miner­ alization–immobilization–turnover route (Barraclough, 1997; Roberts et al., 2007; Geisseler et al., 2010); for these substrates, external min­ eral N provides no stoichiometric benefit, and the full C cost of stress acclimation is expressed as a reduction in aCUE. The low recovery of malic acid in the K2SO4-extractable pool (Supplementary Fig. S2d) likely reflects strong sorption of malic acid to Fe-oxide surfaces of this Ferralsol (Jones and Brassington, 1998), which may slightly underestimate its Fig. 4. Total substrate half-life (t½,total; combining Ccatab and Canab pools) estimated by Newton–Raphson iteration of Eq. (1) for 14C-labeled (a) glucosamine, (b) glucose, (c) glycine, and (d) malic acid in a tropical clayey Ferralsol as affected by granular N fertilizers. Error bars represent the SEM (n = 4). Means sharing common lowercase letters within each panel do not differ at the 5% probability level (Tukey HSD). E. Mariano et al.

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aCUE.

4.3. Effects of P-containing fertilizers on 14C-substrate fate

The two P-containing fertilizers, DAP and struvite, produced re­ sponses to N-rich substrates that departed from the general pattern described above. For glycine, both DAP and struvite reduced catabolic partitioning, increased aCUE by approximately 46% relative to the un­ fertilized control (Fig. 3c), and extended t½,total by an average of 1496% relative to the other N treatments (Fig. 4c). A smaller but consistent effect was observed for glucosamine, where DAP increased aCUE by 32% on average relative to ammonium nitrate, ammonium sulfate, and potassium nitrate (Fig. 3a). For the C-only substrates (glucose and malic acid), no comparable response was detected, and DAP and struvite did not differ from the other N treatments in either aCUE or t½,total (Fig. 3bd; 4bd).

The Ferralsol used here had low extractable P (11 mg kg−1; Table 1), within the range typically considered limiting for microbial growth in tropical soils. Under such conditions, simultaneous supply of N and P alongside an N-rich substrate is expected to relax stoichiometric con­ straints on microbial biosynthesis, allowing a larger fraction of assimi­ lated C to be allocated to growth rather than to respiration (Manzoni et al., 2012, 2017; Sinsabaugh et al., 2013). The selectivity of the response — restricted to the two N-containing substrates and absent for Fig. 5. Main effects of N fertilizer on (a) soil pH and (b) NO3 −-N content of a tropical clayey Ferralsol after 20 d of incubation. Error bars represent the SEM (n = 16). Means sharing common lowercase letters within each panel do not differ at the 5% probability level (Tukey HSD). E. Mariano et al.

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glucose and malic acid — matches this stoichiometric framework, since C-only substrates require concurrent acquisition of N from the soil mineral pool to support biomass synthesis (Geisseler et al., 2010) and would not be expected to benefit from added P alone. Two ancillary observations argue against alternative explanations based on pH or N supply. First, DAP produced only a modest increase in final soil pH (6.6), and struvite a value close to that of the unfertilized control (Fig. 5a), so the divergent response of these two fertilizers cannot be attributed to alkalinization. Second, struvite released very little sol­ uble N over the 20-d incubation (apparent mineral-N recovery of only 2%), yet produced the same direction of response as DAP for glycine, indicating that the effect was not driven by NH4 + concentrations com­ parable to those generated by the soluble ammonium-based fertilizers (Fig. 6c). The longer half-life and higher aCUE of glycine and, to a lesser extent, glucosamine, under DAP and struvite are therefore associated with the additional P supply rather than with the N input or pH change accompanying granule dissolution.

4.4. Fertilizer-specific effects on soil pH and their link to aCUE

The six granular N fertilizers produced strongly contrasting effects on soil pH after 20 d, which helps to explain the observed divergence in aCUE among fertilizers. Urea produced the largest alkalinization (final pH 8.9), in line with the well-known proton-consuming nature of ureasemediated urea hydrolysis (Sommer et al., 2004). DAP produced a more modest pH increase (final pH 6.6), consistent with the buffering action of the H2PO4

−/HPO4

2− pair and with the dissolution chemistry of this fertilizer. Ammonium nitrate and ammonium sulfate decreased pH slightly by 0.2–0.3 units relative to the control, consistent with nitrification proceeding over the 20-d incubation (Robertson and Vitousek, 2009); the nitrate-only salt (potassium nitrate) also pro­ duced a small acidification, which is harder to attribute to a single mechanism from our data alone. Across the fertilizer treatments, lower aCUE for glucose was associated with higher final soil pH, with urea standing out as the combination of the highest pH and the lowest aCUE — a pattern consistent with the pH-dependent respiration response re­ ported by Rousk et al. (2009).

Because pH was measured only at the end of the incubation, we could not follow the transient pH and ionic-strength excursions that are known to be most severe in the first few days after granule dissolution (Yadvinder-Singh and Beauchamp, 1989; Degryse et al., 2017). It is therefore likely that the peak physicochemical stress occurred within the first 48 h, during which the fastest phase of 14CO2 release was observed (Fig. 2). High local concentrations of monovalent cations (NH4 + and K+) and of NH3 near the granule would be expected to impose osmotic and toxicity stresses on microorganisms (Brown et al., 2021, 2022), forcing C allocation toward maintenance rather than biosynthesis. The shorter substrate half-lives observed under urea and ammonium nitrate (Fig. 4) are consistent with increased catabolic turnover under such conditions, although the relative contributions of pH, ionic strength, and NH3 toxicity cannot be disentangled in this experimental design.

4.5. Implications, limitations, and future research

Our data identify a short-term feature of fertilized soils: in the fer­ tosphere, granular N fertilizers reduced the apparent retention of labile C and depressed aCUE for most substrate × fertilizer combinations. Whether this microsite response scales to detectable changes in soil C Fig. 6. NH4 +-N content after 20 d of incubation with 14C-labeled (a) glucosamine, (b) glucose, (c) glycine, and (d) malic acid in a tropical clayey Ferralsol as affected by granular N fertilizers. Error bars represent the SEM (n = 4). BDL = below detection limit. Means sharing common lowercase letters within each panel do not differ at the 5% probability level (Tukey HSD).

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stocks at the field level depends on the spatial frequency and density of fertosphere events and on the long-term adaptation of microbial com­ munities, and cannot be resolved from this experiment alone. Among the fertilizers tested, struvite produced the smallest aCUE depression, in line with its slower dissolution kinetics over the 20-d incubation (Degryse et al., 2017). When N-rich substrates were co-applied with P-contain­ ing fertilizers in this low-P soil, aCUE was instead higher than in the unfertilized control, indicating that P availability can modulate the di­ rection of the fertosphere response.

Some limitations of our experimental design should be acknowl­ edged explicitly. First, we standardized N fertilizers by N content rather than by total ionic concentration; although this was necessary for interpreting mineral N transformations, it means that osmotic effects cannot be disentangled from N-specific effects. Second, microscale physicochemical gradients were not monitored in real time; future work combining isotopic tracing with microelectrode or planar-optode sen­ sors could resolve transient pH and ionic-strength dynamics, and highresolution X-ray imaging could map the physical microstructure of the fertosphere. Third, aCUE as defined here is a kinetic proxy inferred from a two-pool model rather than a direct measurement of biomass C incorporation; complementary approaches such as 18O–H2O incorpora­ tion into microbial DNA (Spohn et al., 2016) or direct 14C tracing into microbial biomass would provide independent validation of our aCUE values. Finally, our observations describe the immediate physiological response of microbial communities that were not pre-adapted to high-N conditions; Bao et al. (2024) reported that, in a long-term mai­ ze–soybean rotation, microbial CUE correlated positively with soil NH4 +, suggesting that long-term N enrichment can select for communities with higher growth efficiency. Reconciling short-term fertosphere responses with long-term community adaptation is an important direction for future research.

5. Conclusion

Granular N fertilizers generally decreased the short-term retention of 14C-labeled LMW substrates at the fertilizer–soil interface. Urea pro­ duced the largest 14CO2 release and the lowest aCUE, concurrent with a pronounced alkalinization of the fertosphere. DAP and struvite, the two P-containing fertilizers, departed from this general pattern: they slowed glycine turnover and increased its aCUE relative to both the unfertilized control and the other N fertilizers, an effect observed selectively for the N-rich substrates and associated with the additional P supply in a soil of low extractable-P status. Substrate chemistry strongly modulated the overall response: glucose and malic acid were preferentially partitioned to the slow-turnover pool, while glycine and glucosamine were prefer­ entially respired, and the fertilizer-induced aCUE reduction was greater for the N-containing substrates. These results indicate that granular fertilizers do not promote the short-term stabilization of labile C in the fertosphere and instead transiently accelerate its mineralization, except for N-rich substrates co-applied with P. The implications for field-scale C cycling will depend on the frequency and spatial density of fertosphere events and on the long-term adaptation of fertilized microbial communities.

Declaration of generative AI and AI-assisted tools During the preparation of this work, the authors used Claude (Opus 4.7) to refine the language and improve conciseness. The authors sub­ sequently reviewed and edited the content, verified all suggested ref­ erences against primary sources, and take full responsibility for the content of the published article.

CRediT authorship contribution statement Eduardo Mariano: Conceptualization, Data curation, Formal anal­ ysis, Investigation, Methodology, Writing – original draft, Writing – review & editing. Ciro A. Rosolem: Funding acquisition, Project administration, Writing – review & editing. J´essica P.Q. Barcelos: Investigation, Writing – review & editing. Bruna Arruda: Investigation, Writing – review & editing. Lílian A. Moreira: Writing – review & editing. David R. Chadwick: Funding acquisition, Writing – review & editing. Davey L. Jones: Conceptualization, Funding acquisition, Methodology, Project administration, Writing – review & editing. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments This work was undertaken as part of NUCLEUS, a virtual joint centre to deliver enhanced N-use efficiency via an integrated soil–plant systems approach for the United Kingdom and Brazil. Funded in Brazil by FAPESP — S˜ao Paulo Research Foundation (grant 2015/50305-8); FAPEG — Goi´as Research Foundation (grant 2015-10267001479); and FAPEMA — Maranh˜ao Research Foundation (grant RCUK-02771/16); and in the United Kingdom by the Biotechnology and Biological Sciences Research Council (grant BB/N013201/1) under the Newton Fund scheme. Eduardo Mariano received a scholarship from FAPESP (grant 2018/14218-1).

Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.soilbio.2026.110215.

Data availability The data and code supporting the findings of this work are openly available in the Zenodo repository at https://doi.

org/10.5281/zenodo.20692296.

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