A dual isotopic ( 32 P and 18 O ) incubation study to disentangle mechanisms controlling phosphorus cycling in soils from a climatic gradient ( Kohala , Hawaii )

Abstract Changes in the isotopic composition of oxygen associated with phosphate can provide information on the impact of phosphatase activity on soil P dynamics, whereas the use of radioactive P delivers information on P fluxes within soil systems. Although these two tracers may provide complementary data, they have rarely been used together to study soil P cycling. We conducted a dual isotopic soil incubation study of one month with soils originating from four sites of a climatic gradient (Kohala, Hawaii), which provides well-controlled geochemical and biological variations on soils derived from the same parent material. Three groups of soils were incubated in parallel, the first group labelled with 32P radioisotopes, the second group labelled with 18O enriched water and the third group not labelled and used for CO2 emission measurements. The dual labelling study informed about three processes controlling P dynamics in soils: those that maintain the bond between P and O and transfer phosphate from one pool to another (category I processes), those that involve the cleavage of the P-O bond and transfer phosphate from one pool to another (category IIa processes), and those that involve the cleavage of the P-O bond but do not transfer phosphate from one pool to another (category IIb processes). The use of 32P showed that the studied soils contained a large amount of P that was isotopically exchangeable with the resin P pool (category I process) and that microorganisms had taken up P, but in much lower amounts, from the resin P pool (category I process). 18O added with water was incorporated into microbial and resin P, but not into the other pools obtained from the modified Hedley extraction. Thus, the turnover of O associated with P within microbial cells (category IIb process) and/or enzymatic hydrolysis of organic P (category IIa process) had occurred and had affected active microbes, which passed the 18O labelled phosphate to the resin pool (category I process). The dual isotopic approach thus provided complementary insights on P cycling processes.


Introduction
Phosphorus (P) transformations in soils are controlled by sorption/desorption, precipitation/dissolution, microbial mineralisation/immobilisation, plant uptake and death, and organic matter dynamics (Frossard et al., 2000;Oberson and Joner, 2005;McLaren et al., 2019). These transformations are most often studied by using sequential chemical extractions, such as the one developed by Hedley et al. (1982). However, information on P distribution among chemical extracts provides a static picture of what is present at a given time; it does not provide information on P cycling processes . Abiotic and biotic P cycling processes are constantly occurring in the soil even if extracted amounts of P stay constant. Approaches that integrate tracers are therefore required to better understand soil P fluxes and transformations.
In this paper, we evaluate processes controlling P dynamics in soils by targeting the isotopic composition of phosphate molecules, in which one P atom is bound to four oxygen (O) atoms.
Diverse process categories are differentiated ( Figure 1): those that involve entire phosphate molecules moving as intact units from one pool to another (category I processes), and therefore, the P-O bonds remain untouched; and those that cleave P-O bonds (category II processes). Within the category II processes, we consider those that lead to a phosphate transfer from one pool to another (category IIa processes) and those that do not lead to a phosphate transfer from one pool to another (category IIb processes). Sorption/desorption, precipitation/dissolution as well as P uptake by organisms and release upon cell lysis involve the transfer of phosphate molecules without the cleavage of a P-O bond and therefore can be classified as category I processes. Enzymatic processes mediated by phosphomono-and phosphodiesterase can be assigned to the category IIa processes as they lead to the cleavage of P-O bonds and the release of phosphate from phosphomono-or phosphodiesters to the soil solution (Blake et al., 2005). Finally, the exchange of O atoms on a phosphate mediated by pyrophosphatase within a cell can be assigned to a category IIb process as the phosphate stays in the same pool . : https://doi.org/10. 1016/j.soilbio.2020.107920 Location Figure 1 Radioactive P isotopes ( 32 P, 33 P) are used to trace the fate of P in soil-plant systems. More specifically, P radioisotopes are used to measure phosphate exchange kinetics between the soil solution and the soil solid phase for assessment of the within a given time isotopically exchangeable phosphate, to estimate the rate of organic P mineralisation, or to quantify the P uptake by a plant or the P recovery from an external source (e.g. a plant residue or a fertilizer) in a specific soil pool (Frossard et al., 2011). Some of these P fluxes are controlled by category I processes (sorption/desorption assessed by isotopic exchange, release of phosphate from dead microbes to the soil solution pool), while other P fluxes belong to category IIa (organic P hydrolysis with P-O bond cleavage and release of phosphate to the soil solution).
In the absence of biological activity and at earth surface temperatures, the P-O bond in phosphate is stable and the isotopic composition of O associated with P (δ 18 O-P) remains constant (Blake et al., 2005;Tamburini et al., 2014). Small changes in δ 18 O-P can occur without cleavage of P-O bonds, for instance during apatite dissolution (Liang and Blake, 2007;Jaisi et al., 2010), during short-term sorption (Jaisi et al., 2010;Melby et al., 2013b), or during P uptake by microorganisms (Blake et al., 2005). Enzyme-mediated reactions have on the contrary a strong impact on δ 18 O-P. They lead to P-O bond cleavage and O atom exchange between phosphate and water (category II processes), which either results in the equilibration of δ 18 O-P with 18 O of water or causes kinetic isotope fractionation effects. Temperature-dependent equilibration can lead to complete O exchange over time and is considered to be mainly intracellular and mediated by pyrophosphatase (Chang and Blake, 2015;von Sperber et al., 2017b). Intra-and extra-cellular hydrolysis of organic P cleave one (monoester) or two (diester) P-O bonds. Oxygen atoms from water are integrated into the newly formed phosphate (Liang and Blake, 2006b;Liang and Blake, 2009;von Sperber et al., 2014) with an enzyme-dependent kinetic fractionation.
Phosphorus radioisotopes and stable O isotopes in phosphate have rarely been considered together. An early study conducted by Larsen et al. (1989) showed that when phosphate : https://doi.org/10. 1016/j.soilbio.2020.107920 labelled with both 18 O and 32 P was added to a soil on which ryegrass was grown, the plant recovered much less 18 O than 32 P compared to what had been added. The authors concluded that the phosphate had lost its 18 O before plant uptake. Furthermore, these authors showed that the phosphate extracted by a resin (plant-available P) at the end of the pot experiment had a lower 18 O/ 32 P ratio compared to the added source. They suggested that the loss of the 18 O label associated with P was related to soil biological activity. These results were subsequently confirmed by Saaby Johansen et al. (1991) and Melby et al. (2013a) who showed that the loss of 18 O from 18 O labelled phosphate added to soils was related to biological activity. Scheerer et al. (2019) supplied beech roots with 18 O labelled phosphate and with 33 P labelled phosphate via addition to the nutrient solution. They could not observe a correlation between root phosphate uptake determined by incorporation of 33 P and by incorporation of 18 O. Thus, the phosphate which had been taken up had lost its 18 O signature due to a rapid turnover of P within roots. Pfahler et al. (2017) supplied soybeans with 33 P labelled phosphate and measured the changes in δ 18 O-P after having stopped or not stopped P inputs. With the 33 P tracing, the authors quantified the transfer of P from older plant leaves to younger plant organs when P inputs to the plants were stopped. Also, they showed the occurrence of organic P hydrolysis in older leaves based on the changes in δ 18 O-P. Further, Helfenstein et al. (2018) assessed the rate of phosphate exchangeability by use of 33 P and measured δ 18 O-P in P pools extracted sequentially from soils of the Kohala climatic gradient on the island of Hawaii. The use of 33 P showed that the soil present at the wettest site had the lowest amount of very slowly exchangeable P, and therefore probably a low concentration of P present in crystallized forms.
The high δ 18 O-P value observed in the HCl extractable P from the sequential extraction measured on this soil showed that the P recovered in this fraction could not be the original apatite derived from the parent material because the O associated with P had been biologically cycled either by plants and/or by microorganisms. The results obtained in the above-mentioned studies show that the use of P radioisotopes and 18 O can provide complementary information related to category I and II processes. However, up to now, no experiment has attempted to simultaneously assess the importance of these processes in controlling soil P dynamics. ): https://doi.org/10.1016/j.soilbio.2020 6 The objective of this study was to assess whether the addition of water labelled with 18 O and of radioactive phosphate to soils would allow us to better understand the importance of category I and II processes during a short-term incubation and how this would improve our understanding of P transformations. This objective was reached by determining P transfer between different P pools with radioactive P ( 32 P), by assessing the incorporation of O from 18 O enriched water into phosphate present in different soil pools, and by relating these patterns to soil properties. The incubation was conducted for 34 days with four soils originating from the Kohala climatic gradient on the Big Island of Hawaii. These soils were chosen as they provide a strong gradient in edaphic properties while deriving from the same parent material (Chadwick et al., 2003;Vitousek and Chadwick, 2013;Peay et al., 2017;Helfenstein et al., 2018).

2.1.
Sampling sites and soil sample collection and preparation Soils from four sites on the Kohala climatic gradient located on the leeward slope of the Kohala Peninsula on the Big Island of Hawaii (USA) were studied (Table SM1). Soils are classified as volcanically derived andosols (Chadwick et al., 2003). The soils developed on a Hawi lava flow produced 150,000 years ago (Spengler and Garcia, 1988). The lava is an alkali basalt and contains large amounts of apatite (Chadwick et al., 2003). Mean annual rainfall of the sites 1 to 4 covers a wide range (275,1578,2163, and 3123 mm y -1 ), whereas mean annual temperature varies in comparison on a smaller range (16.2, 17.8, 19.1, and 23.6 °C) (Giambelluca et al., 2013 to meet the soil dry weight needs of the experiment, this was required due to imposed restrictions on soil sampling and shipment. It was assumed that the soil sampling time and therefore the sample combination does not affect the strong gradient in soil properties, which was the underlying reason for the choice of the sites. However, an impact on microorganisms and available nutrient pools was expected but considered negligible in comparison to changes due to the applied conditions during the incubation experiment. The water content of field moist soil was determined by drying for 20 hours at 105 °C. The water holding capacity (WHC) of field moist soils was determined by first weighing the soils into tared cylinders to a defined height, then saturating the soils with water by putting them in a water bath for 3 hours, and lastly allowing gravitational water to drain by placing the cylinders on a sand bath for 3.75 hours. For the pre-incubation, the water content of the soils was adjusted to 60% max. WHC by drying the soils at 28 °C (soils of sites 3 and 4) and water addition (soils of all four sites).

Experimental design
Separate soil subsamples were used for the two tracer ( 32 P and 18 O) and the respiration experiments. The incubation of labelled soils lasted 34 days, the respiration measurements lasted 36 days. All soils were incubated in the dark at 25 °C and 70% air humidity. After 23 days of pre-incubation at initially 60% WHC, soils were watered to adjust for evaporation losses and mixed before being assigned to either the labelling or the respiration experiments.
Equivalents of 15 g of dry soil were weighed into jars to measure soil respiration, while equivalents of 30 g of dry soil were weighed into 250 ml plastic bottles for the tracer ( 32 P and 18 O) experiments (Table 1). After adding water and labelling solution, all soils were again at 60% WHC.
Location Table 1 Accepted manuscript of : https://doi.org/10.1016/j.soilbio.2020.107920 For the 32 P experiment, each sample was labelled with carrier-free 32 P added in the form of phosphoric acid (Hartmann Analytic GmbH, Braunschweig, Germany) at a rate of 0.034 MBq g -1 for samples of sites 1 and 2, and 0.045 MBq g -1 for samples of sites 3 and 4, respectively.
The rates of 32 P applied were based on pre-tests conducted on test samples in 2015. 32 P was selected due to a delivery shortage of 33 P. The same experiment could have been carried out with 33 P, as there is no significant isotopic fractionation between 31 P, 32 P, and 33 P (Frossard et al., 2011). Four replicates were prepared for each time point and site combination by mixing the soil with the radioactive solution for two minutes in the plastic bottle. During the incubation, the samples were arranged according to a random complete block design with one replicate of each time point and site combination per block. Soils were sampled destructively at five time points (1, 4, 14, 26 and 34 days after labelling). Resin and microbial P contents and radioactive counts were measured at each time point to capture the dynamic isotopic exchange. Further explanatory analyses (total and chloroform-labile C and N, potential acid phosphatase activity, water-extractable P (Pw)) were conducted on these samples at selected time points (Table 1).
This selection occurred as total and chloroform-labile C and N and potential acid phosphatase activity were expected to stay constant over the incubation time and as Presin was considered a more reliable indicator for available P than Pw throughout the incubation. Germany; temperature of combustion 950 °C). Microbial C and N were estimated by the chloroform fumigation-extraction method of Vance et al. (1987). Fresh samples with a dry weight equivalent of 6 g dry soil were fumigated for 24 h. Fumigated and non-fumigated subsamples were extracted with 0.5 M K2SO4 in a 1:4 soil:solution (mass:mass) ratio. The extracts were frozen and then analysed with a TOC/TN analyser for liquid samples (TOC-L, Shimadzu, Kyoto, Japan). The difference between fumigated and non-fumigated subsamples was calculated to estimate microbial C and N. No conversion factors (kC and kN) were applied to correct for inefficient fumigation by chloroform, therefore the terms chloroform-labile C (Cchl ) and N (Nchl) are used instead of microbial C and N.

Soil respiration
Soil respiration was quantified by trapping the released CO2 in 0.2 M NaOH and back-titrating with 0.2 M HCl (Alef, 1995). Measurements were performed at 8, 13, 20, 28 and 36 days after the day of labelling (Table 1).

Phosphatase activity
Potential acid phosphatase activity in soils was determined by using a 4-methylumbelliferon (MUF) substrate in a microplate assay with 6 analytical replicates following the method developed by Marx et al. (2001) and modified by Poll et al. (2006). The assay was conducted for acidic (pH 6.1) phosphatase activity. Fluorescence was measured on a fluorescence plate reader (Biotek FLx800, Fisher Scientific GmbH, Schwerte, Germany). This analysis was performed on a formerly frozen equivalent of 1 g dry soil sampled in the 32 P incubation experiment at 14 days (Table 1).

Soil phosphorus pool concentrations
Soil P pool concentrations were assessed on moist soil samples using a modified Hedley sequential extraction (Moir and Tiessen, 2007). The following steps were performed. First three subsamples were treated in parallel: one subsample was extracted with an anion exchange resin (Presin), the other was fumigated with liquid hexanol and extracted with an anion exchange resin (Phex), and the last was spiked with 31 P and extracted with an anion exchange resin (Pspike). Resins were eluted in 0.1 M NaCl/0.1M HCl by shaking horizontally for 2 hours at 150 rpm. The resin extraction provides an estimation of plant-available P, the difference in P concentration between the resin extract following fumigation and without fumigation provides an estimate of microbial P, while the last sample allows quantifying the sorption of P derived from cell lysis following fumigation (Kouno et al., 1995;Bünemann et al., 2004). The subsample that had been fumigated with hexanol and extracted with a resin was subsequently extracted with 0.25 M NaOH/0.05 M EDTA and the extraction residue was then extracted with 1 M HCl.
NaOH/EDTA is considered to extract inorganic P bound to Fe and Al oxides and organic P, while the HCl is considered to extract insoluble calcium phosphates, such as apatite (Hedley et al., 1982). Each extraction step was conducted by shaking the respective soil solution horizontally for 16 hours at 150 rpm. This sequential extraction was performed before the start of the incubation on each soil with three replicates using a 1:15 soil:solution ratio .
The protocol of Tamburini et al. (2018) was used to analyse the δ 18 O-P in the above-mentioned P pools. This protocol uses a 1:10 soil:solution ratio to collect sufficient P for the measurement of the δ 18 O-P. These analyses were performed before labelling and 34 days after labelling with 18 O enriched water in the 18 O experiment. Additional extractions for Presin, Phex and Pspike and 32 P within these pools were performed at 1, 4, 14, 26 and 34 days after labelling with 32 P on the equivalent of 2 g dry soil with a 1:15 soil:solution ratio on four replicates per site in the 32 P experiment following the protocol of Pistocchi et al. (2018). Added P spikes ranged from 5 to 50 mg P kg -1 during the 32 P experiment depending on soil and sampling time. Water extractable P (Pw) was determined on moist equivalents of 10 g dry soil from the incubation 14 days after labelling with 32 P for four replicates per soil. The concentrations of resin extractable P (Presin), inorganic NaOH-EDTA P, HCl P, total soil P, and water (Pw) were determined colourimetrically (UV-1800, Shimadzu, Canby, USA) with the malachite green method of Ohno and Zibilske (1991). Total P in NaOH-EDTA extracts was measured by ICP-OES (ICPE-9820, Shimadzu, Kyoto, Japan). Organic NaOH-EDTA P was calculated as the difference between total and inorganic P measured in the NaOH-EDTA extract. Microbial P (Pmic, mg P kg -1 dry soil) was calculated as the difference between Phex and Presin subsamples including the correction for sorption (Bünemann et al., 2016): where Phex (mg P kg -1 dry soil) is the resin extractable inorganic P in the fumigated subsample and Presin (mg P kg -1 ) is the resin extractable inorganic P in the non-fumigated subsample, and rec is the recovery of added P spike calculated as: with Pspike (mg P kg -1 dry soil) being the resin extractable inorganic P in the subsample to which the P spike had been added. If recovery of added P was above 1, which happened five times during the 32 P experiment (four times for site 1, once for site 2), rec was set to 1. For the sequential P extraction before the start of the incubation rec was set to 1 for all sites due to amounts of spike P added being not close to actual Pmic values resulting in unrealistically low or high rec values. No conversion factor (kP) was applied to correct microbial P for inefficient fumigation by hexanol, as kP is soil-specific (Oberson and Joner, 2005) and was not determined for the studied soils. Thus, microbial P might be underestimated. Microbial P was found three times to be negative (each time in soils from site 1), in such cases Pmic was set to zero.
2.3.5. Radioisotope tracing in soil phosphorus pools 2.3.5.1. Radioactivity in phosphorus pools Radioactivity in the resin extractable pool (rresin) was directly measured by scintillation counting on a beta-emission counter (Tri-carb 2500 TR, Packard Instruments, Meriden, USA) using 1 ml of sample and 5 ml Ultima Gold TM (Perkin Elmer, USA).
The determination of radioactivity in microbial biomass (rmic) was done after having corrected the release of 32 P from the solid phase of the soil caused by the release of 31 P from the microbial P pool (Bünemann et al., 2016;Schneider et al., 2017). First, a linear correlation was built between Phex and rspike (the amount of radioactivity recovered in the sample spiked with 31 P in Bq g -1 dry soil) which is the corrected radioactivity in spiked subsamples where a and b are the slope and intercept of the linear regression.
And then rspike was introduced in equation 4 to obtain rmic (Bq g -1 dry soil) Negative values were observed 18 times for rmic, 12 of these negative values were recorded for the soils of site 1. These values were probably caused by low microbial P concentrations (Table 2) and lower soil microbial activity ( Figure 2) combined with propagated measurement uncertainties. Thus, negative values were considered as missing values. As a large fraction of the rmic values of site 1 were missing, we do not discuss rmic in the soils of site 1 and focus on the more reliable complementary data.

Radioactivity recovery and specific activity of phosphorus pools
Radioactivity recovery (r/Rpool, %) was calculated for each analysed pool by relating the measured radioactivity r in the pool at a given sampling time (rpool Bq g -1 dry soil) to the decayadjusted introduced radioactivity R (Bq g -1 dry soil, half-life t1/2 = 14.28 days for 32 P): Instead of the commonly used specific activity (SA, Bq mg -1 P), the proportional specific activity in P pools (pSApool, % kg dry soil mg -1 P) was calculated as follows: where r/Rpool is the fraction of recovered radioactivity in the P pool (%) and Ppool is the P concentration of the P pool (mg P kg -1 dry soil). This modification of the calculation was performed to account for the variable amount of introduced 32 P (R) and thus to enable the comparison between sites.

Isotopically exchangeable phosphate
Isotopically exchangeable phosphate was calculated to assess the fluxes of P between the solid phase of the soil and the solution. Since the measurements of Pw were very variable and below the detection limit in the soils of the wetter site 4, we decided to use the resin P pool for the calculation of the isotopically exchangeable phosphate as proposed by Maertens et al. (2004). The amount of isotopically exchangeable resin P (Eresin, mg P kg -1 dry soil) was calculated according to the following equation: where pSAresin is the proportional specific activity in the resin extract.

Estimation of gross phosphorus immobilisation
If we consider that microorganisms take up most of their P from the resin P pool then it becomes possible, using pSAresin and r/Rmic, to calculate the amount of microbial P that is derived from the resin P pool (Pimmo, mg P kg -1 dry soil) using the following equation: Equation 8 provides an estimation of the gross P immobilisation (Bünemann et al., 2012 Phosphate in the resin, hexanol and HCl extracts was purified following the protocol given by Tamburini et al. (2010). For NaOH-EDTA extracts, separation into organic and inorganic P fractions and their respective purification were performed following Tamburini et al. (2018).
Inorganic NaOH-EDTA extracts were processed for sites 1, 2 and 4, while organic ones were processed only for sites 2 and 4. These samples were selected because we expected differences to occur between the extreme sites 1 and 4. As site 1 had not enough P in the organic NaOH-EDTA extract, site 2 was also considered.  (9) where T is the temperature in K during the incubation and δ 18 O-ws (‰) is the O isotopic signature of soil extracted water.
Since temperature-dependent equilibration is mainly occurring intracellularly, the proportion of water, produced during metabolic reactions, and its δ 18 O should be considered for the equilibrium estimation. The contribution of metabolic water to intracellular water depends on the metabolic state of the organism. It reached up to 70% for Escherichia coli during active growth, whereas the contribution in a stationary phase was considerably smaller (Kreuzer-Martin et al., 2005).
Our estimation of the equilibrium considering soil water and metabolic water (Eq-ws+m) was based on two assumptions. First, that intracellular water (δ 18 O-ws+m) consists of 59% soil water and 41% metabolic water, as determined by Li et al. (2016)

Statistical analyses
Statistical analyses were conducted in R version 3.6.0 (R Core Team, 2019

Soil properties
The soil properties varied significantly between sites (Table 2). Water holding capacity, total C and N, chloroform-labile C and N, Pmic, organic NaOH-EDTA P and acid phosphatase activity increased from site 1 to 4. In contrast, Pw, Presin and inorganic NaOH-EDTA P decreased from site 1 to 4.

Soil respiration
Total soil respiration over 36 days was ten times larger for site 4 (2500 mg C kg -1 ) compared to site 1 (259 mg C kg -1 ), with intermediate sites ranging in between and all values being significantly different from each other (Figure 2b). The proportion of total C that was lost by respiration reached 1.6% in the soil of site 1, 1% in site 4, 0.9% in site 2 and 0.8% in site 3.
Daily soil respiration rates decreased during the incubation period, most notably for the soil of 20 A high proportion of introduced 32 P was not detected in either the resin or the microbial P pool already after 1 day of incubation (from 62.0% for site 1 to 89.3% for site 3), indicating a fast disappearance of this tracer towards other P pools.
Location Figure 5 Assuming that microorganisms took up their P from the resin P pool and knowing the specific activity of the resin and microbial pools, we could calculate the amount of P in the microbial biomass derived from the resin pool, (Figure 6, Pimmo, mg P kg -1 dry soil), which is an estimation for the gross P immobilisation. This amount remained low and variable in the soil of site 2 (below 5 mg P kg -1 dry soil), while it increased significantly with time in the soils of sites 3 and 4 reaching 20 mg P kg -1 dry soil at the last sampling point of the soil of site 4. Gross P immobilisation ranged between 2% of microbial P for the first sampling time point of site 4 (2.0 mg P kg -1 for Pimmo compared to 109.5 mg P kg -1 for Pmic) and 27% for the sampling time point at 14 days of site 3 (15.6 mg P kg -1 for Pimmo compared to 58.5 mg P kg -1 for Pmic).
Location Figure 6 3. Taking the metabolic water into consideration led to lower equilibrium values.
Location Table 3 The δ

Incorporation of 18 O into soil phosphorus pools
Incorporation of 18 O from enriched water into phosphate was observable in the resin and microbial P pools, but not in inorganic or organic NaOH-EDTA P or HCl P (Table 4). In the resin and microbial P pools, approximately 1 out of 4 O atoms per phosphate was exchanged during the incubation in the soils of sites 1 to 3. The soil of the wettest site showed higher exchange rates than the other sites, particularly in resin P (41% 18 O incorporation).
Location Table 4 4. Discussion In the introduction, we considered three types of processes controlling P dynamics in soils: those that maintain the bond between P and O and transfer phosphate from one pool to another (category I processes), those that involve cleavage of the P-O bond and transfer phosphate from one pool to another (category IIa processes), and those that involve cleavage of the P-O bond but do not transfer phosphate from one pool to another (category IIb processes). We discuss how far the use of P and O isotopes allows to assess these three types of processes and their roles in soil P dynamics.

Category I pathways: phosphates move as intact units
The use of 32 P allowed assessing the amount of soil P that was isotopically exchanged over the time of incubation (Eresin). The fact that the resin P pool size did not vary significantly with time in each soil, while the fraction of radioactive P in the resin extracts significantly decreased, confirms that the main mechanism controlling 32 P losses from the resin P pool was isotopic exchange rather than a net change in pool size. Soil organic P mineralisation could have contributed to the dilution of the added 32 P, but it was probably not a dominant mechanism. If we assume that C and P are stoichiometrically mineralised, the amount of organic P mineralised during the incubation would be equal to the ratio between the amount of C mineralised during the incubation to total C multiplied by organic P (Achat et al., 2009). The measured organic NaOH-EDTA P was set as organic P. The amount of mineralised C was derived from C emitted as CO2 corrected by the microbial C assimilation efficiency, which was assumed to be 0.4 according to Murphy et al. (2003). This calculation suggests that the amount of mineralised organic P would range between 14 for the soil of site 1 and 41 mg P kg -1 dry soil for the soil of site 4, ergo would reach values smaller or equal to the standard deviations observed for Eresin after 34 days of incubation. Moreover, the changes in Eresin with time can be described for each soil with a simple log/log relation (Eresin(t) = Eresin(1) * t n ), which is similar to the equation used by Fardeau and Zapata (2002) to describe the changes in soil isotopically exchangeable P with time. Isotopic exchange is controlled both by sorption and desorption and by transfer of P from the charged surface to the bulk solution by diffusion and can, therefore, be assigned to the category I processes. The large amounts of soil P isotopically exchangeable with resin P show the importance of category I processes in these soils. Maertens et al. (2004) observed the same type of relationship between Eresin and incubation time. However, in their work, the increase of Eresin with time was several-fold smaller compared to our results. This difference is probably due to the difference in soil types and the difference in total P content as Maertens et al. (2004) presented results on a lixisol and a ferralsol from Kenya, which contained less amorphous minerals and less total P than our andosols. The high rate of P exchangeability found in our andosols confirms the results of Helfenstein et al. (2018) and is consistent with the presence of high amounts of amorphous minerals in these soils (Chadwick et al., 2003).
The use of 32 P allowed the quantification of the amount of P derived from the resin pool that was taken up by microorganisms (gross P immobilisation, Pimmo). These microbial processes probably involved pathways in which phosphate was taken up as a unit (category I processes) and processes during which an O-P bond was cleaved (category IIa and IIb processes), for example, if P was taken up after extracellular enzymatic hydrolysis. If we do not consider the soil from site 1 on which a substantial amount of data was missing due to the very low microbial P content, the amount of P derived from the resin incorporated in the microbial biomass was significantly larger for the soils of sites 3 and 4 than for the soil of site 2, and this amount increased with time for the soils of sites 3 and 4. The highest P uptake rate observed in the soil of site 4 was in agreement with the highest rate of CO2 emission from this soil and the high microbial biomass. Bünemann et al. (2012) and Pistocchi et al. (2018) also used P radioisotopes for the estimation of gross P immobilisation rates during incubation experiments.
Contrary to our approach, their calculations are based on the changes in specific activity in water extracts and not the changes in specific activity in resin extracts. Nevertheless, they observed higher gross P immobilisation (between 40 and 50 mg P kg -1 soil after 11 days in Pistocchi et al. (2018) and between 36 and 49 mg P kg -1 soil after 32 days in Bünemann et al.
(2012)) compared to our study (between 5 and 20 mg P kg -1 soil after 34 days). Since we have not used a correction factor (kP) for calculating microbial P content, the fluxes of P through the microbial biomass are underestimated. Nevertheless, even if we applied a kP factor of 0.8 as in Olander and Vitousek (2005), the resulting values for gross P immobilisation after 34 days of incubation would remain well below the Eresin values. For example, for the soil of site 4 kP corrected gross P immobilisation would only reach 9% of the respective Eresin.
Our results contrast with those presented by Olander and Vitousek (2005), who showed that microbial control on P uptake from the solution was at least as strong as the sorption control in a Hawaiian chronosequence after 48 hours of incubation. But, these results are difficult to compare to ours because i) they estimated available P with the Bray I extractant while we used anion exchange resins, and ii) they added 10 µg P g -1 soil which was a significant input compared to the low amounts of P extracted by the Bray I method from the non-treated soils (smaller than 4.5 µg P g -1 ). We added no 31 P with the 32 P to minimise changes in chemical equilibrium between the soil and the solution. However, based on the assumption that P extracted by the Bray I method and the resin should target a similar P pool (van Raij et al., 2009), we assume that the soils studied in our work (27.6 to 69.3 mg resin P kg -1 , Table 2) had a higher P availability than those studied by Olander and Vitousek (2005) (0.27 to 4.32 mg Bray I P kg -1 ). Our results together with those presented by Olander and Vitousek (2005), by Schneider et al. (2017), and by Pistocchi et al. (2017) suggest that the proportion of P cycling through the microbial biomass increases with decreasing P availability.

Category II pathways: the bond between oxygen and phosphate atoms is cleaved
The δ 18 O-P of the different P pools before labelling with 18 O enriched water (Table 3) were resembling those found for these soils by Helfenstein et al. (2018).  (Chang and Blake, 2015;von Sperber et al., 2017b). However, it is probable that in a heterogeneous medium like soil, within which different types of microorganisms cohabit (Peay et al., 2017), a longer time will be required for O atoms of phosphate to equilibrate with the O atoms of water present within the soil microorganisms. At the end of incubation, in our soils, the mean incorporation of 18 O into microbial P was non-linearly correlated with the mean amount of C emitted as CO2 (r 2 = 0.919). This agrees with Middelboe and Saaby Johansen (1992) and Melby et al. (2013a), who showed that the de-labelling of 18 O from 18 O labelled PO4 was correlated to soil microbial activity.
The resin and microbial P pools had similar δ 18 O-P signatures in the soils of the sites 1, 2 and 3 at day 0 and at the end of the incubation, whereas in the soil of site 4 the δ 18 O-Pmic was lower than δ 18 O-Presin at both time points. This suggests a constant and rapid turnover of P between the resin and the microbial pool for the soils of sites 1, 2, and 3. The resin and inorganic NaOH-EDTA P pools were expected to show similar δ 18 O-P signatures, as NaOH P exchanges relatively rapidly with the P in solution, except in systems that contain P in crystallized minerals such as vivianite (Frossard et al., 1996;Helfenstein et al., 2018). However, this was not the ): https://doi.org/10.1016/j.soilbio.2020 25 case for the soils of sites 1 and 2. The differences can be possibly explained by i) the fact that 34 days were probably not sufficient for complete equilibration between the different inorganic P pools, and ii) the presence of different forms of inorganic P species extracted by NaOH-EDTA. The first explanation agrees with the fact that only 21 to 56% of the inorganic NaOH-EDTA P was exchangeable after 34 days of incubation. In accordance, the estimated mean residence time of P in the NaOH P pool of Andosols is around six months, substantially longer than the duration of our incubation experiment (Helfenstein et al., 2020). More information on the inorganic P species extracted by NaOH-EDTA would be necessary to provide support for the second explanation.

Conclusions
Radioactive P is an established tracer to assess the P fluxes from one soil compartment to another, whereas changes in the isotopic composition of O associated with phosphate are investigated to provide information on the impact of phosphatase activity on soil P dynamics.
In this paper, we showed that the use of 32 P and 18 O in parallel experiments generated results that reinforced each other. For instance, the fact that at the end of the incubation only 21 to 56% of the NaOH-EDTA P was measured as isotopically exchangeable agreed with the fact that the δ 18 O-P of the resin pool was not equal to the δ 18 O-P of the NaOH-EDTA pool. With a longer incubation time, we might reach 100% exchangeability in the NaOH-EDTA P pool and thus, the δ 18 O-P of this pool and of the resin pool would have approached similar values.
Furthermore, the use of 32 P allowed to quantify the fraction of P derived from the resin pool and taken up by microorganisms, while changes of the δ 18 O-P in microorganisms allowed to detect the incorporation of O in phosphate within microorganisms, both aspects being linked to microbial activity. In two cases the δ 18 O-P allowed to follow processes that were not observable with 32 P: i) the incorporation of 18 O in the resin P pool suggested that either some of this P had been released by enzymatic hydrolysis from organic P or that it has been released from microorganisms in which the O associated with P had been turned over, and ii) the difference between δ 18 O-P of the resin and the microbial pool at the end of the incubation in the soil of site 4 suggested that this soil could contain groups of microorganisms exhibiting different levels of activities. Further microbiological analyses would be needed to support this last hypothesis. In any case, the results of this study confirm preceding research which showed that δ 18 O-P cannot be used to quantify the transfer of P from one pool to the next. Nevertheless, we conclude that the two tracers provide complementary information that helps to better understand the soil P dynamics.