Characteristics of phosphorus fractions in the soils derived from sedimentary and serpentinite rocks in lowland tropical rain forests, Borneo

ABSTRACT

Soil organic phosphorus (P) is an important P source for biota especially in P-limited forests. Organic P has various chemical formations which differ in bioavailability and these organic P can be degraded by phosphatase enzymes. Here, we report soil P fractions inferred from solution ³¹P-NMR spectroscopy and soil phosphatase activities of two tropical rain forests on contrasting parent materials; sedimentary and ultramafic igneous (serpentinite) rocks. Compared to the sedimentary soils　and previous studies, P fractions of the serpentinite soils have distinctly high proportions of pyrophosphate and scyllo-inositol hexakisphosphate (scyllo-IP₆). The accumulation of pyrophosphate and scyllo-IP₆ may be related to strong sorptive capacity of iron oxides present in the serpentinite soils, which implies a consequent low P availability in the serpentinite soils. Mean value of soil phosphatase activities was higher in the serpentinite soils than in the sedimentary soils, suggesting that biota in these serpentinite forests depend more on soil organic P as a P source.

KEY WORDS:

• Lowland tropical rain forest
• NMR
• phosphorus
• serpentinite soil
• soil phosphatase activities

1. Introduction

In tropical rain forests on highly weathered soils, biological processes are considered to be limited by phosphorus (P) (Vitousek 1984) because soil P occurs as biologically unavailable forms such as organic and occluded P (Walker and Syers 1976). P-limited plant roots and microbes actively release phosphatase into soils, degrading soil organic P into inorganic P (orthophosphate) to acquire soil P. Indeed, phosphatase activities for plant roots and soils are generally higher in P-limited forests (Allison et al. 2007; Kitayama 2013; Ushio et al. 2015).

Soil organic P occurs in various chemical formations which differ in bioavailability (Turner 2008). Thus, the detection of chemical formations of soil P is important to evaluate P availability. Solution ³¹P-NMR techniques have been applied to soil P fractionation, dividing soil P into several groups, for example, orthophosphate, monoester-P (glucose phosphate, mononucleotide, and inositol phosphate) and diester-P (nucleic acid and phospholipid), with a smaller amount of phosphonates, pyrophosphates, and polyphosphates (Turner and Engelbrecht 2011). ³¹P-NMR has elucidated soil P fractions in various ecosystems, yet only a few studies were conducted in tropical rain forests, where organic P can be an important P source for biota (Vincent et al. 2010). Here, we report soil P fractions with soil phosphatase activities in two tropical rain forests which differ in P availability.

2. Materials and methods

In September 2013, soil sampling was conducted in two lowland rain forests on sedimentary rock and ultramafic igneous (serpentinite) rock around Mount Kinabalu (6° 5ʹ N, 116° 33ʹ E), Sabah, Malaysia. Site data are shown in Table 1. The two forests are located at 700 m and have the same climate, but differ in nutrient status (Kitayama et al. 2000; Kitayama and Aiba 2002). Wagai and Mayer (2007) and Wagai et al. (2009) investigated the soils in our forests and found a much higher content of iron oxides, hydroxides, and oxyhydroxides (collectively called iron oxides hereafter) and specific surface area in the serpentinite soils (Table 1). Their mineralogical characteristics must promote strong P sorption onto soil minerals. Consequently, available P (Bray-I extracted) is much lower in the serpentinite soils (Mori et al. 2016). We laid five transects of 40 m at 10-m intervals and collected 20 soil cores (0–15 cm) at 2-m intervals along each line in each forest. These sampling points cover 40 m × 40 m area in each 1-ha plot. A total of 20 cores per line were homogenized (i.e., five replicates in each site) and taken to laboratory, then sieved with 2-mm mesh.

Table 1. General description on climate, ecosystem, and soil properties of the two forests on sedimentary rock and serpentinite rock on Mount Kinabalu, Borneo.

	Sedimentary site	Serpentinite site
Elevation (m)	650	700
Annual mean temperature (°C) ^a	23.9	23.6
Annual mean precipitation (mm) ^a	2392	2392
Above ground biomass (kg m^−2) ^b	43.7	55.4
pH (H2O) ^c	3.9	4.8
Soil total organic carbon (mg C g^−1) ^c	29.8	30.3
CN ratio ^c	12	15
Dithionite extracted Fe (mg Fe g^−1) ^d	21	309
Oxalate extracted Fe (mg Fe g^−1) ^e	5.3	2.4
Soil-specific surface area (m^2 g^−1) ^f	31.4	96.7
Bray-1 P (µg P g^−1) ^g	2.18	0.94

^aFrom Kitayama (1992).

^bFrom Kitayama and Aiba (2002).

^cFrom Wagai et al. (2008) using 0–10 cm surface mineral horizon.

^dFrom Wagai and Mayer (2007) using 0–10 cm surface mineral horizon.

^eFrom Wagai et al. (2013) using 0–10 cm surface mineral horizon.

^fFrom Wagai et al. (2009) using 0–10 cm surface mineral horizon after organic matter removal.

^gFrom Mori et al. (2016) using the same soil samples as this study.

The extraction procedure is based on Turner and Engelbrecht (2011) with a minor modification. Moist soils (1.5 g) were shaken with 30 ml of 0.25 M NaOH + 50 mM EDTA for 4 h, centrifuged for 30 min at 4830 g, and 20 ml of supernatant was spiked with 1 ml of 100 μg P ml⁻¹ methylene diphosphonic acid (MDPA) solution as an internal standard. Spiked extracts were then frozen and lyophilized. 100 mg of lyophilized extract was re-dissolved in 0.6 mL of 1.0 M NaOH and 0.1 M Na₂EDTA in 10% D₂O solution, and transferred to an NMR tube. ³¹P-NMR spectra were collected on an Alpha 600 FT NMR spectrophotometer (JEOL, Tokyo) at a 242.85 MHz frequency with the following parameters: a pulse width of 15.00 µs (90°), an acquisition time of 0.452 s, a pulse delay time of 2.0000 s, a broadband proton decoupling at 30°C, and scan number of 30,000. We confirmed that the relaxation of ³¹P magnetization was enough to recover (estimated by null method) and P in the standard orthophosphate solutions in the presence of 1.0 M NaOH and 0.1 M Na₂EDTA in 10% D₂O were quantitatively determined at least <1000 P ppm at the measurement condition (data not shown). Signals were assigned to model compounds spiked in NaOH-EDTA as reported by Turner et al. (2003). The spectra were divided manually into five groups: MDPA (17.5 ppm), orthophosphate (6.2 ppm), monoester-P (6.0–4.0 ppm), diester-P (−0.1 ppm), and pyrophosphate (−4.1 ppm). Signal areas were determined by integration and concentration of each P compound was calculated based on the area of MDPA using Alice 2 for windows (JEOL, Tokyo).

Brief explanation of soil phosphatase activities assay is as follows (see Kitayama 2013; Yokoyama et al. 2017 for the details). Moist soils (0.2 g) were incubated in 5-ml MUB buffer solution (pH 5.0) containing 0.04 M p-nitrophenyl phosphate (pNPP) for 1 h. Reaction was terminated with 4 ml of 0.5 M NaOH and 1 ml of 0.5 M CaCl₂. Concentration of p-nitrophenol (pNP) hydrolyzed from pNPP during assay was measured on a spectrometer at 410 nm. The amount of pNP produced during incubation per sample dry weight per incubation time was defined as phosphatase activities.

3. Results and discussions

Obtained NMR spectra are shown in Fig. 1. Proportion of each P fraction in the sedimentary soils was similar to the results of other earlier studies (Turner and Engelbrecht 2011). By contrast, we found distinct characteristics of P fractions in the serpentinite soils (Table 2). Of particular interest was an abundance of pyrophosphate in the serpentinite soils (23.3 µg P g⁻¹ soil, 22.5% in NaOH-EDTA P) despite previous studies which reported a small proportion of pyrophosphate in soils (mostly less than 5% in NaOH-EDTA P). Although pyrophosphate is rapidly degraded by enzymatic hydrolysis in soils (McBeath et al. 2006), pyrophosphate might have accumulated in these serpentinite soils due to an extremely higher iron oxides concentration (Wagai and Mayer 2007) and a greater specific surface area (Wagai et al. 2009). Pyrophosphate can be protected against enzymatic hydrolysis because it is strongly sorbed onto the minerals in the serpentinite soils. In fact, pyrophosphate has a much stronger affinity with soil minerals than orthophosphate (McBeath et al. 2007). As to the origin of pyrophosphate, we suggest that pyrophosphate is supplied from saprophytic as well as mycorrhizal fungal biomass (Makarov et al. 2005; Bunemann et al. 2008). Particularly, mycorrhizal fungi associated with plant roots are known to translocate P in the form of polyphosphate, which is a precursor of pyrophosphate (Bücking and Heyser 1999). However, interestingly, available data suggest ectomycorrhiza biomass (Okada et al. unpublished) or soil fungal biomass (Wagai et al. 2011; Ikeda et al. unpublished) does not differ between the two sites. This again points the possibility that pyrophosphate has preferentially accumulated and been concentrated by sorption in the serpentinite soils via fungal turnover in a long run. Another possibility in addition to the fungal origin is that pyrophosphate is derived also from serpentinite rock itself. Pyrophosphate can be formed during serpentinization under hydrothermal conditions, which might have preceded the formation of adenosine triphosphate (ATP) in a prebiotic environment because it has condensed P as well as pyrophosphate (Holm and Baltscheffsky 2011). Long-term weathering may concentrate pyrophosphate in our serpentinite soils by preferential sorption onto the weathering products, iron oxides. However, this possibility is less likely and needs to be substantiated by investigating other serpentinite soils in the future. Although the origin of pyrophosphate remains elusive, the high accumulation of pyrophosphate in our serpentinite soils implies low P availability to biota in this forest if it is due to strong sorption of pyrophosphate.

Table 2. Mean (±SD) concentration (µg P g⁻¹ soil) and proportion in NaOH-EDTA extracts (%) of each soil P fraction of the sedimentary soils and the serpentinite soils.

	Site	Ortho-phosphate		Monoesters		Diesters		Pyro-phosphate		NaOH-EDTA P
Concentration (µg g^– ^1 soil)	Sedimentary	20.5	(2.1)	48.8	(3.7)	20.7	(2.5)	2.4	(1.3)	92.5	(5.6)
	Serpentinite	14.6	(1.7)	53	(8.1)	12.8	(3.7)	23.3	(5.6)	103.7	(14.6)
Proportion (%)	Sedimentary	22.2	(2.9)	52.8	(2.1)	22.4	(1.8)	2.6	(1.2)	100
	Serpentinite	14.4	(2.8)	51.1	(2.8)	12.2	(2.7)	22.4	(3.1)	100

Figure 1. Examples of solution ³¹P-NMR spectra of NaOH-EDTA extracts of the sedimentary soils and the serpentinite soils. The boxes show the detailed spectra in an orthophosphate and monoester region (6.5–3.0 ppm). Each signal shows A, MDPA (17.5 ppm); B, orthophosphate (6.2 ppm); C, monoester-P (6.0–4.0 ppm); D, diester-P (−0.1 ppm); E, pyrophosphate (−4.1 ppm); and F, scyllo-IP₆ (4.2 ppm). Four vertical lines in each box show the chemical shifts of myo-IP₆ (5.97 ppm: C-2 phosphate on the inositol ring, 5.00 ppm: C-4 and C-6, 4.61 ppm: C-1 and C-3, and 4.50 ppm: C-5). The chemical shifts of myo-IP₆ were estimated from the relative positions of myo-IP₆ and scyllo-IP₆ reported on the literature reference (Cade-Menun 2015) and the chemical shift of scyllo-IP₆ (4.19 ppm) in this study as a criterion. Black vertical lines in each box describe just the ratio of each height of four peaks of myo-IP₆ (1:2:2:1) as a reference but do not represent the observed height of the peaks.

While it is difficult to distinguish each compound in the monoester region of ³¹P-NMR spectra due to dense chemical shifts, we recognized a higher peak at 4.2 ppm in the serpentinite soils (Fig. 1), which can be assumed to be scyllo-Inositol hexakisphosphate (IP₆) based on literature reference of chemical shifts (Turner and Richardson 2004). The peak at 4.2 ppm occurs at the right side of monoester region in these spectra, which has been empirically identified as scyllo-IP₆ or choline phosphate. Choline phosphate, a compound of labile monoester P, appears close to that of scyllo-IP₆, yet choline phosphate is unlikely to be quantitatively important in soils taking into account rapid turnover of labile monoester P (Turner and Richardson 2004). Additionally, choline phosphate can be generated after degradation of phosphatidyl choline during alkali extraction, yet phosphatidyl choline is transformed into α-glycerophosphate or β-glycerophosphate rather than choline phosphate (Doolette et al. 2009). Therefore, the amount of choline phosphate in extraction is expected to be marginal. Scyllo-IP₆ is one of the four stereoisomers of IP₆ found in soils, which are myo-IP₆, scyllo-IP₆, neo-IP₆, and D-chiro-IP₆, and myo-IP₆ and scyllo-IP₆ account for most IP₆ in soils (Turner 2007). Contrary to scyllo-IP6, we could not recognize the clear peaks of myo-IP6 since they were obscured by noises (Fig. 1); therefore, it is impossible to discuss the amount of myo-IP₆ or the total amount of IP₆. Yet at least scyllo-IP₆ is more abundant in serpentinite soils than sedimentary soils. The abundance of scyllo-IP₆ in the serpentinite soils may be explained by the same mechanisms as pyrophosphate accumulation because IP₆ strongly adsorbs to soil minerals and forms IP₆-mineral complexes (Celi and Barberis 2007). Thus, the predominance of scyllo-IP₆ also suggests reduced P availability in the serpentinite soils compared to the sedimentary soils.

Mean value of phosphatase activities in the serpentinite soils was significantly higher than that in the sedimentary soils (t-test, p < 0.05, Fig. 2). Therefore, the potential capacity to degrade soil organic P is greater in P-limited soils. Interestingly, P-use efficiency in the serpentinite forest is almost the same as that in the sedimentary forest (Kitayama and Aiba 2002), implying that trees in the serpentinite forest successfully acquire soil P partly due to prompt degradation of soil organic P.

Figure 2. Mean values of phosphatase activities of the sedimentary soils and the serpentinite soils (n = 5). Error bars are standard deviations; t-test (p < 0.05).

Acknowledgment

We thank staffs of the Sabah Parks for their kind assistance in all aspects. We also thank local coworkers for the assistance in the fieldwork and also staffs in National Institute for Agro-Environmental Sciences, Japan, for the assistance in NMR analysis. This work was supported by JSPS KAKENHI (22255002) to K. Kitayama and by JSPS KAKENHI (JP16J11390) to D. Yokoyama.

Additional information

Funding

This work was supported by JSPS KAKENHI [22255002] to K. Kitayama and JSPS KAKENHI [JP16J11390] to D. Yokoyama.