Acid phosphomonoesterase and β-glucosidase activities in volcanic soils under permanent fertilized pastures: distribution profile and microbial effort toward P acquisition

ABSTRACT

This work focused on the amounts and distribution of organic C (OC), organic phosphorus (P_o), and acid phosphomonoesterase (AcP) and β-glucosidase (βG) activities in volcanic soils, as well as on the effect of inorganic P (P_i) on potential P-acquiring effort. The OC and P_o contents are correlated with the active aluminum (Al_ox) and iron (Fe_ox) and with the stratified gradients of the enzyme activities. The AcP activity does not seem to be suppressed by soil P_i content, showing a positive linear relationship with P_i. High values for the AcP:βG activity ratio are observed, which suggests a high energy allocation (C consumption) to ensure P demand. With the increase of Al_ox and Fe_ox, the AcP:βG ratios increase and the potential C:P acquisition (ln(βG):ln(AcP)) values decrease. Nevertheless, the average C:P molar ratio of the six soils (115) are below the critical value, above which soils may present low P bioavailability. The results suggest that the effort for P acquisition (AcP:βG) and the potential C:P acquisition are both affected by the size of the P_i pools and by Al_ox and Fe_ox contents. If P_i retention increases due to high contents of Al_ox and Fe_ox, energy allocation shifts toward AcP production, in detriment of microbial population and plant nutrition. Considering the OC accumulation in the studied soils, several hypotheses may be considered such as the energy demand toward P acquisition, and the ability of Al_ox and Fe_ox metal complexes to protect organic molecules to depress βG activity.

KEY WORDS:

• Soil phosphorus
• Andosol
• acid phosphomonoesterase
• β-glucosidase
• phosphorus acquisition effort

1. Introduction

The recovering process of key nutrients from residue inputs and accumulated organic matter (OM) is an essential microbial function in soils. Generally, these fluxes require the activity of extracellular enzymes to process the more complex compounds of OM into available subunits (Caldwell 2005). Since enzyme activities can be reliably determined in laboratory essays, have microbial ecological significance, are sensitive to environmental stress, and respond rapidly to changes in land management, the use of soil extracellular enzymes, as biological indicators is of great interest (Dick 1997). Nevertheless, laboratory essays measure the potential activity of the enzymes (Vmax) and not their actual activities, since the conditions in the laboratory (pH, temperature, substrate concentration, and diffusion coefficients) are different from those occurring in situ (Nannipieri, Kandeler, and Ruggiero 2002).

Andosols are known to display unique morphological, physical, and chemical properties attributed to the components of their amorphous fraction, like allophane, imogolite, ferrihydrite, and Al/Fe–humus complexes (Shoji, Nanzyo, and Dahlgren 1993), combined with a high capacity to adsorb P, and may affect their agronomic use. Volcanic ash soils usually also contain high amounts of OM (Batjes 1996). Although comprising only 0.84% of the global land area, these soils store approximately 5.0 % of the global soil C (Eswaran, Berg, and Reich 1993; Dahlgren, Saigusa, and Ugolini 2004). The hypotheses that can explain OM accumulations in Andosols are: (i) P is a rate-limiting factor for OM mineralization (Buurman, Peterse, and Almendros 2007) attributed to the high P-fixing capacity of these soils; (ii) biodegradation of OM is limited by its complexation with Fe, Al, and allophanic constituents (Matus et al. 2014); (iii) enzymes and microbial by-products may be deactivated by adsorption on short-range order mineral surfaces of andic soils (Saggar et al. 1994); (iv) the microbial decomposition of OM may be limited by low pH values and Al toxicity (Tate and Theng 1980) although the formation of metal-humus complexes leaves little or no exchangeable Al available to limit microbes and plants in Andosols (Shoji and Masui 1972).

OM fuels the metabolic activity beneath soil surface (Bauer and Black 1994) and is an important indicator of soil quality and productivity (Reeves 1997). The potential activities of extracellular enzymes are important indicators of nutrient availabilities in soils. Since the synthesis of extracellular glucosidases and phosphatases represents a large metabolic investment, their production is induced when labile C and/or P_i, respectively, are in short supply (Zheng et al. 2015). Consequently, enzyme production and release from cells can be repressed or induced by genetic expression according to environmental signals (Sinsabaugh and Shah 2012). Soil P concentrations regulate the phosphatase activity through P_i signal transduction pathway (PHO), a mechanism linked to cellular P homeostasis (Dick, Dos-Santos, and Meyer-Fernandes 2011). Phosphatase synthesis is regulated throughout the growth phase of microbial cells and population, being influenced by exogenous and endogenous P pools (Persson et al. 2003). Phosphatase activity is repressed in non-limiting P medium, but once exogenous P content became deficient, phosphatase mRNA and phosphatases synthesis are induced (Dick, Dos-Santos, and Meyer-Fernandes 2011).

The exoenzyme β-glucosidase (βG) plays a major role in degradation of carbohydrates in soils and its synthesis is induced by the products of cellulose breakdown, including cellobiose, glucose, and their metabolites (Stewart and Leatherwood 1976). Research has shown that βG is the most abundant and easily detectable of the three enzymes (endo-1,4-glucanase, exo-1,4-glucanase and β-D-glucosidase) involved in cellulose degradation in soil, and is rarely substrate limited, thus making it ideal to examine the importance of physico-chemical controls on the turnover of SOM (Turner et al. 2002). Therefore, βG activity has been used to closely monitor rapid changes in soil OC associated with management practices (Bandick and Dick 1999). On the other hand, βG contributes to provide energy for soil microorganisms and is directly related to soil OC content (Eivazi and Tabatabai 1988; Bandick and Dick 1999). This enzyme catalyzes the cleavage of one mole of cellobiose in two moles of glucose, regulating the supply of energy for microorganisms, which are unable to take up cellobiose directly. Alef and Nannipieri (1995) referred that the cellulose breakdown is a slow process, dependent on the concentration, location and mobility of cellulose-degrading enzymes. Cellulases activities, such as βG, are susceptible to the substrate crystallinity, associated substances, and surface area, also to the environmental conditions such as pH, temperature, and water content.

Regarding phosphatases, the name includes a broad group of enzymes that catalyze the hydrolysis of both esters and anhydrides of phosphoric acid (Schmidt and Laskowsky 1961). Acid phosphomonoesterase and alkaline phosphomonoesterase are phosphoric monoester hydrolases with optimal activities in acid and alkaline environments, whereas phosphodiesterase is a phosphoric diester hydrolase. All these three enzymes act on ester bonds (Deng and Tabatabai 1997). Phosphatases play a key role in soil P cycling by catalyzing mineralization of organic P and contributing to the increasing of soil P bioavailability. Like any exoenzyme molecule, phosphomonoesterases diffuse poorly into the soil matrix (Lefebvre et al. 1990) and their activities are dependent on different parameters such as soil characteristics, microorganism, plant cover, inputs, and the presence of inhibitors or activators (Speir and Ross 1978). Generally, it is assumed that phosphomonoesterases activity is dependent on the enzymes associated with viable or lysed microbial cells, root-released enzymes into the rhizosphere and detritosphere (Nannipieri 1994), although root-released enzymes probably mineralize organic P from sloughed off or damaged cells rather than from native soil organic P (Lefebvre et al. 1990). Nevertheless, the major contribution to its activity, in soil, is due to the extracellular enzymes stabilized by colloids (Nannipieri et al. 2011). In volcanic soils, minerals such as allophane and ferrihydrite showed evidences of enzymes protection, namely AcP and βG and other enzymes appear to be sorbed in the soil minerals without discrimination of the enzyme type (Allison 2006).

Fertilization affects soil biochemical and biological properties (Liang et al. 2014), and the influence of external nutrient inputs on soil microbial ecology has been frequently emphasized (Ai et al. 2012). As an example, PO₄^3- is a competitive inhibitor of both phosphomonoesterases in soils (Juma and Tabatabai 1978), although the absence of response of phosphatase activities to P addition has been also reported (Schneider et al. 2001). On the other hand, N fertilization generally increases the activity of phosphatases in soils (Marklein and Houlton 2012).

Regarding the equilibrium between C and P Sinsabaugh, Hill, and Shah (2009, 2010) found an average value of 1.6 for the ratio of AcP to βG activities in 40 different soils with no andic characteristics. Nevertheless, in a long-term field experiment on an Andosol, Moro, Kunito, and Sato (2015) observed an increase from 3.9 to 9.5 of the same ratio when no P was added or when P was limiting plant growth. Accordingly, the value of this ratio seems to reflect, even for crops, the variable readiness for acquiring phosphorus in soils with different P retention capacity or with different mineral P (or N) fertilizations.

Mineral fertilizers are widely used in the Azores region to enhance soil fertility and improve pasture production, particularly P fertilization due to the high contents of active fractions of Al and Fe and consequent high P retention capacity of the soils. Consequently, it was hypothesized that the abundant presence of Al and Fe active minerals may present a specific role on the relationships between soil OC, soil P enrichment, and the activity of free AcP and βG enzymes in these soils. Therefore, the specific aims of this work were: (i) to evaluate the stratification βG and AcP activities in the 15 cm superficial layer of six Andosols collected in Azores (Pico Island) under cattle grazed pasture and fertilization over the past 20 years, and; (ii) to study the relationships among both enzymes, OM accumulation, and P_i and (iii) assess the effect of Al and Fe active minerals on βG and AcP potential activities and on the indices of P acquisition.

2. Materials and methods

2.1. Soil classification and land management

Six soils, from commercial farms, in Pico island of the Azores archipelago, located between 200 and 700 m of altitude were selected. Soil I is a Haplic Andosol, II and III are Placic Andosol and IV, V and VI are Leptic Vitric Andosol, according to WRB (2006). Brief dicriction: Vitric Andosols are characterized with lower degree of weathering (Alox + 1/2 Feox above 0.4%) and minimum concentrations of glass content (above 10%), bulk density above 0.9 kg dm⁻³, and PR% above 25%. Haplic and Placic Andosols are soils with higher degree of weathering than Vitric Andosols (Al_ox + 1/2 Fe_ox above 2.0%) and minimum concentrations of glass content (below 10%), bulk density below 0.9 kg dm⁻³, and PR% above 70%. Placic Andosols tend to be highly weathered with high metal-humus content.

Soils I and VI were never tilled and the remaining ones have not been tilled for more than 20 years. All soils are under pastures use, being fertilized annually (Table 1).

Table 1. Soils management practices

	management
soil	tillage	fertilization		use
I	never tilled	annually	70 N:18P:31 K*	cut and grazing^c
II, III, IV, and V	no-tillage^a	annually^b	60 N:22P:42 K*	cut and grazing^d
VI	never tilled	not every year	14 N:6P:11 K*	grazing^c

^afor more than 20 years, ^b since 2008, ^c spontaneous, ^d seeded

*xN:yP:zK stands for kg of x/y/z per hectare

2.2. Soil sampling

Superficial layers of 15 cm were selected in each location and then subdivided in 5 sub-layers (0–1, 1–2, 2–5, 5–10, and 10–15 cm) making a total of 30 soil samples. Samples were air-dried, sieved through a 2-mm screen, and stored until analysis, which were conducted in duplicate.

2.3. Soil analysis

Phosphate retention (PR, %) was assessed according to Blakemore, Searle, and Daly (1987), organic carbon (OC) through near-infrared detection with an elemental analyzer (Primac SC, Skalar; Breda, NL), and pH in a 1:2.5 soil-water suspension.

Active Fe_ox and Al_ox were extracted according to the 0.2 M (NH₄)₂(COO₂)₂.H₂O – 0.2 M (COOH₂)₂.2H₂O pH 3.0 method (Schwertmann 1964) and quantifications in the extracts were performed by atomic absorption spectrometry (Thermo Scientific ICE 3000 Series AA Spectrometer).

2.4. P characterization

Total P (P_t) was determined after ignition (550°C/2 h), by extracting 2 g of air-dried soil (1:25 soil-solution/16 h) with 0.5 M H₂SO₄. Inorganic P (P_i) was determined after extracting 2 g of air-dried soil (1:25 soil-solution/16 h) with 0.5 M H₂SO₄. Organic P (P_o) was obtained by the difference between P_t and P_i, according to Saunders and Williams (1955) modified by Walker and Adams (1958).

Extractable inorganic P (P_i-Olsen) was determined by the Olsen method (Olsen et al. 1954) by shaking air-dried soil with 0.5 M NaHCO₃ pH 8.5 (1:20 soil-solution/30 min). Total P Olsen (P_t-Olsen) was assessed by digesting an aliquot of the previous extract with 11 M H₂SO₄ and 4% (NH₄)₂S₂O₈ (USEPA 1982). Organic P Olsen (P_o-Olsen) was obtained by the difference between P_t-Olsen and P_i-Olsen.

Soil P intensity (P-CaCl₂) was determined according to Houba et al. (1990), by shaking air dried soil with 0.01 M CaCl₂ (1:10 soil-solution/30 min).

P concentration in the soil extracts and digestates were determined by the molybdate-ascorbic acid blue method in a segmented flow analyzer (SanPlus, Skalar, Breda, NL) after a dialysis step to prevent interference from color and solids in suspension (Coutinho 1996).

2.5. Potential enzymatic activity

The potential enzymatic activities of βG and AcP were assessed according to Tabatabai (1994). Briefly, p-nitrophenyl-β-D-glucopyranoside was used as substrate to determine the βG activity in a modified universal buffer (pH 6.0) and incubated for 1 h at 37°C. AcP activity was determined with p-nitrophenyl-phosphate as substrate in a modified universal buffer (pH 6.5) and incubated for 1 h at 37°C. In both enzymatic activity determinations, the final p-nitrophenol (p-NP) concentration, as the reaction product, was determined by molecular absorption spectrophotometry, at 400 nm, in a segmented flow analyzer equipped with a dialysis step (SanPlus, Skalar, Breda, NL). Activities were expressed in µg p-NP g⁻¹h⁻¹ and converted to nmol p-NP g⁻¹ OC h⁻¹ for stoichiometric assessments.

2.6. Statistical analysis

Weighted means in each profile were calculated taking into consideration the different thickness of each layer. Statistical analyses (linear and logarithmical regressions, arithmetic, and weighed averages, ranges, and Pearson correlation coefficients (r)) were carried out using Analysis ToolPak and Solver supplement of Microsoft Excel®. The symbols *, ** and *** indicate significance at < 0.05, < 0.01, and < 0.001 probability (p) levels, respectively. Prior to analysis of the potentials for nutrient acquisition, exoenzymatic activities were log_e transformed to normalize variance, according to the conventions of stoichiometric analyses (Sinsabaugh and Shah 2012).

3. Results and discussion

3.1. Soil properties

The weighted averages of some chemical parameters of the soils under study are shown in Table 2. Concerning OC, soils I and VI had the highest contents, 156 and 178 g OC kg⁻¹, respectively. Despite having the same classification of soils IV and V (Vitric Andosols), soil VI seems to present a better preservation of its OC fraction, which may be attributed to the fact that it was never tilled, just as soil I. The no-tillage management of the soils is expected to be partially responsible for a general decrease on the OC mineralization fluxes (Reeves 1997). Nevertheless, and according to Eswaran, Berg, and Reich (1993), soils derived from volcanic materials tend to accumulate OM, influenced by their andic properties. Inoue and Higashi (1988) observed that the accumulation of OC in the upper 35 cm of andic soils has a strong linear relationship with extractable Al_ox and Fe_ox. In the present study, a significant trend of OC increase was observed only with Fe_ox (r = 0.54**, n = 30) but not with Al_ox. Matus et al. (2014) and Kunito et al. (2016) working with Andosols, pointed out that reduced availability of OM, as a result of Al and Fe binding, may protect a substantial pool of OC from microbial degradation, leading to suppress microbial mediated nutrient cycling.

Table 2. Chemical properties of the studied soils (weighed means of the five assessed layers)

	soil
parameters	I	II	III	IV	V	VI
pH (H2O)	5.1	5.3	5.5	5.4	5.4	5.4
OC (g kg^−1)	156	147	137	44	62	178
Po (g kg^−1)	2.1	2.0	1.6	0.52	0.83	1.9
Pt (g kg^−1)	2.8	2.9	2.2	2.3	3.1	3.1
Po-Olsen (mg kg^−1)	46	35	27	14	10	37
Pi-Olsen (mg kg^−1)	23	19	9	65	77	39
P-CaCl2 (mg kg^−1)	0.04	0.01	0.01	0.64	0.98	0.36
PR (%)	99	98	99	50	60	59
Alox (mol kg^−1)	1.51	1.63	1.90	0.81	0.96	1.05
Feox (mol kg^−1)	0.20	0.16	0.18	0.08	0.07	0.12

In each of the individual soils, the OC contents decrease significantly along the soil profile (Figure 1(a)) and this stratification may be due to the management of the soils, since they have been used as permanent grazing grasslands, not being tilled for the past 20 years and thus subjected to topsoil accumulation of crop residues and feces deposition. The average content of the soils was of 120 g OC kg⁻¹ for the whole profile and of 175 g OC kg⁻¹ in the top 0–1 cm layer.

Figure 1. Soil profile distribution of (a) organic carbon (OC) and (b) organic phosphorus (P_o) contents

The mean value for P_t contents of the studied soils is 2.7 g P kg⁻¹, where P_o represents about 54.3 % of the total, which are in accordance with the range of 30 to 70% for the P_o pool on P_t referred by Amano (1981) for Andosols. Nevertheless, the extreme repartition values of 23.0% and 75.3%, observed in soils IV and I respectively, are outside of the range referred by that author. Gudmundsson, Gudmundsson, and Thorvaldsson (2014) also found higher rates of P_o than P_i at superficial bulk layer of 15 and 20 cm of andic soils from Iceland, after long-term inorganic fertilization of grasslands. Considering individual profiles, only soils IV and V presented higher P_i content when compared to P_o, soils where the lowest contents of OC, Al_ox and Fe_ox were also observed (Table 2).

The overall P_o mean of the six soils was 1.5 g P kg⁻¹ in the top 15 cm, decreasing from 2.0 to 1.3 g P_o kg⁻¹ in depth. As a component of the SOM, P_o is highly correlated with OC (r = 0.91***, n = 6 and r = 0.96***, n = 30), and represents about 0.72% of the soil OM fraction. Soils I and IV had the highest and the lowest contents, 2.1 and 0.52 g P_o kg⁻¹, respectively. The lower amounts of P_o and their percentages on P_t in Soils IV and V may be due to their vitric character. In Andosols from Japan, Otani and Ae (1999) found that organic forms of P were tightly bound to Al and Fe compounds, thus not easily mineralizable by microorganisms, since active Al and Fe may bind labile P_o in a similar way to labile P_i.

Considering that both soils IV and V have lower contents of Al_ox and Fe_ox, it is expectable that the OC, and subsequently P_o, also present lower values, as observed in Table 2, probably due to a faster mineralization, or due to the relative youngness of the Vitric Andosols with shorter periods of OC accumulation. Nevertheless, and despite being also a Vitric Andosol, soil VI presents a better preservation of its OC and P_o contents, which may be attributed to the fact that it was never tilled, reinforcing the effect of an easier mineralization in soils IV and V. Apart from these same soils (IV and V), P_o contents decrease significantly along each soil profile (Figure 1(b)). In Otani and Ae (1999, 1997) did not observe an accumulation or stratification of P_o, even after the addition of OM, but they observed a clear stratification for P_i. Cade-Menun et al. (2010) observed that labile P_i concentration was significantly higher under no-till than in tilled soils at 5–10 cm layer, after a long-term study comparing conservation tillage and conventional tillage, but did not observe significant variation in P_t and P_o. Rodrigues et al. (2016), studying tropical soils under conservative tillage and mineral fertilization, reported a P_o built up at the surface layers. No-tillage systems led to an accumulation of P_o, despite representing less than 20% of soil P_t. An increase of labile and non-labile P_i fractions was also observed, particularly in the 0–5 cm soil layers, when compared to conventional tillage. Al_ox + Fe_ox contents presented positive correlations with P_o (r = 0.51***, n = 30), which may indicate the protective effect of Al and Fe constituents on P mineralization (Otani and Ae 1997). As Al_ox is less correlated with P_o (r = 0.48*) than Fe_ox (r = 0.67***), the results may also suggest that P_o forms in this type of soils present a higher affinity for Fe than for Al.

Regarding the P_o fraction extracted by the Olsen method, soils I and V present once more the highest and the lowest P_o-Olsen contents, respectively, 46 and 10 mg P kg⁻¹ (Table 2). The top 2 cm presented an average content of 49 mg P kg⁻¹, about 2.1 times higher than the sub-superficial layers (5–15 cm), where the rhizosphere is primarily developed and nutrients are absorbed. As observed for P_o, soils IV and V do not show a clear decreasing stratification also for P_o-Olsen (data not shown).

The overall mean of P_o-Olsen represents nearly 1.9% of the total P_o, although with variations among the samples. In fact, P_o-Olsen is correlated with both P_o (r = 0.95**) and OC (r = 0.91*) when the weighted means of the six soils are considered, since the accumulation of OC in the soil leads to the accumulation of P_o and subsequently of potentially labile P_o-Olsen. But when the relationships are searched among the individual values of the 30 samples, two linear regressions can be established between P_o-Olsen and P_o or OC. An expected positive slope and a high level of correlation are observed for soils I, II, III, and VI, but negative slopes are observed for soils IV and V (Figure 2). In these relationships, P_o presents a better correlation (r = 0.90***, n = 20) than OC (r = 0.72***, n = 20) in soils I, II, III, and IV. On the opposite, soils IV and V show a negative slope and weaker correlation for both parameters, significant with OC (r = 0.65*, n = 10) but not significant with P_o.

Figure 2. Relationships between (a) organic carbon (OC) versus Olsen extractable organic phosphorus (P_o-Olsen), (b) organic phosphorus (P_o) versus extractable organic phosphorus (P_o-Olsen)

Generally, these results do not fully agree with the data presented by Turner and Haygarth (2005) for temperate pastures in non-volcanic soils, since the authors did not find any correlation between P_o-Olsen and soil OM, although P_o-Olsen and P_o were correlated (r = 0.54**). Soils with different characteristics, after disturbance, undergo changes in the native OC (Cannell and Hawes 1994) and the same may happen with native P_o, with consequences on the P_o-Olsen:P_o ratio. In the present study, although this ratio does not seem to depend on the absolute content of soil OC (r = 0.35^ns, n = 30), a significant relationship was observed with pH values (r = −0.63***, n = 30), with the ratio P_o-Olsen:P_o decreasing with soil acidity. On the other hand, whereas P_o-Olsen (NaHCO₃) is considered as labile form of P_o and available P is closely related to these P_o fractions (Tiessen, Stewart, and Cole 1984), its specific quantification and further study may contribute to advances in soil test procedures and interpretation. In fact, labile P_o is undetectable in the molybdate-reactive P in the Olsen extract, but P_o-Olsen values, in some of our samples, are higher than P_i-Olsen.

3.2. Soil enzyme activities

The weighted means of βG activity (Table 3) vary from 111 (soil III) to 316 µg p-NP g⁻¹ h⁻¹ (soil V), being generally wider than the range of 48 and 169 µg p-NP g⁻¹ h⁻¹ reported by Dick, Breakwell, and Turco (1996) for air-dried soils. The Vitric Andosols under study have a higher average βG activity, presenting approximately twice the activity (296 µg p-NP g⁻¹ h⁻¹) when compared to Haplic and Placic Andosols (143 µg p-NP g⁻¹ h⁻¹).

Table 3. Weighted average ratios of different soil parameter related with enzyme activity and P acquisition effort

parameters	soil
	I	II	III	IV	V	VI
C:P	142	132	160	50	52	149
βG (µg p-NP g^−1 h^−1)	171	148	111	303	316	270
AcP (µg p-NP g^−1 h^−1)	1650	1592	1443	1278	1719	1551
AcP:βG ^a	10.7	10.7	14.7	4.6	5.8	6.4
ln(βG) (nmol p-NP g^−1 C) ^b	8.9	8.8	8.6	10.4	10.2	9.1
ln(AcP) (nmol p-NP g^−1 C) ^b	11.2	11.2	11.2	11.9	11.9	11.0
ln(βG):ln(AcP) ^c	0.79	0.79	0.76	0.87	0.85	0.83

^amicrobial effort for enzyme production/exudation; ^b potential activity of C and P acquisition; ^c potential activity of C:P acquisition

Concerning the weighted means of AcP activity in each soil, values ranged from 1278 (soil IV) to 1719 µg p-NP g⁻¹ h⁻¹ for AcP, again in soil V. Between the two soils groups (Vitric Andosols versus Haplic and Placic Andosols), the averages of this enzyme activity are more similar than the averages of βG activity. On the other hand, the observed values tend to be at the upper limit of the range reported for air-dried soils (Dick, Breakwell, and Turco 1996) or temperate soils under permanent pasture (Turner and Haygarth 2005), 1112 and 1694 µg p-NP g⁻¹ h⁻¹ respectively. Borie and Zunino (1983) have already reported that AcP activities are usually very high in andic soils due to: (i) protective effect of Al and Fe compounds on AcP and (ii) the low P intensity in soil solution. Later, Kunito et al. (2012) also observed, in Japanese Andosols, high AcP activity, referring that microbial communities in these soils produce large amounts of phosphatases to ensure an adequate P supply.

In all the six soils, the 0–2 cm layers presented the higher activity. The gradient of the βG and AcP potential activities for each soil are shown in Figure 3(a) and 3(b). Along the soil profiles, their gradients show similar trends to those of OC and P_o previously referred. Eivazi and Tabatabai (1990), when assessing the factors that affect βG of the 100 cm superficial layer of agriculture soils in Iowa, also found a decrease in the potential activity along the soil depth. For Iowa soils, the same observation for AcP activity was reported by Juma and Tabatabai (1978). In a forest andic soil, Redel et al. (2008) observed higher values of AcP activity on the 0–2 cm layer (9700 to 25300 µg p-NP g⁻¹ h⁻¹) than in the remaining layers to a depth of 20 cm (6000 to 18200 µg p-NP g⁻¹ h⁻¹). Šnajdr et al. (2008) also reported a gradient in the AcP and βG activities along the depth in a forest soil.

Figure 3. Soil profile distribution of (a) β-glucosidase (βG) activity and (b) acid phosphomonoesterase (AcP) activity

Although βG and AcP are highly correlated (r = 0.87***, n = 30) with each other (Figure 4(a)), there were no correlations with OC or P_o when the 30 samples were compared. However, when the linear correlations for the 5 layers of each of the individual soils were considered, relationships were much closer, with most of their r values presenting a high or very high level of significance (Figure 4(b) and 4(c)). Eivazi and Tabatabai (1990) and Juma and Tabatabai (1978) also found a close relationship between potential soil enzymatic activity and the respective substrate (OM and P_o) in each of the studied soils.

Figure 4. Relationships between (a) acid phosphomonoesterases (AcP) versus β-glucosidase (βG); (b) β-glucosidase (βG) activity versus organic C (OC) content; and (c) acid phosphomonoesterases (AcP) versus organic P (P_o) on the six studied soils

Concerning the contents of Al_ox and Fe_ox (Figure 5(a) and 5(b)), inverse correlations with βG or AcP activities were observed, suggesting a repressing effect promoted by these soil minerals, as reported by Allison (2006) on Hawaiian Andosols. Moreover, it seems that these soil characteristics affect more βG activity than AcP (Figure 5(a) and 5(b)). In fact, both the relationship of potential enzymatic activities versus the Al_ox and Fe_ox content were inverse, but the βG versus Al_ox + Fe_ox was better correlated (r = 0.71***, n = 30) than the AcP versus Al_ox + Fe_ox (r = 0.46*, n = 30). Yan et al. (2010) in a silty clay Stagnic Anthrosol, reported that the immobilization of βG on inorganic soil colloids may have depressed its free activity until about 73%, suggesting that the active sites of the enzyme were hindered by the soil colloidal surfaces, resulting in its inaccessibility for the substrate or in modifications of the active sites. Therefore, the high contents of organic matter usually reported for Andosols (WRB 2006) may be also related with the reduced βG activity due to the high contents of Al and Fe of these soils. In fact, the higher slopes presented by both soils IV and V (Vitric Andosols) for the relationship between βG and OC, about 6 times higher than for other soils (Figure 4(b)), suggest a higher substrate availability in the former soils, which also present lower OC contents as a possible consequence of their lower richness in extractable Al_ox and Fe_ox (Table 2).

Figure 5. Logarithmical relationships of the enzymatic activities versus Al_ox and Fe_ox content: (a) β-glucosidase (βG) and (b) acid phosphomonoesterases (AcP)

Moreover, both enzymes seem to be more affected by Al_ox than by Fe_ox. βG activity presented a r = −0.65*** with Al_ox and r = −0.59** (n = 30) with Fe_ox, while AcP activity presented a r = −0.42* with Al_ox and no significant correlation with Fe_ox (n = 30). Kunito et al. (2016) reported the repression of the carbon degrading enzymes in soils with high Al_ox and Fe_ox contents. Rao and Gianfreda (2000) and Rao, Violante, and Gianfreda (2000), studying the interaction between soil AcP enzymatic activity with Al and Fe, observed a 45% reducing effect on its activity when interacting with Al and no effects when interacting with Fe.

The present results do not elucidate if OC accumulation, in soils derived from volcanic material is favored by: (i) the protection of soil OM by the Al and Fe compounds, as pointed out by Matus et al. (2014) and Kunito et al. (2016), lowering its availability for mineralization; (ii) or by the inhibition of βG expression by Al and Fe, lowering the microbial mineralization efficiency. However, it is clear, in both hypotheses, the determinant role of Al and Fe compounds on OC accumulation in the studied volcanic soils.

Although soils are subjected to periodic mineral P fertilization, the AcP activity seems not being inhibited by the applied P. As shown in Figure 6, unexpected positive linear regressions of AcP with the total or available pools of inorganic P (P_i or P_i-Olsen respectively) are observed when all the 30 samples are considered. When the regressions are established with the weighted averages of the 6 soils, no relationship is observed. These results seem to contradict the reported inhibition of AcP expression by the presence of phosphate anion (Dick, Dos-Santos, and Meyer-Fernandes 2011) and the observations of Juma and Tabatabai (1978), authors who reported an inhibition effect of soil AcP expression due to the presence of soil applied P. Nevertheless, this apparent contradiction may be explained by the fact that the observations made by Juma and Tabatabai (1978) were done within the same soil, while the present results respect to six different soils and 30 different samples. On the other hand, the soil characteristics of both studies are substantially different, since soils derived from volcanic ashes present a high P retention capacity. Therefore, different P retention capacities of soils may overlap the inhibition of AcP expression promoted by P_i, since applied P_i in andosols can be readily fixed by Al and Fe compounds, thereby reducing its bioavailability. Once more, the lower slopes presented by Haplic and Placic Andosols for the relationship between AcP and P_o, of about 7 times less than for Vitric Andosols (Figure 4(c)), suggest a lower substrate availability (P_o) in the former soils, as a possible consequence of their higher content in extractable Al_ox and Fe_ox (Table 2). Otani and Ae (1997) reported that labile P_o in andic soils can be also fixed by Al and Fe in a similar manner to labile P_i.

Figure 6. Relationships between (a) acid phosphomonoesterases (AcP) versus inorganic P (P_i) and (b) acid phosphomonoesterases (AcP) versus extractable inorganic P (P_i-Olsen), for all samples (n = 30) and for the weighted average of each soil (n = 6)

3.3. P acquisition effort

Nutrient availability in soil can be expressed by calculating the acquisition effort by microbial extracellular enzymatic activity. The ratio of P to C enzyme activities (AcP:βG), the so-called P acquisition effort, translates the energy allocation made by microorganisms to acquire P through extracellular enzymes (Moro, Kunito, and Sato 2015), providing information about the soil nutrient availability (Sinsabaugh and Shah 2012). Haplic and Placic Andosols have a higher ratio than Vitric Andosols, 12 and 5.6, respectively, showing nearly twice of the P acquisition effort (Table 3). Moro, Kunito, and Sato (2015) assessed values with the same magnitude for soils derived from volcanic materials, although the values of both studies are much higher than those indicated by Sinsabaugh, Hill, and Shah (2009, 2010) in soils that covered 40 ecosystems from the river Mississippi, Missouri, Ohio to the Great Lakes (an average of 1.6). The AcP:βG ratio presents a high inverse correlation with P_i, P_i-Olsen and P-CaCl₂ (r = −0.76***, −0.78*** and −0.71***, n = 30; Figure 7(a), 7(b) and 7(c)), meaning that comparing soil P richness among different soils, the increase of their richness leads to the decrease on P acquisition effort, but not necessarily decreases the values of AcP activity (Table 3). These observations support the results obtained by Moro, Kunito, and Sato (2015) when relating the AcP:βG ratio versus soil P_i-Olsen and the results recently presented by Fujita et al. (2017) when relating AcP:βG ratio with P_i availability assessed by different methods in forest Andosols and Cambisols. But a high positive correlation (Figure 7(d)) is observed with active Al_ox + Fe_ox contents (r = 0.85***, n = 30), meaning that P acquisition effort increases with the capacity of the soil to retain P.

Figure 7. Logarithmical (ln) regression of acid phosphomonoesterases (AcP) to β-glucosidase (βG) ratio (P acquisition effort) versus: (a) inorganic P (P_i); (b) extractable inorganic P (P_i-Olsen); c) soil solution inorganic P (P-CaCl₂); and (d) linear relationship of acid phosphomonoesterases (AcP) to β-glucosidase (βG) ratio versus Al_ox + Fe_ox content

These results suggest that the effort for the acquisition of P decreases, through the allocation of energy to produce AcP enzymes, when available P exists at levels able to supply microorganism and plants homeostasis and full development needs. So, when Al and Fe are present at higher levels (Haplic and Placic Andosols), high values of AcP:βG ratio are expected, since P retention is higher, resulting in low P_i availability, leading to a more intense production and exudation of AcP to mineralize soil P_o. In acidic Andosols from Japan, Kunito et al. (2012) have already reached equivalent conclusions, observing that the high AcP activity is due to low P availability. In these soils, microbial communities produce large amounts of AcP to ensure an adequate P supply, resulting in an increase of activity in P-limited soils. Relating soil AcP:βG ratio with plant P availability and uptake, Moro, Kunito, and Sato (2015) reported values between 3.9 and 9.5 for cabbage field plots subjected to P fertilization. Their results indicate the potential usefulness of this index also for agronomic purposes, since the higher values of AcP:βG were observed on plots where no P was applied, where the crop also presented the lower yield and the lower P contents in the aerial biomass.

Based on the same principles, another approach has been proposed by Sinsabaugh and Moorhead (1994) and Sinsabaugh and Shah (2012), using the concepts of potential activity for C acquisition and for P acquisition, ln(βG) and ln(AcP), respectively, and defining the potential of C:P acquisition as the ratio ln(βG):ln(AcP) ratio. This potential of C:P acquisition measures the different energy resource partition between P_o acquisition and microbial growth (Sinsabaugh et al. 2008). As expected from AcP and βG relationship (Figure 4(a)), ln(βG) versus ln(AcP) are highly correlated (r = 0.90***, n = 30), with a slope of 1.40. Studying nutrient acquisition in soils and Sinsabaugh, Hill, and Shah (2009, 2010) indicated a slope of 1.16. Waring, Weintraub, and Sinsabaugh (2014) performed a meta-analysis to explore energetic and stoichiometric constraints on enzyme production in tropical soils with low P bioavailability. These authors also found a high linear correlation (r = 0.80***, n = 57) between potential C acquisition and potential P acquisition, with a slope of 1.18. The soil type may also play a key role on the resource allocation for P-acquiring enzymes synthesis, as recently also referred by Fujita et al. (2017). Therefore, the higher value achieved in our study is consistent with the low bioavailability P in soils derived from volcanic materials.

Concerning the potential of C:P acquisition (ln(βG):ln(AcP)), its value is expected to be higher when lesser energy effort is directed to P acquisition (AcP:βG), being both parameters highly correlated (r = −0.93***, n = 30) in our soils. Therefore, the potential of C:P acquisition is positively affected by the P_i pools and negiatively affected by Al_ox and Fe_ox contents, in accordance with the results of the present study referred previously (Figure 7) and with Allison (2006) and Sinsabaugh and Shah (2012) observations. In the case of the regression of ln(βG):ln(AcP) with Al_ox + Fe_ox, the relationship is much closer (Figure 8, r = 0.90***, n = 30) than with AcP: βG (Figure 7(d)). Considering that active Al and Fe bond to soil OM (Dahlgren, Saigusa, and Ugolini 2004), repressing the activity of AcP (Kunito et al. 2016), the results suggest that active Al and Fe are involved in the decreasing of the potential C:P acquisition activity (r = −0.86***, n = 30) on these soils. Sinsabaugh, Hill, and Shah (2009, 2010) also indicated the average of 0.95 for soils (n = 929) as the threshold value below which the energy resource allocation shifts toward P_o mineralization on microbial population growth. In the present study, all the observed values are much lower (Figure 8), either for the weighed mean of the Haplic and Placic Andosols or Vitric Andosols, with values of 0.66 and 0.75, respectively (Table 3), as for each individual layer (between 0.74 and 0.91). Therefore, these lower ratios indicate, once more, the low P_i bioavailability of these soils, which leads to an intense AcP production and tends to shift the energy partition from growth toward P acquisition and homeostasis maintenance, especially on the Haplic and Placic Andosols, with higher levels of extractable Al_ox and Fe_ox. Thus, the limitation on the size of the soil microbial biomass through the energy restriction for microbial growth may also contribute for the OC accumulation in these soils.

Figure 8. Linear relationship of potential of C:P acquisition (ln(βG):ln(AcP)) versus Al_ox + Fe_ox extractable content, n = 30; the threshold value for energy shift for P acquisition is represented by the dashed line

Cleveland and Liptzin (2007) pointed out an average value of 186 (166 for pasture soils) for the molar C:P ratio, assessed on molar basis of total content of each Sinsabaugh, Hill, and Shah (2009, 2010) consider this value as the threshold value above which a P limitation may occur. In our work, it was observed that Vitric Andosols show a value of 84 for the molar C:P ratio, lower than the average value of 145 observed for the Haplic and Placic Andosols (Table 3). Vitric Andosols show lower P acquisition effort, when compared to Haplic and Placic Andosols that have higher Al and Fe contents, being related to the observed lower C:P ratio in Vitric Andosols (Table 3). Therefore, it seems that the average C:P ratio of 186 as a general threshold value, pointed by the former authors, needs to be adjusted for soils derived from volcanic materials. In these conditions, the abundance of Al_ox and Fe_ox compounds are responsible for an intense P retention and a lower effective availability of mineral P, their high contents on P_t and P_i.

4. Conclusions

Based on the available results, the following conclusions may be presented:

• The management of the soils for the past 20 years (no-till and grazing) led to a strong increment of all the analyzed parameters (OC, P_o, P_o-Olsen and βG and AcP expression) on the 2 cm top layers, when compared with the 10–15 cm layers;

• Activity of soil βG is repressed by both Al_ox and Fe_ox, being the effect of Al_ox much closer related to enzyme expression than that of Fe_ox;

• The expression of soil AcP seems to be affected by Al_ox but not by Fe_ox.

• The presence of abundant and growing P_i in itself is not a condition for the decrease of soil AcP, since its expression in different soils also depends on their capacity for P retention;

• The effort for P acquisition (AcP:βG activity ratio) and the potential of C:P acquisition (ln(βG):ln(AcP)) are both affected by the P_i pools and by Al and Fe contents. Therefore, to maintain the homeostasis condition, the allocation of energy to produce and exudate AcP enzymes decreases, when available P_i exist at levels able to supply microbial growth requirements. On the contrary, when Al and Fe minerals are abundant and P_i retention is high, the energy allocation shifts toward AcP synthesis enzymes, which may affect the microbial growth rate;

• The general C:P molar ratio threshold value for soil biomass resource energy partition between growth and P acquisition probably needs to be adjusted for soils derived from volcanic materials.

Acknowledgments

Rui Bajouco was funded by a fellowship granted by the Regional Directorate for Agriculture. The authors would like to thank UTAD Soil and Plants Laboratory staff for technical assistance, and Dr David Walker and Dr Cristina Bajouco for reviewing the English version of this manuscript.

Disclosure statement

No potential conflict of interest was reported by the authors.