Microbial extracellular enzymes can remain active in soil for a long time bound and protected by humic substances. The ecological importance of humic-enzyme complexes is that their activity is not affected by microbial stresses, therefore playing an important role in a large number of biochemically mediated processes under adverse conditions for soil microorganisms (Nannipieri et al., 1996; Doni et al., 2012). Humic-bound enzymes extracted from soils or other organic materials can be separated by isoelectric focusing (IEF) on the basis of the isoelectric point of the humic substances. IEF did not modify either the enzyme activity or the molecular structure of the complexes (Benítez et al., 2000; Ceccanti et al., 2008).

Beta-glucosidase activity has been proposed as a good indicator for soil functions because its critical role on soil-carbon cycle (Nannipieri et al., 2012). The importance of extracellular β-glucosidase lies in the capacity of the soil to hydrolyse C substrates too large or insoluble for direct absorption by microbial cells. The characterization of stable humic–β-glucosidase compounds could assess the ability to fulfil essential functions in a highly vulnerable −and economically vital− agroecosystem as rainfed olive farming. Within this framework, the aim of this work was to determine the effects of soil management on the stable pool of soil carbon cycling enzymes as indicators of essential functions.

Humic–β-glucosidase complexes were extracted and then characterized from soils under four agricultural managements in a rainfed olive orchard representative of semi-arid Mediterranean habitats. The main characteristics of the experiment and soils are described in Moreno et al. (2009). The soil has been classified as an Anthropic Regosol (FAO, 1998). The study was conducted for 30 years in Jaen (south-eastern Spain) by designing a random-block of four treatments and four replicates (plots). The treatments tested were:

Two samples were collected in the centre of each plot by a modified soil-sample ring kit (Eijkelkamp) which includes a cylinder of 20 cm (depth of sampling) specifically manufactured for this purpose and then bulked. The samples were stored at 4ºC until analyses were made.

Humic substances were extracted from soil at 37 °C for 24 h under shaking, using Na₂P₄O₇ (0.1 M, pH 7.1) in a 1:10 w/v dry soil/extract ratio (Ceccanti et al., 1978). Then, the suspension was centrifuged (12,400×g) and filtered through a 0.22-μm Millipore membrane. The C-derived fraction >10⁴ Da was obtained through ultrafiltration on AMICON PM10 membrane of the C extract. The C content was determined by acid digestion with K₂Cr₂O₇ and H₂SO₄ at 140 °C for 2 h and then quantified according to Yeomans & Bremner (1988). This C fraction was used to determine extracellular β-glucosidase activity as described by Garcia et al. (1993), and it is based on the use of 0.05 M 4-nitro-phenyl-β-D-glucanopyranoside (PNG) as a substrate (Hayano & Tubaki, 1985). The p-nitrophenol (PNP) released by β-glucosidase activity was extracted and determined spectrophotometrically at 398 nm (Tabatabai & Bremner, 1969).

Isoelectric focusing was carried out in cylindrical gel rods (0.5×8 cm) containing polyacrylamide gel (5% w/v) and carrier ampholines in the pH 4–6 range (Bio-Rad Laboratories, Richmond, CA, USA) at a final concentration of 2% (Ceccanti et al., 1986; 1989). N,N,N′,N′-tetramethyl-1,2-diaminomethane and ammoniumperoxy-disulfate were also added in gel solution at a concentration of 0.03%. Organic material (100 μL of pyrophosphate extract derived fraction >10⁴ Da) at 4.4% of glycerine was applied at the top of the gel rod (cathode). A little amount (5 μL) of glycerine at 2.2% was added on the top of the sample to avoid the mixing with the cathodic solution (0.02 N NaOH); 0.01 M H₃PO₄ was used for the anodic cell. A pre-run of 1 h at the same current intensity and voltage used for the samples run were carried out for each gel tube (1.5 mA for each tube, 100-800 V); subsequently, the samples run was carried out for 2 h or longer until a stable IEF banding was reached. The electrophoretic bands were scanned by a Bio-Rad GS 80 densitometer, obtaining a typical IEF profile for each soil investigated. The IEF peak area was determined for each soil IEF profile, assuming as 100% the area under the entire IEF profiles (representative of the total loaded C). Gel pH was measured at 0.5 cm intervals with an Orion microprocessor (model 901, Orion research) connected to a microelectrode gel-pHiler (Bio-Rad Laboratories, Richmond, CA, USA). To determine β-glucosidase activity of the humic bands obtained by IEF, the gel was gently removed from the glass tubes. The bands were cut and pre-washed for 1 h with 2 mL of 0.1 M phosphate buffer, pH 6.4, at 37 °C (pre-washing removes the carrier ampholytes, salts, and other impurities from the gel, without releasing the gel-trapped humic matter). After removal of the buffer, 2 mL of fresh 0.1 M phosphate buffer, pH 6.4, and 0.5 mL of 0.05 M PNG were added and the mixture was incubated at 37 °C under shaking for 17 h.

Results are the means of 12 replicates (three per plot). The results were subjected to a factorial analysis of variance (ANOVA) using the STATISTICA software program (StatSoft Inc., Tulsa, OK, USA). The post hoc Tukey HSD test in a one-way ANOVA was used. to be evidence of statistical significance p-values of under 0.05 were considered.

Extracellular β-glucosidase was higher in soils under cover vegetation (Fig. 1), especially when these were managed with mechanical methods, evidencing the effect of sustainable managements on the soil biochemical activity. No significantly differences were found between soils where spontaneous vegetation was regularly eliminated by tillage (T) or herbicide applications (NC). The above agrees with previous experimental works where extracellular enzyme production has been linked to those agricultural managements increasing the quantity, quality and chemical diversity of cover crops residues (Tiemann et al., 2015).

The humic carbon in the bands after IEF humic carbon calculated from the IEF peak areas focused in the different selected bands (1, 2, 3 and 4) is shown in Table 1. The sum of the humic carbon in the IEF bands represent the C>10⁴ Da fraction of high molecular weight heteropolymers traditionally described as humic acids. CM soils showed the greater levels of humic substances, both total and those corresponding to each IEF band. Humic C focused at pH 4.2 was higher in all the treatments, confirming the stability of the humic substances isolated (Govi et al., 1994; Doni et al., 2012).

The β-glucosidase activity was also quite higher in all the four humic bands of CM soils (Fig. 2a). For T, NC and CH soils, the higher β-glucosidase activity was measured in the lower pH where band focalized. The trend was just the opposite for CM soils, and the higher activity was found at pH 4.5, pH range where enzymatic proteins generally are stabilized by humus molecules Ceccanti & Masciandaro (2003). However, the specific β-glucosidase activity, i.e. the ratio between β-glucosidase activity and C>10⁴ Da, was higher at this pH except for soil where tillage was the main management (Fig. 2b).

In this work, the stable pool of soil β-glucosidase enzymes were used to compared four long-term contrasting agricultural managements. Maintaining cover crops through fall, winter and early spring provoked a more stable and active pool of extracellular β-glucosidase in soils only if spontaneous vegetation was managed with mechanical methods. When herbicides were used during 30 years, the pattern of the molecular composition and activity of humus-β-glucosidase complexes were similar in covered and nude soils, although higher activity was retrieved in the former. This fact is probably related with the higher vegetal biomass incorporated and also with the different mode of action of the herbicides applied during the whole experiment, with different effects on both microbial activity and CO₂ fixation (Fedtke, 1982; Riah et al., 2014; Kane, 2015). Tillage management increased carbon mineralization. As a consequence, the level of humic substances and the activity of β-glucosidase humic-bound were quite lower than in the rest of treatments. Given the ecological role of extracellular soil carbon cycling enzymes, the characterization of humus β-glucosidase complexes could be an adequate indicator of sustainability of agricultural management systems.