Microbial counts were performed according to Italian official methods (Picci and Nannipieri 2003). Briefly, soil samples (10 g) were shaken for 30 min in 90 ml of physiological solution containing 0.162 g of tetrasodium pyrophosphate to detach the bacteria from soil particles. After soil particles were allowed to settle for 15 min, the solution was diluted tenfold in a series. Selected populations of soil microbial community were detected at 28°C by using the Surface Spread Plate Count Method (aerobic bacteria) and the Pour Method (anaerobic bacteria). Three plates were used per each dilution. Total heterotrophic aerobic bacteria were counted in Plate Count Agar (Oxoid Ltd., Oxford, UK). The plates were incubated for 3 days.
Mould and yeast were cultivated on Malt Agar (Oxoid Ltd., Oxford, UK) supplied with chloramphenicol (100 mg L-1) for 3 days (Allievi and Quaroni 2003).
For the isolation of actinomycetes, Starch-Casein Agar (10 g soluble starch, 0.30 g casein, 2 g KNO3, 2 g NaCl, 2 g K2HPO4, 0.01 g FeSO4, 0.05 g MgSO4, 0.02 g CaCO3, 1,000 ml distilled water, 17 g bacteriological agar, pH 7.0) was used (Kuster and Williams 1964). The medium also contained cycloheximide at 100 mg ml-1 to minimize fungal contamination. The plates were incubated for 14 days.
The medium used for aerobic and anaerobic cellulolytic bacteria was composed by 5 g L-1 carboximethylcellulose (CMC) (Sigma-Aldrich Chemie GmbH, Steinheim, Germany), 1 g L-1 (NH4)NO3, 1 g L-1 yeast extract, 50 ml L-1 standard salt solution, 1 ml L-1 trace elements solution, 15 g L-1 bacteriological agar, at pH 7.0. The plates, incubated in aerobic or anaerobic (Oxoid's Anaerogen™ System) (Allievi and Moller 1992) conditions for 7 days, were stained with Congo red (0.1%) for 20 min and bleached with NaCl (5 M) for 20 min to put in evidence cellulolytic activities by developing clear haloes around the colonies (Kluepfel 1988). All microbial counts were carried out in triplicate and microbiological data were expressed as CFU g-1 of dry soil.
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