 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.plant-soil.com J. Plant Nutr. Soil Sci. 2011, 174, 381–394 DOI: 10.1002/jpln.200900267 381 Multivariate analysis of soils: microbial biomass, metabolic activity, and bacterial-community structure and their relationships with soil depth and type Antonio Gelsomino 1 * and Arianna Azzellino 2 1 Dipartimento di Biotecnologie per il Monitoraggio Agroalimentare ed Ambientale (BIOMAA), Università Mediterranea di Reggio Calabria, Salita Melissari, 89124 Reggio Calabria, Italy 2 Dipartimento di Ingegneria Idraulica Ambientale Infrastrutture Viarie, Rilevamento (DIIAR), Politecnico di Milano, Piazza Leonardo da Vinci 32, 20133 Milano, Italy Abstract A multivariate statistical approach based on a large data set of abiotic and biotic variables was used to classify four contrasting-land-use soils. Soil samples were collected at increasing depth from a calcareous agricultural soil, a temperate upland grassland soil, a moderately acidic agri- cultural soil, and an acidic pine forest soil. Analytical investigations were carried out by using a combination of conventional physical, chemical, and biochemical methods coupled with denatur- ing gradient gel electrophoresis (DGGE) community fingerprinting of PCR-amplified 16S rRNA gene-coding fragments from soil-extracted total-community DNA. The data set of soil physical, chemical, and biochemical variables was reduced in dimensionality by means of a principal- component-analysis (PCA) procedure. Compositional shifts in soil bacterial-community structure were analyzed through a clustering algorithm that allowed identifying six main bacterial-commu- nity clusters. DGGE fingerprinting clusters were further analyzed by discriminant analysis (DA) using extracted PCA components as explanatory variables. Soil organic matter–related pools (TOC, TN) and functionally related active pools (microbial biomass C and N, K 2 SO 4 -extractable C) significantly decreased with soil depth, and resulted statistically linked to one other and posi- tively related to enzymatic activities (acid phosphatase, arylsulfatase, b-glucosidase, dehydro- genase, hydrolysis of fluorescein diacetate) and silt content. Besides organic-C gradients, pedo- genetic-driven physico-chemical properties, and possibly soil thermal and moisture regimes seemed to play a key role in regulating size and energetic ecophysiological status of soil micro- bial communities. DGGE analysis showed that contrasting horizons were conducive to the domi- nance of particular bacterial ribotypes. DA revealed that the bacterial-community structure was mainly influenced by organic matter–related variables (TOC, TN, CEC, C flush ,N flush , Extr-C), chemical properties such as pH, CaCO 3 , and EC, together with textural properties. Results indi- cate that, beyond land use or plant cover, pedogenetic-driven physico-chemical conditions chan- ging with soil type and depth are the key factors regulating microbial size and activity, and deter- mining the genetic structure of bacterial community. Key words: depth gradients / DGGE community fingerprinting / energetic ecophysiological indices / multivariate statistics / pedogenetic horizons / soil enzymes Accepted June 28, 2010 1 Introduction The functioning of soil, as a complex and spatially structured biological system, depends on the interplay of three general factors: environment, biological community structure, and biological activity (Griffiths et al., 1997). This basic concept has greatly stimulated investigations in search of significant relationships among biological properties and microbial-com- munity composition in relation to soil type (Girvan et al., 2003; Marschner et al., 2004; Ritz et al., 2004), crop (Buckley and Schmidt, 2001; Smit et al., 2001; Buyer et al., 2002; Johnson et al., 2003), and management practices (Ibekwe et al., 2002; Marschner et al., 2003; Bending et al., 2004). Indeed, it is well-known that the microbial numbers and bio- chemical activities are higher within the 25 cm topsoil and they are more rapidly responsive to environmental impacts (Joergensen and Emmerling, 2006). Needless to say, soils are comprised of differing pedogenetic layers that may sup- port a large number of microorganisms also residing in sub- surface horizons, which play an important role in soil forma- tion, ecosystem bio-geochemistry, contaminant degradation, and maintenance of groundwater quality (Fierer et al., 2003). In recent years, several authors have attempted to examine the variations in microbial-community composition and in bio- logical properties through increasing soil depths. Changes in the vertical distribution of various components of microbial communities in different terrestrial ecosystems were observed by using direct counting methods (Zvyagintsev , 1994; Ekelund et al., 2001). Other authors found a decrease in microbial-biomass size and in metabolic activities with increasing soil depth (Jörgensen et al., 2002; Taylor et al., 2002; Castellazzi et al., 2004; Sanesi and Certini, 2005). Compositional shifts in microbial-community composition in differing soil horizons were also assessed by using a combi- * Correspondence: Dr. A. Gelsomino; e-mail: agelsomino@unirc.it