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Soil is a complex and dynamic biological system with up to 90% of the processes in soil mediated by microbes. The microbial population in soil is diverse and contains up to 6000 bacterial genomes per gram of soil (Nannipieri et al., 2003). Furthermore, it has recently been reported that soil microbial presence far exceeds currently accepted values and large contributions of microbial peptide/protein are found in the humic substance fraction of soils (Simpson et al., 2007). Investigating microbial diversity is important as it is believed that differences in microbial community composition may also influence the chemical composition of organic materials in soil (Makarov et al., 2002). Moreover, the structure of SOM is significantly impacted by the carbon input source, since the microbial and the plant derived biomass residues are believed to differ significantly in their molecular structures (Kindler et al., 2009). In these contexts, the study of soil microbiology ecology is vital to our understanding of natural processes such as C and N cycling and their associated impacts on agricultural productivity and climate change.
Molecular markers, such as the 16S rRNA gene, have been extensively applied to detect, identify and measure microbial diversity from environmental samples (Peixoto et al., 2002). In most cases, the 16S rRNA is amplified from total DNA extracted from a sample by employing polymerase chain reaction (PCR). Universal primer pair 27f and 1525r (Lane, 1991), that allow amplification of nearly complete 16S rRNA genes from the majority of known bacteria have been used to study the types of bacteria present (composition), the number of types (richness), and the frequency of distribution or the relative abundance of types (structure) in a diverse range of habitats (Dunbar et al., 199; Osborne et al., 2005). In this chapter, the near complete 16S rRNA gene sequences of 40 isolates were determined and analysed for two soils used in subsequent studies.
2.2 Materials and Method
2.2.1 Soil and sampling
Soil samples were collected from two Irish field sites (Church and Big Bull Park) at the Teagasc, Oakpark Crops Research Centre, Carlow in February 2007. The soils, a light clay-loam (Big Bull Park) and heavy clay-loam (Church) were under similar management practices and agricultural regimes, and have been subjected to intensive tillage for over 20 years. The agricultural management practices and physico-chemical properties of these fields are summarized in Table 188.8.131.52. Sampling was carried out according to a modified version of the protocol described by Joseph et al. (2003). Composite samples (each composite sample composed of three samples) were collected at eight locations along transect lines following a ‘Z’ pattern. A 25-mm-diameter clean metal core was used to sample 100-mm long soil cores from the A horizon, which were transferred to sterile polyethylene bags and sealed at the collection sites. Soil cores were transported at the ambient temperature and processed within 24 h of collection. The upper 30 mm of each core was discarded, and large pieces of roots and stones were removed from the remainder, which was sieved through a stainless steel sieve with a 2-mm aperture (IMPACT Laboratory Test Sieve, UK). Sieved samples were pooled, homogenized and stored at 4°C at its field moisture content for further analysis.
Characteristics Oakpark soils
Origin Church Big Bull Park
Management history Intensive tillage for over 20 years
% sand 38.0 45.0
% silt 22.0 25.0
% clay 40.0 30.0
Total C (%) 5.33 4.17
Total N (%) 0.42 0.14
pH in H2O 6.9 6.7
Table 184.108.40.206: Origin, management history and physico-chemical properties of soils for microbial characterization. Information provided by Oakpark Crops Research.
2.2.2 Media and growth conditions
Microbial cultivation was carried out according to a modified version of the protocol described by Janssen et al. (2002). Approximately 1 g of either soil was added to 100-ml aliquots of sterile distilled water in 250-ml conical flasks and dispersed by stirring with Teflon-coated magnetic bars (8 mm in diameter, 50 mm long) on a magnetic stirrer for 15 min at 400 rpm. One-millilitre aliquots of soil suspension were added to 9-ml portions of dilute nutrient broth (DNB), containing gL-1: Lab-Lemco’ Powder 1.0; Yeast Extract 2.0; Peptone 5.0; and Sodium Chloride 5.0, at a concentration of 0.08 g per litre of distilled water (Oxoid Ltd., Hampshire, England). Diluted soil suspensions were mixed by vortexing at approximately 150 rpm for 10 s, and used to prepare serial dilutions containing 10-2 to 10-4 g of soil suspension. One hundred-microlitre (100 µl) aliquots of each dilution series was plated on duplicate LB agar plates containing 0.5% dripstone, 0.25% yeast extract, 0.1% D-glucose, 0.25% NaCl and 1.5% agar. Serially inoculated LB plates were incubated at RT for 48 h and all isolated colonies were selected from the 10-4 dilution of each soil type and used to inoculate 3.0 ml of LB broth. Cultures were incubated at RT for 48 h. All samples were done in duplicates.