Extraction of soil bacteria from a Ferralsol

Abstract

Extraction of intact bacteria from soil by dispersion and density gradient centrifugation may facilitate analyses of soil bacterial communities which are otherwise hampered by soil particles. Reasonable cell yield and representative fractions with sufficient purity can be extracted from most soils, but highly weathered, clayey and acidic tropical soils like Ferralsols represent a challenge to the method due to low cell yields. At an early stage of our studies with Ferralsols we also found substantial contamination of the extracted bacterial fractions by soil material, measured as total Al and Fe. We have adapted the method to a Ferralsol (pH 4.9, 64% clay, 21% silt, 15% sand) by factorial combinations of pH modification and salt (NaCl) concentration during soil dispersion, and evaluated the yield (microscopic cell counts), purity (optical density and Al + Fe content), and compared the composition of the extracted bacteria (phospholipid fatty acid analysis and 16S rDNA-denaturing gradient gel electrophoresis) versus that of the intact soil community. The cell yield was increased with a factor 2–3 by increasing the pH to 7.5, while it was decreased to a similar extent by adding NaCl (8 g l −1). However, NaCl removed more than 99% of the Al + Fe contamination of the bacterial extracts, and the combination of modified pH and NaCl addition secured reasonable cell yield (4.6% of total number) and low contamination. The observed effects of pH and NaCl are probably due to changes in variable charge (by pH) and ion distribution (NaCl) around interacting particles (soil and bacteria), thus affecting their flocculation. Phospholipid fatty acid and denaturing gradient gel electrophoresis analysis indicate that the bias of the bacterial extracts compared to direct soil extracts increases with the addition of NaCl as well as by pH manipulation. Nevertheless, representativeness was acceptable as indicated by a Bray–Curtis similarity index (bacterial extracts versus soil) of 70% and 87% for phospholipid fatty acid profiles and denaturing gradient gel electrophoresis, respectively. Overall, the results reveal a trade-off among yield, purity and representativeness. Thus, depending on application and analyses, future users can choose the right treatment according to their specific purpose.