Increased culturability of soil bacteria from Marcellus shale temperate forest in Pennsylvania

Abstract

Soil bacteria comprise a largely untapped resource with only 1-10% of bacterial species predicted to live in soil being culturable in the laboratory. Establishing culture-dependent protocols that identify unique operational taxonomic units (OTUs) is an important research topic in soil bacterial ecology. The culturability of soil bacteria may be improved by employing different culture media due to inherent preferences of growth substrate utilization. Soil-extract agar, R-2A agar, and 1% nutrient agar were used in this study to isolate bacteria obtained from soil samples collected in winter months to increase the understanding of bacterial diversity in Abernathy Field Station, a Marcellus shale temperate forest in Washington, Pennsylvania. Changes in bacterial diversity can be used to assess the early impact of anthropological factors, such as hydraulic fracturing in the Marcellus shale region, which may lead to severe environmental problems. For the purpose of long term ecological monitoring, data obtained from this year’s sample collection were analyzed in conjunction with previous years’ assessments. Bacterial isolates were analyzed taxonomically and phylogenetically. Unique OTUs were identified through comparative analysis of 16S rDNA. The Shannon-Weaver and Simpson’s diversity indices ranked isolates on soil-extract agar highest for species richness, and rarefaction analysis suggests that sampling saturation of OTUs identified on soil-extract agar has not yet been reached. Each culture medium studied supported isolates of four common phyla: Actinobacteria, Bacteroidetes, Firmicutes, and Proteobacteria. Soil-extract agar supported the greatest proportion of pigmented colonies including a Cyanobacterium which exhibited intra-16S rDNA polymorphism. Each culture medium supported the growth of unique OTUs and genera with Bacillus, Flavobacterium, Pseudomonas, Rhizobium, and Streptomyces found on each. This study suggests that utilizing different culture media can increase the culturability of soil bacteria.