Isolating Unique Bacteria from Terra Preta Systems: Using Culturing and Molecular Techniques as Tools for Characterizing Microbial Life in Amazonian Dark Earths.
Brendan O'Neill1, Julie Grossman1, Siu Mui Tsai2, Jose Elias Gomes2, Carlos Eduardo Garcia2, Dawit Solomon3, Biqing Liang3, Johannes Lehmann1, and Janice Thies1. (1) Cornell Univ, Dept of Crop and Soil Science, Bradfield Hall, Ithaca, NY 14853, (2) Centro de Energia Nuclear na Agricultura (CENA), Av. Centenário, 303, Piracicaba, 13400-970, Brazil, (3) Cornell University, Department of Crop and Soil Science, Bradfield Hall, Ithaca, NY 14853
The greater fertility of Terra Preta (TP) soils is thought to be due to their high black carbon (BC) content, which contributes to increased nutrient and moisture retention, and increased pH. The unique chemistry of BC may also result in distinct microbial communities involved in nutrient cycling and organic matter turnover. TP soils offer an excellent model system for studying soils containing elevated and stable BC fractions in comparison to adjacent soils, because state factors, such as mineralogy, precipitation and climate, are the same between soils at a given site. We compared the microbial communities in TP soils with those in background soils adjacent to TP sites at four locations in the Brazilian Amazon. We used a combination of culture-based and molecular techniques to characterize and identify the key members of the bacterial communities in these soils. We found that culturable bacteria were more numerous in TP soils than in adjacent background soils. Bacteria were grown on soil extract agar prepared from TP and adjacent soils and, by cross-cultivating on the two media, bacteria uniquely suited to growth on TP soil substrates were isolated. All isolates were screened by use of RFLP fingerprinting and then the 16S rDNA of unique isolates was sequenced. We hypothesized that the TP soils would contain bacteria that are uniquely adapted to soils high in BC and that these bacteria would have more phylogenetic similarity to each other across TP sites than in comparison to bacteria from their corresponding adjacent soils. Clustering analysis of RFLP fingerprints indicated that isolates obtained from TP soils were more closely associated with each other than with bacterial isolates from adjacent soils within the same site. Of the bacterial isolates sequenced, most fell within the Bacterial divisions: Firmicutes, High G+C Gram positives, alpha-Proteobacteria and gamma-Proteobacteria, however only 18% of the sequences matched sequences in the Ribosomal Database Project II database at or above 97% and only 4% of the sequences matched reported sequences at or above 99% similarity. Lastly, we compared phylogenies of sequences obtained from individual soil isolates with those obtained from cloning and sequencing DNA from PCR-DGGE gels. Results from both approaches show a greater homology between sequences obtained from the four TP sites than between sequences obtained from adjacent and TP soils from the same site. These results indicate that commonalities in the chemistry of the TP soils, regardless of the background soil type (oxisol, latosol or spodosol), are influencing bacterial population structure in these soils. By combining culture-based and culture-independent molecular techniques we are obtaining a more complete analysis of the suites of organisms adapted to soils rich in BC. Black carbon is widespread in the environment and, once created, persists over long time scales. Knowledge of the ecology of TP soils may contribute to a broader understanding of the behavior of BC in natural environments and inform its possible future use in agricultural systems to improve soil fertility.