1. Isolating Unique Bacteria from Terra Preta Systems: Using Culturing and Molecular Techniques as Tools for Characterizing Microbial Life in Amazonian Dark Earths Brendan O’Neill 1, Julie Grossman 1, Siu Mui Tsai 2, Jose Elias Gomes 2, Carlos Eduardo Garcia 2, Dawit Solomon 1, Biqing Liang 1, Johannes Lehmann 1 and Janice Thies 1 (1) Cornell University, Department of Crop and Soil Science, Ithaca, NY, (2) Centro de Energia Nuclear na Agricultura (CENA) Piracicaba, Brazil Results Introduction Methods Conclusions Most probable number (log10) of bacteria colony forming units (CFU) g-1 ODW soil growing in R2 liquid medium by site, depth and soil (anthrosol = gray bar, adjacent soil = brown bar). MPN was calculated using MPNES software from Woomer et al. (1990), CI(0.95). Terra preta soils are anthropogenic soils created by pre-Colombian indigenous cultures through the incorporation of organic-rich material into existing highly weathered soil. The resulting anthrosols are highly fertile, due, in part, to high charcoal (black carbon) content, which also leads to their distinct, blackish color. These anthrosols likely harbor a unique microbial ecology which contribute to their sustained fertility. We examined anthrosols (Anth) and adjacent (Adj) background soils from four sites in the Amazon Basin: Hatahara (Hat), Dona Stella (DS), Lago Grande (LG) and Açutuba (Acu). We hypothesized that 1) bacterial populations were higher in Anth than Adj. soils 2) Using culturing and molecular techniques, bacterial populations would be more similar between anthrosols than within the same site on two soil types. Extracts from anthrosols and adjacent soils were used to inoculate: 1) 5 replicate tubes at 6 dilutions of liquid R2 media and incubated for 45 days at 30ºC. 2) R2A and soil extract (SE) solid media, incubated for 120 days at 30ºC. Liquid media tubes were scored for positive growth and used to calculate most probable number (MPN). Colonies were isolated from solid media plates, and cross-cultivated onto alternate media (R2A for colonies forming on SE and SE for colonies forming on R2A). After media screening, 16S rDNA region was amplified using colony PCR, and resulting fragments digested with restriction endonucleases to determine unique isolates. Select 16S rDNA regions were sequenced based on RFLP pattern and results from media screening. From soil DNA extraction, a culture-independent DNA fingerprint, DGGE, was used to compare portions of 16S rDNA in soil types. Select bands from DGGE were sequenced and compared. Summary of bacterial isolates Summary DNA sequencing – family level Source Soil Isolate medium + alt. medium growth % Isolates from soil type % Isolates with unique RFLP Anth SE+R2A 85.526 Adj65.914 Anth SE only 14.550 Adj34.125 Anth R2A+SE 66.736 Adj62.718 Anth R2A only 33.318 Adj37.333 Source of unique RFLP pattern % Anthrosol48.1 Adjacent Soil29.8 Common to both22.1 MPN enumeration on liquid R2 medium DGGE of community DNA by site and soil type Phylogeny of 16S rDNA from Isolates Sequence Source No. of Families Bacterial families DGGE - Anth2 Verrucamicrobiaceae, Acidobacteriaceae DGGE - Adj0 no families unique to this method and soil type Culture - Anth10 Caryophanaceae, Enterobacteriaceae, Hyphomicrobiaceae, Intrasporangiaceae, Microbacteriaceae, Micrococcaceae, Myobacteriaceae, Phyllobacteriaceae, Streptomycetaceae, Xanthomonadaceae Culture - Adj4 Burkholdariaceae, Bacillales, Crenotrichaceae, Flexibacteraceae Both soil types3 Pseudamonadaceae, Bradyrhizobiaceae Flexibateraceae Both methods and soils3 Bacillaceae, Sphingomonadaceae, Paenibacillaceae MPN calculation shows that for every site, anthrosols have as higher or a culturable bacteria population than adjacent soils. The majority of isolate diversoty in termsn of unique RFLP types was derived from anthrosol isolates (52.8%) and only a third of unique RFLP types came from adjacent soils. In spite of a smaller percentage of isolates forming initially and growing exclusively on SE agar from anthrosols (14,5%) compared to adjacent soils (34.1%), anthrosols have twice the number of unique RFLP types. On a community level basis using DGGE, anthrosols are more similar to each other than different soil types form the same site. Sequencing reveals much higher family-level diversity in anthrosols, and for screening purposes, culturing proved useful for identifying unique community members.