Isolating Unique Bacteria from Terra Preta Systems: Using Culturing and Molecular Techniques as Tools for Characterizing Microbial Life in Amazonian Dark Earths Brendan O

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Isolating Unique Bacteria from Terra Preta
Systems Using Culturing and Molecular Techniques
as Tools for Characterizing Microbial Life in
Amazonian Dark Earths Brendan ONeill1, Julie
Grossman1, Siu Mui Tsai2, Jose Elias Gomes2,
Carlos Eduardo Garcia2, Dawit Solomon1, Biqing
Liang1, Johannes Lehmann1 and Janice Thies1
(1) Cornell University, Department of
Crop and Soil Science, Ithaca, NY, (2) Centro de
Energia Nuclear na Agricultura (CENA) Piracicaba,
Brazil
Results
Introduction
Phylogeny of 16S rDNA from Isolates
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.
MPN enumeration on liquid R2 medium
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).
Summary of bacterial isolates
Source of unique RFLP pattern
Anthrosol 48.1
Adjacent Soil 29.8
Common to both 22.1
Source Soil Isolate medium alt. medium growth Isolates from soil type Isolates with unique RFLP
Anth SER2A 85.5 26
Adj SER2A 65.9 14
Anth SE only 14.5 50
Adj SE only 34.1 25
Anth R2ASE 66.7 36
Adj R2ASE 62.7 18
Anth R2A only 33.3 18
Adj R2A only 37.3 33
Methods
Summary DNA sequencing family level

• 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.

Sequence Source No. of Families No. of Families Bacterial families
DGGE - Anth DGGE - Anth 2 Verrucamicrobiaceae, Acidobacteriaceae
DGGE - Adj DGGE - Adj 0 no families unique to this method and soil type
Culture - Anth Culture - Anth 10 Caryophanaceae, Enterobacteriaceae, Hyphomicrobiaceae, Intrasporangiaceae, Microbacteriaceae, Micrococcaceae, Myobacteriaceae, Phyllobacteriaceae, Streptomycetaceae, Xanthomonadaceae
Culture - Adj Culture - Adj 4 Burkholdariaceae, Bacillales, Crenotrichaceae, Flexibacteraceae
Both soil types Both soil types 3 Pseudamonadaceae, Bradyrhizobiaceae Flexibateraceae
Both methods and soils Both methods and soils 3 Bacillaceae, Sphingomonadaceae, Paenibacillaceae
DGGE of community DNA by site and soil type

• 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.

Conclusions

• 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.