Pearson correlation [0.0%-100.0%]

DGGE

100

90

80

70

60

50

40

30

20

DGGE

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

Lago Grande

Acutuba

Lago Grande

Acutuba

Acutuba

Acutuba

Lago Grande

Lago Grande

Hatahara

Hatahara

Dona Stella

Dona Stella

Dona Stella

Hatahara

Hatahara

TP

TP

TP

TP

Background

Background

Background

Background

TP

TP

TP

Background

Background

Background

Background

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 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

0-30 40-70 80-120 Buried/50+ 0-8 10-40 0-30 50-80

Hat DS LG Acu

Site and sample depth (cm)

Lo

g C

FU

/od

w

Anthrosol Adj. Soil

103

104

105

106

107

108

109

Firmicutes

LG Anth 17-2 (63-70) SE/R2A AB110497 Dyella japonica

Hat Adj 7-14 (0-10) SE/R2A AY741332 Burkholdaria cepacia

Hat Anth 1-8 (0-30) R2A/SE AF063219 Pseudamonas stutzeri

Hat Anth 1-13 (0-30) SE AY946283 Enterobacter sp.

Hat Adj 7-22 (0-10) R2A/SE Hat Anth 2-13 (43-69) R2A

AB033949 Sphingomonas sp. Hat Anth 2-3 (43-69) SE/R2A

Hat Anth 2-10 (43-69) SE/R2A AF508207 Mesorhizobium septentrionale

Acu Anth 27-9 (48-83) R2A LG Anth 18-10 (0-16) R2A/SE

Hat Adj 7A-11R (0-10) SE/R2A Hat Anth 2-5R (43-69) SE/R2A

AF510586 Bradyrhizobium sp. LG Anth 17-6 (63-70) SE/R2A

AB035490 alpha proteobacterium LG Anth 18-11 (0-16) SE/R2A

Proteobacteria

Hat Anth 2-24 (43-69) R2A/SE DS Anth 16-11 (Buried) SE/R2A

X95471 Actinomyces sp. Hat Anth 2-6 (43-69) SE X52921 Myobacterium fortuitum

LG Anth 17-3 (63-70) SE Y08853 Terrebacter sp.

Hat Anth 1-17R (0-30) SE/R2A AJ717357 Microbacterium oxydans

LG Anth 17-8 (63-70) R2A/SE AY572475 Anthrobacter sp.

Hat Anth 2-12c (43-69) R2A/SE Hat Anth 2-15 (43-69) SE AY651318 Anthrobacter sp.

LG Anth 19-1 (16-43) SE/R2A Hat Anth 3-9 (43-69) SE/R2A Hat Anth 3-6 (78-100) SE/R2A Hat Anth 2-22(*9)R (0-30) R2A/SE

LG Anth 17-10 (63-70) R2A/SE Hat Anth 2-7 (43-69) SE/R2A LG Anth 18-1 (0-16) SE/R2A

Actinobacteria

AY238335 Flexibacter sp. LG Adj 22-7 (0-8) R2A/SE

Hat Adj 11-14 (80-120) SE/R2A AF361187 Flexibacter sp.

Bacteroidetes

X64372 Isosphaera pallida AJ862839 Methanoculleus thermophilus

100

100

4799

100

100

52

502960

92

94

100

97

5886

100

7694

99

36109

96

48

100

7498

933753

76

51

99

61

100

100

100

80

65

56

45

57

70

40

96

0.05

ResultsIntroduction

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

SourceSoil

Isolate medium + alt. medium growth

% Isolates from soil type

% Isolates with unique RFLP

AnthSE+R2A

85.5 26

Adj 65.9 14

AnthSE only

14.5 50

Adj 34.1 25

AnthR2A+SE

66.7 36

Adj 62.7 18

AnthR2A only

33.3 18

Adj 37.3 33

Source of unique RFLP pattern

%

Anthrosol 48.1

Adjacent Soil 29.8

Common to both 22.1

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

FamiliesBacterial families

DGGE - Anth 2 Verrucamicrobiaceae, Acidobacteriaceae

DGGE - Adj 0 no families unique to this method and soil type

Culture - Anth 10Caryophanaceae, Enterobacteriaceae, Hyphomicrobiaceae, Intrasporangiaceae, Microbacteriaceae, Micrococcaceae, Myobacteriaceae, Phyllobacteriaceae, Streptomycetaceae, Xanthomonadaceae

Culture - Adj 4 Burkholdariaceae, Bacillales, Crenotrichaceae, Flexibacteraceae

Both soil types 3 Pseudamonadaceae, Bradyrhizobiaceae Flexibateraceae

Both methods and soils 3 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.