New strategies to harness the potential of Amazonian Dark...

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Cell and Molecular Biology Laboratory

Center for Nuclear Energy in Agriculture

University of São Paulo

TSAI SIU MUI Fabiana Cannavan, Fernanda Nakamura, Mariana Germano, Maria Júlia Brossi, Amanda Lima, Aline de França, Clovis Daniel Borges, Ademir Durrer, Beatriz Maria Ferrari Borges, Rosineide de Souza, Andressa Venturini, Lucas W. Mendes,

Rodrigo Taketani

Newton Falcão, Wenceslau Teixeira, Eduardo Neves, Dirse Kern, Maria de Lourdes

New strategies to harness the potential of Amazonian Dark Earth microbiomes

1

Causes of deforestation in Amazonia

PASTURE – 60%

CROP PRODUCTION

(SMALL SCALE) – 33%

BURNING, MINING, URBANIZATION, ROAD AND DAM

CONSTRUCTIONS – 3% (>) TIMBER (LEGAL AND ILEGAL) – 3%

COMMERCIAL CROP PRODUCTION

(LARGE SCALE) – 1%

Source: Cattle Management in Amazon – trends and implications for environmental conservation . IMAZON, 2005.2

Conservation Practices and Crop Productivity in in the Tropics are Feasible Only if Sustainable Crop Management is Applied

3

The Below Ground Microbial World:

New Perspectives for Improvement of Soil

Quality and Crop Production in the Tropics

4

Uatumã River (2006)

Balbina (2005, 2006)

AMAZONIAN DARK EARTH AS MODEL SOIL

Black Gold

Terra Preta Nova

Wim Sombroek(40 years of study)

5

Brazilian Native Populations andTerra Preta Sites (0.1-0.3% ?)

6

Terra Mulata Terra PretaLeisure

Habitation

Forest

Terra Preta da Amazonia (Amazonian Dark Earth)

• Deep Horizon A with dark color

• Litic artifacts and ceramics

• High levels of P, Ca, Zn, Mg

• Elevated Organic Matter

• High pH

• Presence of black carbon = charcoal7

(2006-2008)

Montenegro and Sítio Santa RitaHatahara and Rio Preto da Eva

(2009-2010)

ADE PROFILE

8

Photo Thies © 2003

Hatahara Site

Partial Pyrolysis (400oC to 500oC)

9

Bacterial Community Composition in Brazilian Anthrosols and Adjacent Soils Characterized Using Culturing and Molecular Identification

FIRST STUDIES

Hatahara Lago Grande Açutuba

Hatahara Lago Grande Açutuba

22.1Commons (1)(2)

29.8Adjacent Soil (2)

48.1Anthrosol (1)

%Pattern (RFLP)

O´Neill et al. Microb. Ecol. (2009) 58:23-35 10

ELF – ELISA FLUORESCENCE

Alkaline Phosphatase

+ Biochar

3 hr 4 hr

β – D

glucuronidase

Phosphatase

Control

4 hr

11

Fluorescence Associated to Enzymes

β-D-glucuronidasePhosphatase

12

Hyphae are present on the surface of C-pyrogenic particle

Green dye indicates the presence of live organisms

Grossman J.M., O’Neill B.E., Gomes J. E, Tsai, S.M, Lehmann, J., Liang B., Thies J.E. (2010)

Black Carbon (Biochar) found in Amazonian Dark Earth

FIRST STUDIES

13

HAT I

Bacillaceae

8%

Burkholderiaceae

30%

Neisseriaceae

8%

Oxalobacteraceae

8%

Pseudomonadaceae

22%

Comamonadaceae

8%

Moraxellaceae

8%

Rhizobiaceae

8%

Terra Preta de Índio

Sinorhizobium

Burkholderia

R. tropici

Hatahara - Iranduba

16S rRNA Clone Libraries

HAT II

Moraxellaceae

21%

Comamonadaceae

20%

Micrococcaceae

16%

Pseudomonadaceae

11%

Bacillaceae

16%

Rhizobiaceae

5%

Burkholderiaceae

11%

Adjacent Soil

14

hyphavesicles

Vesicular Arbuscular Mycorrhiza in Leguminous Trees

0

5

10

15

20

25

30

35

40

45

50

55

60

% C

olo

niz

atio

n

ADE ADJ ADE ADJ

Site 1 Site2

River Uatumã

High P content does not inhibitVAM infection

Collembola are present, but not ants

The Amazonian Dark Earth microbiomes:

• Culture Dependent - Selective Media and Environmental Control

• Culture Indepent - Biochemical and Molecular Techniques

16

Community StructuresWho are in soil charcoal?

• Phylogenetic analyses• Sequencing the 16S rRNA gene• Image analyses of communities

Genetic PotentialWhat can the microbes do?

• Metagenome• Functional genes• Gene Neural network

Gene ExpressionWhat are the roles of the microbes?

• Metatranscriptome

• Metaproteome

Functional Characterization

• New methods of cultivation• Stable isotope probing (SIP)• NanoSIMS• Card FISH

NEW METHODS ARE AVAILABLE FOR THE DISCOVERY AND CARACTERIZATION OF THE MICROBIAL DIVERSITY

Cardenas & Tiedje (2008 )Current Opinion in Biotechnology 2008,19:544–549

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Lagoa Balbina

Se

qu

en

ce

Nu

mb

er

6873

19 18

12

51 1 0

79

27

75

33

1014

10

04

0

10

20

30

40

50

60

70

80

90

Terra Preta

Charcoal

ADE

Charcoal

Two 16S rRNA gene libraries were constructed from samples taken from a site that was undisturbed for over two decades and sequenced.

Ribosomal Database Project (RDP)

CLONE LIBRARY OF 16S rRNA GENEADE and Biochar

18

PRODUCTION OF ANTIBIOTICS AND PEPTIDES

150 Isolates from ADE

Siderophore production:

35 Pseudomonas 50% +

30 Arthrobacter 20% +

14 Bacillus 65% +

Antibiotics: Q-TOF (inhibition halos)

Phenazine (Pseudomonas sp.)

Pseudomonine (P.putida) – 330 Da

NH

N

N

O

O

NH

O OHNH

N

N

O

O

NH

O OH

Pseudomonine

Extract AE-control

Extract AE - TP16

20

0

200

400

600

800

1000

1200

1400

10

0

30

0

50

0

70

0

90

0

11

00

13

00

15

00

17

00

19

00

21

00

23

00

25

00

27

00

29

00

31

00

33

00

35

00

37

00

39

00

41

00

43

00

45

00

47

00

49

00

51

00

53

00

55

00

57

00

59

00

61

00

TPI

Carvão

Nu

mb

er

of

OTU

s o

r Sp

eci

es

Number of Sequences

Rarefaction Curve (Mothur)

HATAHARA SITE

Biochar

ADE

Amazonian Dark Earth and its Biochar

21

HATAHARA SITE

s

Biochar – Pyrogenic Charcoal (CAR) ADE – Amazonian Dark Earth

BACTERIA PYROSEQUENCING 16S rRNA gene (454 Roche)

ADE and its Biochar

22

In vitro Test: Antimicrobial Compound

A

B

Test with the isolate BCM 20

A) Culture media – extracted in chloroform (control)

B) Supernatant extracted in chloroform

C) Test against Rhizobium sp CB1809

ESI of isolate BCM20

Siderophore

hidroxamate = CATECOL

C

23

ARHD primers for large subunit (catalytic domain) of aromatic dioxygenases.

FUNCTIONAL DIVERSITY OF ALPHA-ARHD GENE IN AMAZONIAN DARK EARTH AND BLACK CARBON

24

N°

ob

serv

ed

OTU

s

N° of valid clones

0

5

10

15

20

25

30

0 20 40 60 80 100 120 140 160 180

BLACKCARBON

AMAZONIANDARK EARTH


25

BIOCHAR: BACTERIAL bph GENE

26

0 cm

30cm

30cm

03º16.883’S60º07.762’O

03º15.770’S60º06.023’O03º13.799’S60º16.150’O

03º05.829’S60º21.340’O

03º16.494’S60º12.340’O

03º15.964’S60º10.013’O

BIOCHAR MICROBIOME

27

DNA ExtractionSequencing Analysis

Amplification16s rRNA, alk e bph

Sequence Analyses(MEGA 5.0)

SE BHI TSA

AIAGA

RDP EZTaxon-e

BLASTnBLASTx

Fungene

Sequencing

MicroaerophylicIncubation 5 Different Culture Media

BiocharExtraction

ADE Sampling

CULTIVATION AND IDENTIFICATION OF ISOLATES FROM BIOCHAR

28

Orbital Agitation320C, 42h

NB mediumO.D. A600nm

All wells: BH culture mediaDCPIP indicatorSubstrates

O.D. and Cell Counts

Color MeasurementDCPIP A620nm

2h, 4h, 8h, 16h, 24h

CN: no inoculumCP: DSMZ type strainsSamples: isolates

Agitation at roomtemperature in the dark

Biodegration rates

of Each Isolates

Agitation andcentrifugation

Collection afterdiscolouration

Cell collection O.D. A600nmCell Growth Rates

Elution in water

Centrifugationand water

elution

Cell Counts

BIODEGRADATION IN VITRO TESTS

29

PahAc [2189972] Pseudomonas putida OUS82

0.1

Type IV

Type IIIαβ

Type IV

Type V

Type Iαβ

Type II

Type Iα

Type IIIα

Type ?


30

-2 -1,5 -1 -0,5 0 0,5 1 1,5 2

2h 4h

8h 16h

24h

Positive Type Strains Diesel

DSM6899 Pseudomonas putida 24h

DSM11192 Gordonia sp. 16h

ADE Isolates

42GA Leifsonia sp. JN872512.1 (99% 885bp) (Laguinho/GA) 2h

43A Arthrobacter woluwensis AB244289.1 (98% 707bp) (Laguinho/GA) 2h

30A Uncultured Pseudomonas HM996802.1 (98% 914bp) (Barroso/AIA) 2h

12B Bacillus pseudomycoides ACMX01000133 (99,45% 1447bp) (Barroso/BHI) 2h

62A Arthrobacter nicotinovorans X80743 (97,05% 901bp) (Hatahara Pasture/AIA) 4h

3GA Achromobacter aegrifaciens HF586507 (97,7% 949bp) (Hatahara Pasture/GA) alkB 8h

9B Uncultured Pseudomonadaceae (100% 1441bp) (H.Capoeira/BHI) 16h

7GA Uncultured Alcaligenaceae KC179063.1 (93% 1408bp) (Hatahara Pasture/GA) 16h

31E Uncultured Variovorax JQ770029.1 (97% 808bp) (Hatahara Sugarcane/ES) 16h

12GA Uncultured Gammaprotebacteria JF772106.1 (98% 762bp) (Laguinho/GA) 16h

8A Acinetobacter sp. KC257028.1 (97% 798bp) (Lago Grande/AIA) alkB 16h

17GA Enterobacter ludwigii KC139450.1 (99% 882bp) (C.Açutuba/GA) 16h

1E Pseudomonas nitrireducens HM246143 (97,27% 1449bp) (C.Açutuba/ES) 16h

1GA Ralstonia mannitolilytica KF751574.1 (99% 871bp) (Hatahara Sugarcane/GA) 16h

30E Burkholderia caribensis GU144372.1 (98% 1435bp) (Barroso/ES) 16h

45GA Pantoea anthophila EF688010 (95.00%_841bp) (Lago Grande/GA) 16h

5E Enterobacter ludwigii KF475862.1 (99% 1446bp) (C.Açutuba/ES) 16h

6GA Burkholderia ubonensis EU024179 (99,32% 916bp) (Hatahara Pasture/GA) 16h

4B Bacillus methylotrophicus EU194897 (99,23%_1441bp) (Barroso/BHI) 16h

40E Streptomyces tanashiensis AJ781362 (99,23% 1428bp) (Hatahara Sugarcane/ES) 16h 32

PAH BIODEGRADATIONPositive Type Strains Biphenyl Diesel Phenanthrene

DSM6899 Pseudomonas putida 16h 24h 24h

DSM11192 Gordonia sp. 24h 16h 16h

DSM6612 Arthrobacter oxydans 24h - -

Isolates

42GA Leifsonia sp. JN872512.1 (99% 885bp) (Laguinho/GA) 4h 2h -

43A Arthrobacter woluwensis AB244289.1 (98% 707bp) (Laguinho/GA) 8h 2h -

30A uncultured Pseudomonas HM996802.1 (98% 914bp) (Barroso/AIA) 16h 2h -

12B Bacillus pseudomycoides ACMX01000133 (99,45% 1447bp) (Barroso/BHI) 16h 2h -

62A Arthrobacter nicotinovorans X80743 (97,05% 901bp) (Hatahara Pasture/AIA) 16h 4h 16h

3GA Achromobacter aegrifaciens HF586507 (97,77% 949bp) (Hatahara Pasture/GA) alkB 24h 8h -

9B uncultured Pseudomonadaceae (100% 1441bp) (Hatahara Capoeira/BHI) 16h 16h 16h

7GA uncultured Alcaligenaceae KC179063.1 (93% 1408bp) (Hatahara Pasture/GA) 16h 16h 16h

31E uncultured Variovorax JQ770029.1 (97% 808bp) (Hatahara Sugarcane/ES/ES) 16h 16h 16h

12GA uncultured Gammaprotebacteria JF772106.1 (98% 762bp) (Laguinho/GA) 16h 16h 16h

8A Acinetobacter sp. KC257028.1 (97% 798bp) (Lago Grande/AIA) alkB 16h 16h 16h

17GA Enterobacter ludwigii KC139450.1 (99% 882bp) (C.Açutuba/GA) 16h 16h 16h

1E Pseudomonas nitrireducens HM246143 (97,27% 1449bp) (C.Açutuba/ES) 16h 16h 16h

1GA Ralstonia mannitolilytica KF751574.1 (99% 871bp) (Hatahara Pasture/GA) 16h 16h 16h

30E Burkholderia caribensis GU144372.1 (98% 1435bp) (Barroso/ES) 16h 16h 16h

45GA Pantoea anthophila EF688010 (95.00%_841bp) (Lago Grande/GA) 16h 16h 16h

5E Enterobacter ludwigii KF475862.1 (99% 1446bp) (C.Açutuba/ES) 16h 16h 16h

6GA Burkholderia ubonensis EU024179 (99,32% 916bp) (Hatahara Pasture/GA) 16h 16h 16h

4B Bacillus methylotrophicus EU194897 (99,23%_1441bp) (Barroso/BHI) 16h 16h 16h

40E Streptomyces tanashiensis AJ781362 (99,23% 1428bp) (Hatahara Sugarcane/ES) 16h 16h 16h 33

41E

Cu

pri

avid

us

nec

ato

rC

P0

02

87

8(9

94

4 1

43

5b

p)

64A unclassified Emterobacteriaceae(100 901bp)

34

BiocharBest Degraders

Maximum Likelihood TreeMega 5

34

SiteIdentity

(Phylum, Genus)Nº

isolatesCultureMedia

Similarity Seq (pb)Presencegene alk

Laguinho Firmicutes; Bacillus sp. 2 ES/BHI 100% 595pbIsolate

10E

Barroso Actinobacteria; Arthrobacter sp. 1 ES 99,6% 538pb -

Barroso Firmicutes; Bacillus sp. 3 BHI 100% 615pbIsolates4B, 14B

Barroso Firmicutes; Lysinibacillus sp. 1 BHI 100% 597pb -

Barroso Firmicutes; Paenibacillus sp. 1 BHI 100% 477pb -

Lago Grande Firmicutes; Bacillus sp. 1 AIA 94% 448pb -

Lago Grande Firmicutes; Paenibacillus sp. 1 ES 100% 449pb -

Costa do Açutuba

Proteobacteria; Pseudomonas sp. 1 ES 98% 551pbIsolate

3E

Costa do Açutuba

Firmicutes; Bacillus sp. 1 BHI 100% 589pb -

HataharaCapoeira

Actinobacteria; Arthrobacter sp. 1 BHI 98,4% 467pb -

HataharaSugarcane

Firmicutes; Bacillus sp 2 BHI 100% 660pb -

HataharaSugarcane

Proteobacteria; Burkholderia sp. 1 BHI 100% 439pb -

HataharaSugarcane

Proteobacteria; Ralstonia sp. 1 ES 100% 468pb -

HataharaSugarcane

Actinobacteria; Streptomyces sp. 1 ES 100% 626pb -

HataharaPasture

Filo Firmicutes; Bacillus sp. 4 ES/BHI 100% 627pbIsolate

46E35

20A alkB (C.Açutuba/AIA) 549bp

8A alkB Acinetobacter sp. (Lago Grande/AIA) 504bp

32T alkB Acinetobacter sp. (C.Açutuba/TSA) 423bp

35T alkB Arthrobacter sp. (C.Açutuba/TSA) 498bp

28B alkB partial unclassified bacteria (Hatahara Pasture/BHI) 286bp

9A alkM partial Paenibacillus sp. (Lago Grande/AIA) 299bp

3E alkB Pseudomonas sp. (C.Açutuba/ES) 543bp

2E alkB (C.Açutuba/ES) 523bp

gi|554751491|gb|ESK56380.1| alkane 1-monooxygenase Acinetobacter tjernbergiae DSM 14971 CIP 107465

3GA alkB Pantoea sp. (Hatahara Sugarcane/GA) 576bp

30GA alkB Pseudomonas sp. (Hatahara Sugarcane/GA) 465bp

37GA alkB (Hatahara Sugarcane/GA) 499bp

gi|551226365|emb|CDG78736.1| alkane 1-monooxygenase Acinetobacter baumannii 107m

gi|306996337|gb|ADN21388.1| AlkB-2 Alcanivorax dieselolei

16GA alkB (Laguinho/GA) 541bp

gi|240867807|gb|ACS65467.1| Alkane 1-monooxygenase Ralstonia pickettii 12D

gi|358072705|emb|CCE49828.1| Alkane-1 monooxygenase Burkholderia cenocepacia H111

gi|387997712|gb|EIK59041.1| alkane 1-monooxygenase Pseudomonas fluorescens SS101

gi|341603180|emb|CCC65858.1| probable transmembrane alkane 1-monooxygenase alkB Mycobacterium bovis

BCG str. Moreau RDJ2A alkB partial (C.Açutuba/ES) 581bp

gi|260066438|gb|ACX30751.1| AlkB5 Rhodococcus ruber

99

99

65

74

33

26

17

22

71

18

100

21

5

14

15

0.1

Gene alk – Maximum Likelihood Tree

3 substrates

2 substrates

36

PHAs Degradation Among 86 isolates:

46 isolates 0,05%

phenanthrene61 isolates 8% diesel76 isolates 0,05% biphenyl

14 isolates 1 substrate26 isolates 2 substrates40 isolates 3 substrates

ABILITY TO DEGRADE POLY-AROMATIC HYDROCARBON

37

SiteIdentity

(Phylum, Genus)Nº

isolatesCultureMedia

Similarity Seq (pb)Presencegene alk

Laguinho Firmicutes; Bacillus sp. 2 ES/BHI 100% 595pbIsolate

10E

Barroso Actinobacteria; Arthrobacter sp. 1 ES 99,6% 538pb -

Barroso Firmicutes; Bacillus sp. 3 BHI 100% 615pbIsolates4B, 14B

Barroso Firmicutes; Lysinibacillus sp. 1 BHI 100% 597pb -

Barroso Firmicutes; Paenibacillus sp. 1 BHI 100% 477pb -

Lago Grande Firmicutes; Bacillus sp. 1 AIA 94% 448pb -

Lago Grande Firmicutes; Paenibacillus sp. 1 ES 100% 449pb -

Costa do Açutuba

Proteobacteria; Pseudomonas sp. 1 ES 98% 551pbIsolate

3E

Costa do Açutuba

Firmicutes; Bacillus sp. 1 BHI 100% 589pb -

HataharaCapoeira

Actinobacteria; Arthrobacter sp. 1 BHI 98,4% 467pb -

HataharaSugarcane

Firmicutes; Bacillus sp 2 BHI 100% 660pb -

HataharaSugarcane

Proteobacteria; Burkholderia sp. 1 BHI 100% 439pb -

HataharaSugarcane

Proteobacteria; Ralstonia sp. 1 ES 100% 468pb -

HataharaSugarcane

Actinobacteria; Streptomyces sp. 1 ES 100% 626pb -

HataharaPasture

Filo Firmicutes; Bacillus sp. 4 ES/BHI 100% 627pbIsolado

46E

38

BIOCHAR53,49% 0,05% de phenanthrene70,93% 8% diesel88,37% 0,05% biphenyl

ADE SOIL77,7% 0,05% phenanthrene86,48% 8% diesel

39

Cell and Molecular Biology and the Terra Preta Group

CENA-USP

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New strategies to harness the potential of Amazonian Dark...

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