Unlocking Microbial Unlocking Microbial Communities in Communities in Terra Terra PretaPreta

Nucleic acid extraction and purification Nucleic acid extraction and purification as keys to characterizing biology in black as keys to characterizing biology in black

carbon soilscarbon soilsBrendan O’NeillBrendan O’NeillJulie GrossmanJulie Grossman

Janice ThiesJanice Thies

Cornell University Cornell University Department of Crop and Soil SciencesDepartment of Crop and Soil Sciences

Ithaca, NYIthaca, NY

Microbiology in TPMicrobiology in TP

Noted both for their high black carbon (BC) Noted both for their high black carbon (BC) content and fertility.content and fertility.

Soils high in BC appear to harbor distinct Soils high in BC appear to harbor distinct microbial populations microbial populations ((PietikäinenPietikäinen, , KiikkiläKiikkilä et al. 2000).et al. 2000).

Terra Terra PretaPreta (TP) soils may possess a large and (TP) soils may possess a large and unique microbial community unique microbial community (Thies and Suzuki 2003).(Thies and Suzuki 2003).

Preliminary data suggest high microbial Preliminary data suggest high microbial populations compared to adjacent (nonpopulations compared to adjacent (non--TP) TP) soils.soils.

TP compared to adjacent soilsTP compared to adjacent soils

Higher C mineralization rates Higher C mineralization rates in TP compared to adjacent in TP compared to adjacent ferralsolsferralsols (Glaser et al., 2003)(Glaser et al., 2003)

Microbial communities clearly Microbial communities clearly differ differ (Thies and Suzuki, 2003)(Thies and Suzuki, 2003)

Natural experimental design Natural experimental design and unique opportunity for and unique opportunity for studystudy

We have shown DNA difficult We have shown DNA difficult to extract and purifyto extract and purify

Abundance of culturable bacteriaAbundance of culturable bacteria(Most Probable Number (Most Probable Number –– MPN)MPN)

MPN enumeration on liquid R2A Media - TP and Oxisol

1.00E+00

1.00E+01

1.00E+02

1.00E+03

1.00E+04

1.00E+05

1.00E+06

1.00E+07

1.00E+08

1.00E+09

1 2 3 4 5 6 7 8 9

Site

MPN

TPOX

(n=5)

Hatahara Lago Grande Acutuba

Extracting nucleic acids from soilExtracting nucleic acids from soilKits available for soilKits available for soil

1.1. Cell extraction or Cell Cell extraction or Cell lysislysis2.2. Protein precipitationProtein precipitation3.3. PurificationPurification

Problems and PitfallsProblems and Pitfalls–– Lysis efficiency of cells Lysis efficiency of cells

differs.differs.–– HumicHumic acids/organic acids/organic

matter may decrease matter may decrease extraction efficiency. extraction efficiency.

What is the effect of the What is the effect of the chemistry of black carbon chemistry of black carbon on DNA extraction?on DNA extraction?

Methods for improving DNA Methods for improving DNA extraction from soilextraction from soil

Direct lysis of community DNADirect lysis of community DNA–– Bead beating Bead beating (Miller et al., 1999)(Miller et al., 1999)

–– Heating and freezing Heating and freezing ((BrunsBruns and Buckley, 2002)and Buckley, 2002)

Purification Purification –– HexadecyltrimethylammoniumHexadecyltrimethylammonium (CTAB), (CTAB),

polyvinylpolypyrrolidonepolyvinylpolypyrrolidone (PVPP) (PVPP) (Zhou et al., 1996)(Zhou et al., 1996)

–– Chemical flocculation AlNHChemical flocculation AlNH44(SO(SO44))22, MgCl, MgCl22(Braid et al., 2003)(Braid et al., 2003)

HypothesesHypotheses

HH11: DNA recovery is low due to poor cell : DNA recovery is low due to poor cell lysislysisTP soil matrix may protect cells from TP soil matrix may protect cells from lysislysis

HH22: Cell : Cell lysislysis occurs but DNA binds to the soil occurs but DNA binds to the soil Black carbon in Black carbon in terra terra pretapreta may bind with may bind with DNA DNA in solution and reduce yield or purity.in solution and reduce yield or purity.

Tests for this StudyTests for this Study

Improving cell Improving cell lysislysis

-- Varying bead beating time (30s/150s)Varying bead beating time (30s/150s)

Reducing DNA binding to the soil matrix.Reducing DNA binding to the soil matrix.-- Varying DNA extraction kit (MoBio/Bio101) Varying DNA extraction kit (MoBio/Bio101) -- Adding chemical flocculants Adding chemical flocculants

-- MgClMgCl22-- AlNHAlNH44(S0(S044))22

-- 5X phosphate buffer5X phosphate buffer

Methods: Methods: Quantification and PCRQuantification and PCR

QuantificationQuantification–– Stain nucleic acids, image and compare to a DNA Stain nucleic acids, image and compare to a DNA

standard curve. standard curve. –– Run on an Run on an agaroseagarose gel. gel. Measures quantity of raw DNA extract.Measures quantity of raw DNA extract.

PCR PCR –– Polymerase chain reaction is used to amplify Polymerase chain reaction is used to amplify

community DNA fragments. community DNA fragments. –– Can be used to evaluate suitability of DNA extracts Can be used to evaluate suitability of DNA extracts

for molecular analysis.for molecular analysis.Measures purity of DNA extract.Measures purity of DNA extract.

MPN enumeration on liquid R2A Media - TP and Oxisol

1.00E+00

1.00E+01

1.00E+02

1.00E+03

1.00E+04

1.00E+05

1.00E+06

1.00E+07

1.00E+08

1.00E+09

1 2 3 4 5 6 7 8 9

Site

MPN TP

OX

Microbiological enumeration Microbiological enumeration ––sites chosensites chosen

Hatahara AcutubaLago Grande

(n=5)

Results: Initial DNA extraction using Results: Initial DNA extraction using two commercial kitstwo commercial kits

020

4060

80100120

140160

180200

TP-1 OX-1 TP-2 OX-2 TP-3 OX-3

Nuc

leic

Aci

d (n

g/ul

)

Bio101-ControlMoBio-Control

Bio101

MoBio

Hatahara Lago Grande Acutuba

Results: Bead beating and DNA yieldResults: Bead beating and DNA yieldNucleic Acid Yield with 30 and 150 Seconds Bead

Beating for Cell Lysis

0

50

100

150

200

250

300

TP-1 OX-1 TP-2 OX-2 TP-3 OX-3

Nuc

leic

aci

d (n

g/ul

)

Control - 30Control - 150

30 150High Molecular Weight

Low Molecular Weight

Hatahara Lago Grande Acutuba

Results: PCR with different chemical treatments

(+) (-) C | P | M | A | | C | P | M | ATP OX

OX-1

OX-2TP-2

TP-1

C = Control (no addition to commercial kit) P = 5 x Phosphate bufferM = MgCl2 A = AlNH4(SO4)2

Results: Effect of chemical Results: Effect of chemical treatments on community DNA yieldtreatments on community DNA yield

DNA Yield Using Chemical Treatments - TP and Adjacent Soils

0

50

100

150

200

250

300

TP-1 OX-1 TP-2 OX-2 TP-3 OX-3

Nuc

leic

Aci

d (n

g/ul

)

Control5 x PhosphateMgCl2AlNH4(SO4)2

AcutubaLago GrandeHatahara

ConclusionsConclusions

Some extraction kits are better than others Some extraction kits are better than others for particular soils.for particular soils.

In some cases, increased bead beating In some cases, increased bead beating time does increase DNA yield, but it can time does increase DNA yield, but it can increase DNA shearing, which may impact increase DNA shearing, which may impact downstream molecular analyses.downstream molecular analyses.

Some additions and modifications of kits Some additions and modifications of kits may improve PCR. MgClmay improve PCR. MgCl22 is the more is the more promising among them, but not best for all promising among them, but not best for all soils.soils.

Further work and optimizationFurther work and optimization

Use microscopy to Use microscopy to assess efficiency of cell assess efficiency of cell lysislysis..Determine best purifiers Determine best purifiers for each sample to for each sample to improve downstream improve downstream molecular molecular analysesanalysesCharacterize microbial Characterize microbial community in community in terra terra pretapretaand adjacent soils using and adjacent soils using molecular methods such molecular methods such as Tas T--RFLP and DGGE.RFLP and DGGE.

Still more Still more digging to get digging to get to the bottom to the bottom of of terra terra pretapreta!!

Thank YouThank You

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