unlocking microbial communities in terra preta files/boneill.pdf · terra preta (tp) soils may...
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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
ReferencesReferencesBraid MD, LM Daniels, CL Kitts. 2003. Removal of PCR inhibitors Braid MD, LM Daniels, CL Kitts. 2003. Removal of PCR inhibitors from soil from soil DNA by chemical flocculation. Journal of Microbiological MethodsDNA by chemical flocculation. Journal of Microbiological Methods 52:38952:389--393.393.BrunsBruns MA and DH Buckley. 2002. Isolation and purification of microbiaMA and DH Buckley. 2002. Isolation and purification of microbial l community nucleic acids from environmental samples community nucleic acids from environmental samples –– soil, rhizosphere soil, rhizosphere and and phyllospherephyllosphere..Miller DN, JE Bryant, EL Madsen and WC Miller DN, JE Bryant, EL Madsen and WC GhiorseGhiorse. 1999. Evaluation and . 1999. Evaluation and optimization of DNA extraction and purification procedures for soptimization of DNA extraction and purification procedures for soil and oil and sediment samples. Applied and Environmental Microbiology 65:4715sediment samples. Applied and Environmental Microbiology 65:4715--4724.4724.PietikäinenPietikäinen J, O J, O KiikkiläKiikkilä and H and H FritzeFritze. 2000. Charcoal as a habitat for . 2000. Charcoal as a habitat for microbes and its effect on the microbial community of the underlmicrobes and its effect on the microbial community of the underlying humus. ying humus. OikosOikos 89:23189:231--242.242.Thies JE, K Suzuki. 2003. Amazonian dark earths: biological Thies JE, K Suzuki. 2003. Amazonian dark earths: biological measurements, measurements, InIn J. J. LehmannLehmann, D.C. Kern, B. Glaser, W.I. Woods, ed. , D.C. Kern, B. Glaser, W.I. Woods, ed. Amazonian Dark Earths: Origin, Properties, Management. Amazonian Dark Earths: Origin, Properties, Management. KluwerKluwer Academic Academic Publishers, The Netherlands.Publishers, The Netherlands.Zhou J, MA Zhou J, MA BrunsBruns and J and J TiedjeTiedje. 1996. DNA recovery from soils of diverse . 1996. DNA recovery from soils of diverse composition. Applied and Environmental Microbiology 62:316composition. Applied and Environmental Microbiology 62:316--322.322.