DNA Extraction from Biochar Amended Soils ASA, CSSA, SSSA 2014 Annual Meeting, Long Beach, CA

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1 DNA Extraction from Biochar Amended Soils ASA, CSSA, SSSA 2014 Annual Meeting, Long Beach, CA Lauren Hale and David Crowley University of California, Riverside

2 DNA Provides Vast Insight Into Soil Biology Diversity Kim et al. 2007

3 DNA Provides Vast Insight Into Soil Biology Diversity Kim et al. 2007

4 DNA Provides Vast Insight Into Soil Biology Diversity Activity Kim et al Harter et al 2014

5 DNA Provides Vast Insight Into Soil Biology Diversity Activity Kim et al Harter et al 2014

6 DNA Provides Vast Insight Into Soil Biology Diversity Activity Pathogen detection Kim et al Harter et al 2014

7 DNA Extraction From Soils Can Be Problematic Hindrance by Extraction inhibitors Contaminants Sorption of DNA via cation bridging Protective pore spaces DNA Humic acid Switchgrass Biochar, 500 C (Brewer, et al 2009)

8 Previous Work Jin (2010) demonstrated reduced extraction effieciency when soils were amended with biochar. Harter et al (2014) PowerSoil, no bias from DNA extraction (loamy sand and 700 C greenwaste biochar) Leite, et al (2014) demonstrated good yield and purity of DNA when using PowerSoil kit with biochar-amended sand

9 Strategies Employed During Extraction Cell lysis Chemical (ie, Detergents including guanidinium thiocyanate and SDS) Enzymatic (Proteinase K, lysozyme) Mechanical (Freeze-thawing, bead beating) Inhibitor removal Prior to cell lysis by washing samples (PBS, EDTA, Tween-20) or flocculating contaminants (Ie CaCO 3 ) Phase separation during cell lysis (ie CTAB, TritonX-100, PVPP, CaCl 2, chloroform) Separation from extracted DNA (CsCl density centrifugation, gel electrophoresis, Sephandex G-200 columns) Post Extraction DNA Clean-up (Sepharose gel purification or spin columns) Reduce effects of inhibitors by diluting DNA or treating with BSA

Methods: DNA Extraction Techniques Traditional (Griffiths, et al 2000)

CTAB, phenol:chloroform:isoamyl Preciptated with polyethylene glycol then

detergent and bead-beating via vortex adapter Purification via spin columns and

Phenol:cholorform:isoamyl alcohol (24:24:1) added to chemical detergent and

10 Methods: DNA Extraction Techniques Traditional (Griffiths, et al 2000) Bead-beating w FastPrep system, glass and zirconia/ silica beads Treatment with CTAB, phenol:chloroform:isoamyl Preciptated with polyethylene glycol then ethanol Suspended in 60 µl water Treatment with RNase MoBio PowerSoil Chemical detergent and bead-beating via vortex adapter Purification via spin columns and wash steps Elution with 60 µl buffer PCI-modified PowerSoil Phenol:cholorform:isoamyl alcohol (24:24:1) added to chemical detergent and bead-beating via FastPrep Additional wash steps, which include ethanol Elution with 60 µl buffer Treatment with RNase

11 Methods: Soils and Biochars Clay and sand soil Biochar application rate: 3% Spiked with GFP-tagged Pseudomonas putida UW4 Biochar ph SSA Shell ± ± 20.9 Palm ± ± 16.3 Nut ± ± 0.02 Pine ± ± 0.8

12 Sand Sand shell600 Sand palm600 Sand nut300 Sand pine300 Clay Clay shell600 Clay palm600 Clay nut 300 Clay pine300 Sand Sand shell600 Sand palm600 Sand nut300 Sand pine300 Clay Clay shell600 Clay palm600 Clay nut 300 Clay pine300 Sand Sand shell600 Sand palm600 Sand nut300 Sand pine300 Clay Clay shell600 Clay palm600 Clay nut 300 Clay pine300 Results: DNA Traditional PowerSoil provided PowerSoil - PCI Modified

13 Results: Purity 260/280 Ratios

14 Results: Purity 260/230 Ratios

15 Results: Yield NanoDrop

16 Results: Yield, Double-stranded DNA

17 Results: DNA Quantification, qpcr

18 Useful Findings Traditional method and both PowerSoil protocols show no bias from presence of any biochar type DNA concentration and purity greatly improved with both PowerSoil protocols GFP copy number determined for qpcr reflected significant differences among all protocols tested

19 Recommendation Both PowerSoil protocols resulted in high yields of qpcr-quality DNA The PCI-modified PowerSoil protocol resulted in the greatest yield and purity of DNA This may be most useful for low-yielding or contaminant-prone soils

20 Acknowledgements Thank you to Dr. David Crowley and Crowley lab members, Funding source: EPA STAR fellowship FP

21 References Brewer, C. E., Schmidt Rohr, K., Satrio, J. A., & Brown, R. C. (2009). Characterization of biochar from fast pyrolysis and gasification systems. Environmental Progress & Sustainable Energy, 28(3), Griffiths, R. I., Whiteley, A. S., O'Donnell, A. G., & Bailey, M. J. (2000). Rapid method for coextraction of DNA and RNA from natural environments for analysis of ribosomal DNA-and rrna-based microbial community composition. Applied and Environmental Microbiology, 66(12), Harter, J., Krause, H.-M., Schuettler, S., Ruser, R., Fromme, M., Scholten, T., Kappler, A., Behrens, S., Linking N2O emissions from biochar-amended soil to the structure and function of the N-cycling microbial community. ISME J. Jin, H. (2010) Characterization of microbial life colonizing biochar and biochar amended soils, NY, USA (PhD Thesis, Cornell University). Kim, J.-S., Sparovek, G., Longo, R.M., De Melo, W.J., Crowley, D., Bacterial diversity of terra preta and pristine forest soil from the Western Amazon. Soil Biol. Biochem. 39, Leite, D. C. A., et al. "Comparison of DNA extraction protocols for microbial communities from soil treated with biochar." Brazilian Journal of Microbiology 45.1 (2014):

22 Questions?

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