Cornell University 2009 ASA-CSSA-SSSA Meetings High C/N ratio Refugia pH & aeration Physico- chemical sorption Surface change Microbes Nutrients Amending soils with biochar is a currently popular strategy to improve soil health and better use limited natural resources. Biochar added to soil typically:  improves soil structure and the availability of water and nutrients to plants  is recalcitrant to decomposition and appears to slow the turnover of labile organic matter  increases the capacity of soils to sequester C  reduces GHG emissions  sorbs many organic contaminants, thus reducing their off-site movement Many of these processes are microbially mediated, hence, it is critical to begin to characterize the interaction between biochar and soil microbial community composition and its function. Biochar as a soil amendment strategy Biochar specific properties make the interactions between biochar and microorganisms quite complicated. On one hand, the quality and quantity of the biochar used to amend the soil may affect soil microbial community composition and activity. On the other hand, microorganisms may have a biodegrading effect and/or alter the surface properties of the biochar. Figure 1 depicts the potential effects of biochar on soil microbial community dynamics. Characterizing microbial community abundance, activity and composition frequently depend on extracting cells, their chemical constituents or metabolic end products from soil. The adsorption properties of biochar strongly reduce extraction efficiencies and may lead to biased results, thus complicating their interpretation. Molecular profiling, in particular, may be significantly influenced by low recovery of target nucleic acids (see Fig. 3). Challenges to characterizing microbial communities in biochar-amended soils T-RFLP fingerprinting was used to investigate: 1. The effects of three DNA extraction protocols on the efficiency of DNA extraction from biochar-amended soils and resulting analyses of bacterial and fungal community composition. 2. Changes in bacterial and fungal community composition in rhizosphere and bulk soils in response to increasing rates of biochar added to field soils. The basic protocol for PCR-T-RFLP is shown in Fig. 2. Fig. 5. AMMI analyses of T-RFLP fingerprints of the bacterial (A) and fungal (B) community composition in biochar- amended soils (RE=Hha1). Each point represents one separately analyzed replicate field soil sample. Points are gradiently-colored according to increasing biochar application levels and in shape according to DNA extraction method. II: The presence of biochar significantly decreased DNA recovery efficiency, regardless of extraction protocol (Fig. 4). The Ultraclean TM soil DNA extraction kit was the least efficient. I: Adding biochar dramatically reduced DNA extraction efficiency (Fig. 3).  WARNING: DNA is difficult to extract from biochar-enriched soil samples.  The modified LaMontagne et al. (2002) protocol and the MO BIO Powersoil ® DNA extraction kit extracted DNA more efficiently from biochar-amended soils than the MO BIO UtraClean ® kit.  The choice of DNA extraction method did not significantly influence T-RFLP fingerprint analyses of bacterial or fungal community composition.  The MoBio Powersoil ® soil DNA extraction kit is our DNA extraction method of choice.  Higher rates of biochar (12 and 30 t ha -1 ) and sampling location (bulk or rhizosphere) significantly affected soil microbial community composition.  Work is ongoing to further characterize these changes in community composition to determine the main shifts in taxonomic groups to inform investigations on changes in soil function. III: The choice of DNA extraction method did not significantly influence the outcome of either the bacterial (Fig. 5A) or fungal community T-RFLP fingerprint analyses (Fig. 5B). Fig. 6. AMMI analysis of bacterial and fungal community T-RFs (RE=HhaI ) derived from field samples. IV: Both rate of biochar added and sampling location (bulk and rhizosphere) significantly influenced bacterial and fungal community composition. Fig. 2 T-RFLP protocol A field experiment with varying rates of biochar amendment was established at Cornell Musgrave Farm, Aurora, NY, in May, Corn stover charcoal was incorporated into the soil at rates of 0, 1, 12, and 30 t ha -1, denoted as CT, Low, Medium and High respectively, to each plot before planting corn seeds at a density of 32,000 seeds acre -1. Corn rhizosphere and bulk soil samples were taken in October, 2008, and used to characterize soil bacterial and fungal communities. Extracted soil community DNA PCR with fluorescently labeled 16S rRNA forward primer Restriction digest of PCR products A B C Sizing of terminal restriction fragments (T-RFs) Relative fluorescence Fig. 1 Potential interactions between biochar and soil microorganisms MW ladder DNA only DNA + Biochar Fig. 3 Soil microbial genomic DNA extracted from soils with and without biochar added using the Powersoil T® soil DNA extraction kit (MO BIO Laboratories, Inc., Carlsbad, CA). Lane 1 : DNA MW ladder. Lane 2 : 10 µg of purified bacterial DNA Lane 3: 10 µg of purified bacterial DNA with 0.5 g of biochar added Fig. 4 Effect of biochar on DNA recovery using three extraction protocols. Light columns = DNA extracted without biochar added Dark columns = DNA extracted in the presence of biochar. Method I: Protocol modified from LaMontagne et al. (2002) Method II: Powersoil ® kit (MO BIO). Method III: UltraClean ® kit (Mo BIO).