Ashley Campbell edited Results & Discussion.tex  almost 10 years ago

Commit id: e5ba8d9717bc3dc72561f46c581797c5df98bcdf

deletions | additions      

       

Whole phylum responses were not detected for xylose or cellulose yet utilization of these substrates spanned many phylogenetically diverse groups. However, substrate utilization within each phylum was demonstrated at the clade or single taxa level. In a study that amended forest soils with single C substrates in the presence of 3-bromodeoxyuridine (BrdU), they determined that more than 500 taxa responded to labile C but occured predominantly in two phlya (Proteobacteria and Actinobacteria)\cite{Goldfarb_2011}. On the other hand, the soils amended with more recalcitrant C such as cellulose and lignin were noted for spanning eight phyla, but were limited to a small number of taxa within each of those phyla that were responders \cite{Goldfarb_2011}. It has previously been suggested that all taxa within a phylum are unlikely to share ecological characteristics \cite{Fierer_2007}, and furthermore, within a species population \cite{Choudoir_2012,Preheim_2011,Hunt_2008}. Habitat traits of coastal Vibrio isolates were mapped onto microbial phylogeny revealing discrete ecological populations based on seasonal occurrence and particulate size fractionation \cite{Preheim_2011, Hunt_2008}. These data would suggest that portraying the response of a few OTUs or clades as a phylum level response would be overreaching.   \textbf{Conclusions.} We have demonstrated how next generation sequencing-enabled SIP gives a taxa level resolution for substrate utilization. Using this technique, we are able to resolve discrete OTUs that would otherwise be missed using bulk community sequencing efforts. Additionally, this technique provides greater taxonomic resolution than previous techniques (cloning, TRFLP, ARISA) used to determine substrate utilizing community members. Traditionally, SIP is performed with a single time point in an attempt to minimize or eliminate cross-feeding of the substrate. However, in a temporal food web mapping study, this cross-feeding is an exciting advantage of this system because it will enable us to track substrates of interest (i.e. cellulose) through many trophic cascades. Observed organisms will exhibit a range of ^{13}C \textsuperscript{13}C  labeling, 0-100\%, with primary consumers being the most enriched and subsequent trophic levels being less enriched (Morris 2002, McDonald 2005, Ziegler 2005). This relationship of trophic level consumption to dilution of label will facilitate tracking C as it moves through various operational taxonomic units (OTUs) in the soil. While we are currently able to resolve highly responsive OTUs, there is still a need to resolve taxa that are partially responsive which we cannot differentiate from noise with confidence at this time. Yet, given the ability to resolve partially responsive taxa, the ecology would still be difficult to discern. For example, a generalist utilizing many substrates including the ^{13}C-labeled \textsuperscript{13}C-labeled  substrate may exhibit the same partial labeling that a specialist utilizing both the ^{13}C-substrate and the same substrate (unlabeled) that is inherent in the soil. Additionally, partially labeled taxa could be further down the trophic cascade including predators or secondary consumers of waste products from primary consumer microbes that were highly labeled. Our study is consistent with carbon degradative succession that has previously been demonstrated (ref). We demonstrate a rapid decrease in the labile carbon, xylose, confirmed by its ^{13}C \textsuperscript{13}C  label incorporation into the microbial community DNA during the first 7 days of the experiment, after which, the label is not detectable in the DNA. Subsequently our data demonstrates a slow degradation of the more recalcitrant, polymeric carbon demonstrated by ^{13}C-cellulose \textsuperscript{13}C-cellulose  label incorporation into the microbial community DNA at 14 and 30 days. We did not observe the ^{13}C-cellulose \textsuperscript{13}C-cellulose  signal leave the DNA within the time limits, 30 days, of our experiment. This degradative succession is also confirmed by isotopic analysis of the soil from the microcosms (Table S1). NMDS demonstrates microbial succession and based on xylose and cellulose treatments separating away from the control, but in opposing directions indicating different microbial community members are responsible for degradation of the two C substrates. It is likely that the slow degradation of cellulose can be attributed to the energy-taxing process of synthesizing cellulolytic enzymes and exporting them, as cellulose is broken down externally (Schimel & Schaeffer 2012, Lynd et al 2002). As a result, microorganisms responsible for the synthesis of cellulases preferentially shuttle energy towards enzyme synthesis rather than biomass until cellulose hydrolysis begins (Schimel & Schaeffer 2012). This accounts for the delay in growth and ultimately the slow decomposition of cellulose (Perez et al 2002, Schimel & Schaeffer 2012).