The succession hypothesis of decomposition predicts a succession from microbial types that use labile C to those that use recalcitrant polymeric C over time CITE. Cellulose degraders succeeded labile C degraders as predicted. But, in response to \(^{13}\)C-xylose, Firmicutes phylotypes were succeeded by Bacteroidetes which were then succeeded by Actinobacteria representing a nested succession (Figure \ref{fig:xyl_count}). We found that \(^{13}\)C substrate responders changed as much as XX-fold in relative abundance over time (Figure \ref{fig:time}).
The xylose responders demonstrate a smaller change in BD than the cellulose responders suggesting that xylose responders assimilate multiple C sources (labeled and unlabeled) consistent with a generalist response, while cellulose responders are more heavily labeled suggesting that cellulose is their main source of C, a response more consistent with a specialist lifestyle. Xylose responders include many taxa, such as spore-fomers, known for the ability to respond rapidly to an influx of new nutrients while cellulose responders include many OTUs that are common in soil but uncultured.
Xylose and cellulose utilization were demonstrated across 7 phyla each revealing a high diversity of bacteria able to utilize these substrates. The high taxonomic diversity may enable substrate metabolism under a broad range of environmental conditions \citep{Goldfarb_2011}. Other studies of microbial communities have observed a positive correlation with taxonomic or phylogenetic diversity and functional diversity \citep{Fierer_2012,Fierer_2013,Philippot_2010,Tringe_2005,Gilbert_2010,Bryant_2012}. The data presented here supports that specific functional attributes can be shared among diverse taxa and closely related taxa may have very different physiologies \citep{Fierer_2012,Philippot_2010}. This information adds to the growing collection of data suggesting that community membership is important to biogeochemical processes.
In the future deeper sequencing will enable us to increase coverage and assess C use by more community members. We can expand our knowledge of soil C use dynamics to a wide array of C substrates and increase our grasp on specific community member contributions to the soil C cycle. ARE OUR RESULTS CONSISTENT WITH SUBSTRATE SPECIFICITY STUDIES? C substrate specificity can be assessed by measuring the BD shift of OTU DNA upon \(^{13}\)C incorporation CITE. OTUs that incorporate more \(^{13}\)C per unit DNA have greater specificity for the labeled substrate than OTUs that incorporate less \(^{13}\)C per unit DNA. \(^{13}\)C-cellulose incorporating OTUs as a group displayed greater substrate specificity than \(^{13}\)C-xylose incorporating OTUs (p-value XX, Wilcoxon Rank Sum test). This suggests that polymeric C-degraders tend to be specialists tuned to particular C-substrates such as cellulose or lignin whereas labile C-degraders are generalists able to assimilate C from many different soluble, labile sources. Although we observed a succession of \(^{13}\)C-xylose responders (Figure \ref{fig:l2fc} and \ref{fig:xyl_count}), there was no discernible difference in substrate specificity between \(^{13}\)C-xylose responders at days 1, 3 or 7.
Although presence in heavy fractions can indicate \(^{13}\)C labeling, not all DNA in heavy fractions is \(^{13}\)C-labeled. Some DNA is heavy due to high G+C. With lower resolution fingerprinting techniques the banding pattern of SSU rRNA gene sequences can look similar across the entire density gradient CITE, however, high throughput sequencing of density gradient fractions shows light and heavy fractions are statistically different even when input DNA is entirely unlabeled (Figure \ref{fig:time_class}, and CITE). Hence, DNA-SIP studies that do not incorporate controls wherein amendments contain only \(^{12}\)C substrates, may confuse high G+C organisms with organisms that incorporated \(^{13}\)C into biomass.
marine and atmospheric SOM represents more C than the marine and atmospheric C reservoirs combined and approximately 80% of SOM flux is mediated by microorganisms CITE. SOM decomposition is more sensitive to temperature changes than primary productivity CITE. Climate change can affect terrestrial microbial communities on a global scale CITE Garcia-Pichel. Therefore, climate change has the potential to influence SOM flux and storage. Knowing how temperature changes the rates at which C decomposes in soil is essential to predict how climate change will affect SOM flux and storage. Additionally we need to observe how climate change alters the abundance and activity of key microbial players. Bur first, we must identify which microbial phylogenetic types decompose different SOM C components.
’Heavy’ fraction amplicon pools from samples that received \(^{13}\)C-xylose diverged from corresponding controls on days 1 through 7 . Furthermore, amplicon pool composition varied across these days indicating dynamic changes in \(^{13}\)C-xylose assimilation with time. At days 14 and 30 heavy fractions from \(^{13}\)C-xylose labeled samples are no longer differentiated from corresponding controls indicating that \(^{13}\)C is no longer detectable in DNA. The decline in \(^{13}\)C-labelling of DNA is likely due to isotopic dilution resulting from assimilation of unlabeled C and/or due to cell turnover resulting from mortality.
There were 6 shared responders among all unique responders identified in both the xylose and cellulose treatments (n = 72); Stenotrophomonas, Planctomyces, two Rhizobiaceae, Comamonadaceae, and Cellvibrio. Of these, Stenotrophomonas and Comamonadaceae are the only taxa that are among the top ten l2fc responses measured in both treatments. On the other hand, the only shared responder that is not among the top ten responders for either the cellulose or xylose treatment is Rhizobiaceae. Two of the shared responders corresponded in time between the two treatments
many taxa important to cellulose cycling are present in the rarer fraction of the overall microbial community.
Patterns of carbon use vary dramatically within phylum. Dynamic patterns of \(^{13}\)C-assimilation from xylose and cellulose occur at discrete, fine-scale taxonomic units . Responders for xylose and cellulose are widespread across 6 and 7 phyla, respectively . There are 5 phyla containing responders for both treatments; of all the responder OTUS detected within those phyla for either xylose or cellulose, there are only six OTUs that respond to both xylose and cellulose (discussed previously). This result suggests that phyla do not represent coherent ecological units with respect to the soil C-cycle, that is, taxa within phyla exhibit differences in substrate use, level of substrate specialization, and dynamics of incorporation.
In this study, we have identified Actinobacteria responders for both substrates. Although there were no shared Actinobacteria OTUs that responded to both xylose (Microbacteriaceae, Micrococcaceae, Cellulomonadaceae, Nakamurellaceae, Promicromonosporaceae, and Geodermatophilaceae) and cellulose (Streptomycetaceae and Pseudonocardiaceae). This information may suggest that while Actinobacteria exhibit an ability to utilize an array of carbon substrates, substrate use may be more clade specific and not widespread throughout the phylum . Similarly, Bacteroidetes responders were identified for both substrates, yet, at a finer taxonomic resolution there is a clear differential response for xylose (Flavobacteriaceae and Chitinophagaceae) and cellulose (Cytophagaceae).
Whole phylum responses were not detected for xylose or cellulose yet utilization of these substrates spanned many phylogenetically diverse groups. Within each phylum we observed substrate utilization at the clade or single taxa level with each exhibiting a unique pattern of \(^{13}\)C-assimilation over time. It has previously been suggested that all taxa within a phylum are unlikely to share ecological characteristics \citep{Fierer_2007}, and furthermore, within a species population \citep{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 \citep{Preheim_2011,Hunt_2008}. Yet, it has been proposed that the microbial community functionality responsible for soil C cycling appear at the level of phlya rather than species/genera \citep{Schimel_2012}. The traditional phylum level assignment conventions could in part be due to limitations in finer scale taxonomic identifications or methodological limitations (i.e. sequencing depth). Our data in concert with others \citep{Goldfarb_2011,Fierer_2007,Choudoir_2012,Preheim_2011,Hunt_2008} would suggest that assigning substrate utilization of a few OTUs or clades as a phylum level response is not accurate.
Conclusions. We have demonstrated how next generation sequencing-enabled SIP gives an OTU 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. 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. Although, if we could identify partially responsive taxa, their contributions to the C-cycle would still be difficult to discern. For example, a generalist utilizing many substrates including \(^{12}\)C substrates and the \(^{13}\)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.
OTUs that assimilate xylose and those that assimilate cellulose are largely mutually exclusive. Those OTUs that assimilate xylose are labeled within 1-7 days, while those that assimilate cellulose are labeled primarily after 2-4 weeks. The xylose responders demonstrate a smaller change in BD than the cellulose responders suggesting that xylose responders assimilate multiple C sources (labeled and unlabeled) consistent with a generalist response, while cellulose responders are more heavily labeled suggesting that cellulose is their main source of C, a response more consistent with a specialist lifestyle. Xylose responders include many taxa, such as spore-fomers, known for the ability to respond rapidly to an influx of new nutrients while cellulose responders include many OTUs that are common uncultivated soil organisms. Finally, xylose responders are more abundant in the community while cellulose responders are, on average, more rare as indicated by their rank abundance within the soil community. These results indicate that different bacteria in soil have distinct physiological and ecological responses which govern their interactions with soil C pools.
We did not observe consistent C utilization at the phylum level although both xylose and cellulose utilization were observed across 7 phyla each revealing a high diversity of bacteria able to utilize these substrates. The high taxonomic diversity may enable substrate metabolism under a broad range of environmental conditions \citep{Goldfarb_2011}. Other studies of microbial communities have observed a positive correlation with taxonomic or phylogenetic diversity and functional diversity \citep{Fierer_2012,Fierer_2013,Philippot_2010,Tringe_2005,Gilbert_2010,Bryant_2012}. The data presented here supports that specific functional attributes can be shared among diverse, yet distinct, taxa while closely related taxa may have very different physiologies \citep{Fierer_2012,Philippot_2010}. This information adds to the growing collection of data suggesting that community membership is important to biogeochemical processes. Furthermore, it highlights a need to examine substrate utilization by discrete microbial taxa within a whole community context to better understand how specific community members function within the whole.
The sensitivity of SIP-NGS provides a means to elucidate substrate utilization by discrete microbial taxa with the hope that we can begin to construct a belowground C food web. We obtained enough information to conclusively determine isotope incorporation for 61% of the more than 6,000 OTUs detected. For those OTUs with enough information (n = 3,825), approximately 2% (n = 72) significantly assimilated \(^{13}\)C from either xylose or cellulose. In the future deeper sequencing will enable us to increase coverage and assess C use by more community members. Using the informations we gain from SIP-NGS, we can expand our knowledge of specific C-cycling OTUs by taking a targeted metagenomic approach in the nucleic acid pools of ’heavy’ fractions. Furthermore, we can now expand our knowledge of soil C use dynamics to a wide array of C substrates and increase our grasp on specific community member contributions. Illuminating these microbial contributions associated with decomposition in soil are important because as environments change, there are measurable and functional changes in soil C \citep{Grandy_2008} which could cumulatively have large impacts at a global scale.