We highlight two key results with implications for understanding structure-function relationships in soils, and for applying DNA-SIP in future studies of the soil-C cycle. First, cellulose responders were members of physiologically undescribed taxonomic groups with few exceptions. This suggests that we have much to learn about the diversity of structural-C decomposers in soil before we can begin to assess how they are affected by climate change and land management. Second, the response to xylose was characterized by a succession in activity from Paenibacillus OTUs (day 1) to Bacteroidetes (day 3) and finally Micrococcales (day 7). Notably, Paenibacillus have been previously shown by DNA-SIP to metabolize glucose \citep{Verastegui_2014}, also a common sugar in plant biomass. This activity succession was mirrored by relative abundance profiles and may mark trophic-C exchange between these groups. While trophic exchange has been observed previously in DNA-SIP studies \citep{lueders2004b} most applications of DNA-SIP focus on proximal use of labeled substrates. However, with increased sensitivity, DNA-SIP is well suited to tracking C flows throughout microbial communities over time and is not limited only to observing the entry point for a given substrate into the soil C-cycle. Trophic interactions will critically influence how the global soil-C reservoir will respond to climate change \citep{Crowther2015} but we know little of biological interactions among soil bacteria. Often bacteria are cast as a single trophic level \citep{Moore1988} but it may be appropriate to investigate the soil food web at greater granularity. Additionally, our results show that DNA-SIP results can change dramatically over time suggesting that multiple time points are necessary to rigorously and comprehensively describe which microorganisms consume \(^{13}\)C-labeled substrates in nucleic acid SIP incubations.
Bacteria that consumed \(^{13}\)C-cellulose were seldom related closely to any physiologically characterized cultured isolates but were members of cosmopolitan phylogenetic groups in soil including Spartobacteria, Planctomycetes, and Chloroflexi. Often cellulose responders were less than 90% related to their closest cultured relatives showing that we can infer little, if anything at all, of their physiology from culture-based studies. Notably, many Spartobacteria were among the cellulose responder OTUs. This is particularly interesting as Spartobacteria are globally distributed and found in a variety of soil types \citep{Bergmann_2011}. These lineages may play important roles in global cellulose turnover (please see SI note 1 for further discussion of the phylogenetic affiliation of cellulose responders).
The turnover of cellulose and plant-derived sugars in soil has been studied previously using DNA-SIP (e.g. \citep{Verastegui_2014}). Similar to our study, phylotypes among the Chloroflexi, Bacteroidetes and Planctomycetes have all been previously implicated in soil cellulose degradation \citep{Schellenberger_2010}. Additionally, functional metagenomics enabled by DNA-SIP has identified glycoside hydrolases putatively belonging to Cellvibrio and Spartobacteria further suggesting a role for these organisms in cellulose breakdown in soils \citep{Verastegui_2014}. Fungi undoubtedly also contribute to the decomposition of cellulose in soils \citep{boer_2005}, but they are not a focus of this experiment. It should be noted that longstanding hypotheses that delineate the life history strategies of fungi and bacteria on the basis of substrate preference have been recently questioned \citep{rousk_2015}. The approach we describe is also suitable for observing the activity of fungi (by targeting genetic markers in fungi with fungal specific PCR primers) and should prove useful in testing hypotheses that explain the functional traits of both bacteria and fungi as they occur in soils.
In addition to taxonomic identity, we quantified four ecological properties of microorganisms that were actively engaged in labile and structural C decomposition in our experiment: (1) time of activity, (2) estimated rrn gene copy number, (3) phylogenetic clustering, and (4) density shift in response to \(^{13}\)C-labeling. Labile C was consumed before structural C and these substrates were consumed by different microorganisms (Figure \ref{fig:ord}). This was expected and is consistent with the degradative succession hypothesis. Consumers of labile C had higher estimated rrn gene copy number than structural C consumers (Figure \ref{fig:shift}). rrn copy number is positively correlated with the ability to resuscitate quickly in response to nutrient influx \citep{Klappenbach_2000} which may be the advantage that enabled xylose responders to rapidly consume xylose. Both xylose and cellulose responders were terminally clustered phylogenetically suggesting that the ability to use these substrates was phylogenetically constrained. Although labile C consumption is generally considered to be mediated by a diverse set of microorganisms, we found that xylose responders at day 1 were mainly members of one genus, Paenibacillus. Our results suggests that life-history traits such as the ability to resuscitate quickly and/or grow rapidly may be more important in determining the diversity of microorganisms that actually mediate a given process than the genomic potential for substrate utilization (see SI note 2 for further discussion with respect to soil-C modelling). And last, labile C consumers, in contrast to structural C consumers, had lower \(\Delta\hat{BD}\) in response to \(^{13}\)C-labeling. This result suggests that labile C consumers were generalists, assimilating C from a variety of sources both labeled and unlabeled, while structural C consumers were more likely to be specialists and more closely associated with C from a single source.
We propose that the temporal fluctuations in \(^{13}\)C-labeling in the \(^{13}\)C-xylose treatment are due to trophic exchange of \(^{13}\)C. Alternatively, the temporal dynamics could be caused by microorganisms tuned to different substrate concentrations and/or cross-feeding. However, trophic exchange would explain well the precipitous drop in abundance of Paenibacillus after day 1 with subsequent \(^{13}\)C-labeling of Bacteroidetes at day 3 as well as the precipitous drop in abundance of Bacteroidetes at day 3 followed by \(^{13}\)C-labeling of Micrococcales at day 7. Trophic exchange could be enabled by mother cell lysis (in the case of spore formers such as Paenibacillus), viral lysis, and/or the direct indirect effects of predation. Bacteroidetes types have been shown to become \(^{13}\)C-labeled after the addition of live \(^{13}\)C-labeled Escherichia coli to soil \citep{Lueders2006} indicating their ability to assimilate C from microbial biomass. In addition, the dominant OTU labeled in the \(^{13}\)C-xylose treatment from the Micrococcales shares 100% SSU rRNA gene sequence identity to Agromyces ramosus a known predator that feeds upon on many microorganisms including yeast and Micrococcus luteus \citep{16346402}. Agromyces are abundant microorganisms in many soils and Agromyces ramosus was the most abundant xylose responder in our experiment – the fourth most abundant OTU in our dataset. It is notable however, that if Agromyces ramosus is acting as a predator in our experiment, the organism remains unlabeled in response to \(^{13}\)C-cellulose which suggests that its activity may be specific for certain prey or for certain environmental conditions (see SI note 3 for further discussion of trophic C exchange). Climate change is expected to diminish bottom-up controls on microbial growth increasing the importance on top-down biological interactions for mitigating positive climate change feedbacks \citep{Crowther2015}. Currently the extent of bacterial predatory activity in soil, and its consequences for the soil C-cycle and carbon use efficiency is largely unknown. Elucidating the identities of bacterial predators in soil will assist in assessing the implications of climate change on global soil-C storage.
Microorganisms govern C-transformations in soil and thereby influence global climate but still we do not know the specific identities of microorganisms that carry out critical C transformations. In this experiment microorganisms from physiologically uncharacterized but cosmopolitan soil lineages participated in cellulose decomposition. Cellulose responders included members of the Verrucomicrobia (Spartobacteria), Chloroflexi, Bacteroidetes and Planctomycetes. Spartobacteria in particular are globally cosmopolitan soil microorganisms and are often the most abundant Verrucomicrobia order in soil \citep{Bergmann_2011}. Fast-growing aerobic spore formers from Firmicutes assimilated labile C in the form of xylose. Xylose responders within the Bacteroidetes and Actinobacteria likely became labeled by consuming \(^{13}\)C-labeled constituents of microbial biomass either by saprotrophy or predation. Our results suggest that cosmopolitan Spartobacteria may degrade cellulose on a global scale, decomposition of labile plant C may initiate trophic transfer within the bacterial food web, and life history traits may act as a filter constraining the diversity of active microorganisms relative to those with the genomic potential for a given metabolism.