Discovering widespread microbial processes that drive unexpected variation in carbon cycling may improve modeling and management of soil carbon (Prescott, 2010; Wieder et al., 2015a, 2018). A first step is to identify community features linked to carbon cycle variation. We addressed this challenge using an epidemiological approach with 206 soil communities decomposing Ponderosa pine litter in 618 microcosms. Carbon flow from litter decomposition was measured over a 6-week incubation. Cumulative CO 2 from microbial respiration varied twofold among microcosms and dissolved organic carbon (DOC) from litter decomposition varied five-fold, demonstrating large functional variation despite constant environmental conditions where strong selection is expected. To investigate microbial features driving DOC concentration, two microbial community cohorts were delineated as "high" and "low" DOC. For each cohort, communities from the original soils and from the final microcosm communities after the 6-week incubation with litter were taxonomically profiled. A logistic model including total biomass, fungal richness, and bacterial richness measured in the original soils or in the final microcosm communities predicted the DOC cohort with 72 (P < 0.05) and 80 (P < 0.001) percent accuracy, respectively. The strongest predictors of the DOC cohort were biomass and either fungal richness (in the original soils) or bacterial richness (in the final microcosm communities). Successful forecasting of functional patterns after lengthy community succession in a new environment reveals strong historical contingencies. Forecasting future community function is a key advance beyond correlation of functional variance with end-state community features. The importance of taxon richness-the same feature linked to carbon fate in gut microbiome studies-underscores the need for increased understanding of biotic mechanisms that can shape richness in microbial communities independent of physicochemical conditions.

25Microbial biomass is increasingly used to predict respiration in soil organic carbon (SOC) 26 models. Its increased use combined with the difficulty of accurately measuring this variable 27 points a need to directly assess the importance of microbial biomass abundance for carbon (C) 28 cycling. To test the hypothesis that the initial microbial biomass abundance (i.e. biomass 29 abundance on new plant litter) is a strong driver of plant litter C cycling, we manipulated 30 biomass abundance by 10 and 100-fold dilution and composition using 12 source communities 31 on sterile pine litter and measured respiration in microcosms for 30 days. In the first two days of 32 microbial growth on fresh litter, a 100-fold difference in initial biomass abundance caused an 33 average difference in respiration of nearly 300%, but the effect rapidly declined to less than 30% 34 in 10 days and to 14% in 30 days. Parallel simulations with a soil carbon model, SOMIC 1.0, 35 also predicted a 14% difference over 30 days, consistent with the experimental results. Model 36 simulations predicted convergence of cumulative CO 2 to within 10% in three months and within 37 4% in three years. Rapid microbial growth likely attenuates the effects of large initial differences 38 in biomass abundance. In contrast, the persistence of source community as an explanatory factor 39 in driving differences in respiration across microcosms supports the importance of microbial 40 composition in C cycling. Overall, the results suggest that the initial abundance of microbial 41 biomass on litter is a weak driver of C flux from litter decomposition over long timescales 42 (months to years) when litter communities have equal nutrient availability. By extension, slight 43 variation in the timing of microbial dispersal to fresh litter is likely to be a minor factor in long-44 term C flux. 45 46 Importance 3 47Microbial biomass is one of the most common microbial parameters used in land carbon 48 (C) cycle models, however, it is notoriously difficult to measure accurately. To understand the 49 consequences of mismeasurement, as well as the broader importance of microbial biomass 50 abundance as a direct driver of ecological phenomena, greater quantitative understanding of the 51 role of microbial biomass abundance in environmental processes is needed. Using microcosms, 52 we manipulated the initial biomass of numerous microbial communities across a 100-fold range 53 and measured effects on CO 2 production during plant litter decomposition. We found that the 54 effects of initial biomass abundance on CO 2 production was largely attenuated within a week, 55 while the effects of community type remained significant over the course of the experiment.56 Overall, our results suggest that initial microbial biomass abundance in litter decomposition 57 within an ecosystem is a weak driver of long-term C cycling dynamics. 58 59 Introduction 60 Microbial decomposition of plant litter is a key process in terrestrial carbon (C) cycling 61 [1]. Although the dynamics of plan...

Numerous studies have examined the long-term effect of experimental nitrogen (N) deposition in terrestrial ecosystems; however, N-specific mechanistic markers are difficult to disentangle from responses to other environmental changes. The strongest picture of N-responsive mechanistic markers is likely to arise from measurements over a short (hours to days) time scale immediately after inorganic N deposition. Therefore, we assessed the short-term (3-day) transcriptional response of microbial communities in two soil strata from a pine forest to a high dose of N fertilization (ca. 1 mg/g of soil material) in laboratory microcosms. We hypothesized that N fertilization would repress the expression of fungal and bacterial genes linked to N mining from plant litter. However, despite N suppression of microbial respiration, the most pronounced differences in functional gene expression were between strata rather than in response to the N addition. Overall, ∼4% of metabolic genes changed in expression with N addition, while three times as many (∼12%) were significantly different across the different soil strata in the microcosms. In particular, we found little evidence of N changing expression levels of metabolic genes associated with complex carbohydrate degradation (CAZymes) or inorganic N utilization. This suggests that direct N repression of microbial functional gene expression is not the principle mechanism for reduced soil respiration immediately after N deposition. Instead, changes in expression with N addition occurred primarily in general cell maintenance areas, for example, in ribosome-related transcripts. Transcriptional changes in functional gene abundance in response to N addition observed in longer-term field studies likely result from changes in microbial composition. Ecosystems are receiving increased nitrogen (N) from anthropogenic sources, including fertilizers and emissions from factories and automobiles. High levels of N change ecosystem functioning. For example, high inorganic N decreases the microbial decomposition of plant litter, potentially reducing nutrient recycling for plant growth. Understanding how N regulates microbial decomposition can improve the prediction of ecosystem functioning over extended time scales. We found little support for the conventional view that high N supply represses the expression of genes involved in decomposition or alters the expression of bacterial genes for inorganic N cycling. Instead, our study of pine forest soil 3 days after N addition showed changes in microbial gene expression related to cell maintenance and stress response. This highlights the challenge of establishing predictive links between microbial gene expression levels and measures of ecosystem function.

27During plant litter decomposition in soils, carbon has two general fates: return to the atmosphere 28 via microbial respiration or transport into soil where long-term storage may occur. Discovering 29 microbial community features that drive carbon fate from litter decomposition may improve 30 modeling and management of soil carbon. This concept assumes there are features (or 31 underlying processes) that are widespread among disparate communities, and therefore amenable 32 to modeling. We tested this assumption using an epidemiological approach in which two 33 contrasting patterns of carbon flow in laboratory microcosms were delineated as functional states 34 and diverse microbial communities representing each state were compared to discover shared 35 features linked to carbon fate. Microbial communities from 206 soil samples from the 36 southwestern United States were inoculated on plant litter in microcosms, and carbon flow was 37 measured as cumulative carbon dioxide (CO2) and dissolved organic carbon (DOC) after 44 38 days. Carbon flow varied widely among the microcosms, with a 2-fold range in cumulative CO2 39 efflux and a 5-fold range in DOC quantity. Bacteria, not fungi, were the strongest drivers of 40 DOC variation. The most significant community-level feature linked to DOC abundance was 41 bacterial richness-the same feature linked to carbon fate in human-gut microbiome studies. 42 This proof-of-principle study under controlled conditions suggests common features driving 43 carbon flow in disparate microbial communities can be identified, motivating further exploration 44 of underlying mechanisms that may influence carbon fate in natural ecosystems. 45 46

Leaf litter decomposition is a major carbon input to soil, making it a target for increasing soil carbon storage through microbiome engineering. We expand upon previous findings to show with multiple leaf litter types that microbial composition can drive variation in carbon flow from litter decomposition and specific microbial community features are associated with synonymous patterns of carbon flow among litter types. Although plant litter type selects for different decomposer communities, within a litter type, microbial composition drives variation in the quantity of dissolved organic carbon (DOC) measured at the end of the decomposition period. Bacterial richness was negatively correlated with DOC quantity, supporting our hypothesis that across multiple litter types there are common microbial traits linked to carbon flow patterns. Variation in DOC abundance (i.e. high versus low DOC) driven by microbial composition is tentatively due to differences in bacterial metabolism of labile compounds, rather than catabolism of non-labile substrates such as lignin. The temporal asynchrony of metabolic processes across litter types may be a substantial impediment to discovering more microbial features common to synonymous patterns of carbon flow among litters. Overall, our findings support the concept that carbon flow may be programmed by manipulating microbial community composition.

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Microbial biomass is increasingly used to predict respiration in soil organic carbon (SOC) models. Its increased use combined with the difficulty of accurately measuring this variable points a need to directly assess the importance of microbial biomass abundance for carbon (C) cycling. To test the hypothesis that the initial microbial biomass abundance (i.e. biomass abundance on new plant litter) is a strong driver of plant litter C cycling, we manipulated biomass abundance by 10 and 100-fold dilution and composition using 12 source communities on sterile pine litter and measured respiration in microcosms for 30 days. In the first two days of microbial growth on fresh litter, a 100-fold difference in initial biomass abundance caused an average difference in respiration of nearly 300%, but the effect rapidly declined to less than 30% in 10 days and to 14% in 30 days. Parallel simulations with a soil carbon model, SOMIC 1.0, also predicted a 14% difference over 30 days, consistent with the experimental results. Model simulations predicted convergence of cumulative CO 2 to within 10% in three months and within 4% in three years. Rapid microbial growth, evidenced by appearance of visible microbial mats on the litter during the first week of incubation, likely attenuates the effects of large initial differences in biomass abundance. In contrast, the persistence of source community as an explanatory factor in driving differences in respiration across microcosms supports the importance of microbial composition in C cycling. Overall, the results suggest that the initial abundance of microbial biomass on litter is a weak driver of C flux from litter decomposition over long timescales (months to years) when litter communities have equal nutrient availability. By extension, slight variation in the timing of microbial dispersal to fresh litter is likely to be a minor factor in long-term C flux.