Soil microbial communities are intricately linked to ecosystem functioning such as nutrient cycling; therefore, a predictive understanding of how these communities respond to environmental changes is of great interest. Here, we test whether phylogenetic information can predict the response of bacterial taxa to nitrogen (N) addition. We analyze the composition of soil bacterial communities in 13 field experiments across 5 continents and find that the N response of bacteria is phylogenetically conserved at each location. Remarkably, the phylogenetic pattern of N responses is similar when merging data across locations. Thus, we can identify bacterial clades – the size of which are highly variable across the bacterial tree – that respond consistently to N addition across locations. Our findings suggest that a phylogenetic approach may be useful in predicting shifts in microbial community composition in the face of other environmental changes.

Bacterial abundance is a fundamental metric for understanding the population dynamics of soil bacteria and their role in biogeochemical cycles. Despite its importance, methodological constraints hamper our ability to assess bacterial abundance in terrestrial environments. Here, we aimed to optimize the use of flow cytometry (FCM) to assay bacterial abundances in soil while providing a rigorous quantification of its limitations. Soil samples were spiked with Escherichia coli to evaluate the levels of recovery efficiency among three extraction approaches. The optimized method added a surfactant (a tetrasodium pyrophosphate [TSP] buffer) to 0.1 g of soil, applied an intermediate degree of agitation through shaking, and used a Nycodenz density gradient to separate the cells from background debris. This procedure resulted in a high (average, 89%) level of cell recovery. Recovery efficiencies did not differ significantly among sites across an elevation gradient but were positively correlated with percent carbon in the soil samples. Estimated abundances were also highly repeatable between technical replicates. The method was applied to samples from two field studies and, in both cases, was sensitive enough to detect treatment and site differences in bacterial abundances. We conclude that FCM offers a fast and sensitive method to assay soil bacterial abundance from relatively small amounts of soil. Further work is needed to assay differential biases of the method across a wider range of soil types. IMPORTANCE The ability to quantify bacterial abundance is important for understanding the contributions of microbial communities in soils, but such assays remain difficult and time-consuming. Flow cytometry offers a fast and direct way to count bacterial cells, but several concerns remain in applying the technique to soils. This study aimed to improve the efficiency of the method for soil while quantifying its limitations. We demonstrated that an optimized procedure was sensitive enough to capture differences in bacterial abundances among treatments and ecosystems in two field studies.

The vast majority of microbes inhabiting oligotrophic shallow subsurface soil environments have not been isolated or studied under controlled laboratory conditions. In part, the challenges associated with isolating shallow subsurface microbes may persist because microbes in deeper soils are adapted to low nutrient availability or quality. Here, we use high-throughput dilution-to-extinction culturing to isolate shallow subsurface microbes from a conifer forest in Arizona, USA. We hypothesized that the concentration of heterotrophic substrates in microbiological growth medium would affect which microbial taxa were culturable from these soils. To test this, we diluted cells extracted from soil into one of two custom-designed defined growth media that differed by 100-fold in the concentration of amino acids and organic carbon. Across the two media, we isolated a total of 133 pure cultures, all of which were classified as Actinobacteria or Alphaproteobacteria. The substrate availability dictated which actinobacterial phylotypes were culturable but had no significant effect on the culturability of Alphaproteobacteria. We isolated cultures that were representative of the most abundant phylotype in the soil microbial community (Bradyrhizobium spp.) and representatives of five of the top 10 most abundant Actinobacteria phylotypes, including Nocardioides spp., Mycobacterium spp., and several other phylogenetically divergent lineages. Flow cytometry of nucleic acid-stained cells showed that cultures isolated on low-substrate medium had significantly lower nucleic acid fluorescence than those isolated on high-substrate medium. These results show that dilution-to-extinction is an effective method to isolate abundant soil microbes and that the concentration of substrates in culture medium influences the culturability of specific microbial lineages. IMPORTANCE Isolating environmental microbes and studying their physiology under controlled conditions are essential aspects of understanding their ecology. Subsurface ecosystems are typically nutrient-poor environments that harbor diverse microbial communities—the majority of which are thus far uncultured. In this study, we use modified high-throughput cultivation methods to isolate subsurface soil microbes. We show that a component of whether a microbe is culturable from subsurface soils is the concentration of growth substrates in the culture medium. Our results offer new insight into technical approaches and growth medium design that can be used to access the uncultured diversity of soil microbes.

Soil microbial communities and pools of carbon (C) and nitrogen (N) play an important role in ecosystem responses to precipitation variability and N deposition. In southern California, ecosystem vulnerability to these environmental change drivers may differ for grassland versus shrubland vegetation types. We hypothesized that 1) these vegetation types would differ in their responses to precipitation and N manipulation; 2) reduced precipitation ("drought treatment") would have a negative effect on soil microbial abundance and alter microbial community composition, 3) these changes would be associated with reductions in soil C and N pools, 4) N addition would increase microbial abundance as well as soil C and N pools, and 5) combined drought and N deposition would have offsetting effects on soil properties. We tested these hypotheses at the Loma Ridge Global Change Experiment in southern California. Across vegetation types, we found that microbial biomass based on phospholipid fatty acids declined with drought and N addition. Microbial composition differed more strongly by vegetation type than with environmental change treatments. Added precipitation had little effect on microbial biomass but reduced labile C and N pools; these reductions were mitigated by N addition. Drought reduced labile forms of soil C and N, whereas N addition increased labile soil C pools and all soil N pools. Negative effects of drought and N addition were additive for microbial biomass, which could inhibit soil C cycling if both of these environmental changes occur together. Drought interacted with N addition to significantly increase the most labile N pool under the drought+N treatment, which suggests a build-up of available N under these conditions. These results imply that multiple environmental changes may combine non-additively to affect below-ground microorganisms and soil C and N pools, which may have important consequences for ecosystem services such as productivity, biodiversity, and soil quality in Mediterranean climate regimes of North America.

Microbial decomposers mediate the return of CO to the atmosphere by producing extracellular enzymes to degrade complex plant polymers, making plant carbon available for metabolism. Determining if and how these decomposer communities are constrained in their ability to degrade plant litter is necessary for predicting how carbon cycling will be affected by future climate change. We analyzed mass loss, litter chemistry, microbial biomass, extracellular enzyme activities, and enzyme temperature sensitivities in grassland litter transplanted along a Mediterranean climate gradient in southern California. Microbial community composition was manipulated by caging litter within bags made of nylon membrane that prevent microbial immigration. To test whether grassland microbes were constrained by climate history, half of the bags were inoculated with local microbial communities native to each gradient site. We determined that temperature and precipitation likely interact to limit microbial decomposition in the extreme sites along our gradient. Despite their unique climate history, grassland microbial communities were not restricted in their ability to decompose litter under different climate conditions across the gradient, although microbial communities across our gradient may be restricted in their ability to degrade different types of litter. We did find some evidence that local microbial communities were optimized based on climate, but local microbial taxa that proliferated after inoculation into litterbags did not enhance litter decomposition. Our results suggest that microbial community composition does not constrain C-cycling rates under climate change in our system, but optimization to particular resource environments may act as more general constraints on microbial communities.