This study provides important insights into our understanding of the feedback response of soil microbial communities to elevated CO2 and global change. Methods Site, sampling and environmental variable analysis This study was conducted within the BioCON experiment site [6] located at the Cedar Creek Ecosystem Science Reserve, MN, USA. The main BioCON field experiment has 296 plots (2 by 2 m) in six 20-meter-diameter rings, three for an aCO2 concentration of 368 μmol/mol and three for an Ibrutinib elevated CO2 concentration of 560 μmol/mol using a FACE system as described by Reich et al. [6]. In this

study, soil samples without plant root from 24 plots (12 biological replicates from ambient CO2 and 12 biological replicates from elevated NVP-AUY922 CO2. All with 16 native plant species including four C4 grasses,

four C3 grasses, four N-fixing legumes and four non-N-fixing herbaceous species, and no additional N supply) were collected in July 2007. The aboveground and belowground biomass, plant C and N concentrations, soil parameters, and in situ net N mineralization and net nitrification were measured as previously described [6, 32]. More detailed information about sampling is provided in Additional file 13. GeoChip analysis DNA extraction, amplification and labeling, as well as the purification of labeled DNA, were carried out according the methods described by Xu et al. [23]. GeoChip 3.0 [26] was used to analyze the functional structure of the soil microbial communities. Details for GeoChip hybridization, image processing and data pre-processing

are described in Additional file 13. Statistical analysis Pre-processed GeoChip data were further analyzed with different statistical methods: (i) detrended correspondence analysis (DCA) [48], combined with analysis of similarities (ANOSIM), non-parametric multivariate analysis of variance (Adonis) and Multi-Response ifoxetine Permutation Procedure (MRPP), for determining the overall functional changes in the microbial communities; (ii) microbial diversity index, Significant Pearson’s linear correlation (r) analysis, analyses of variance (ANOVA) and response ratio (RR) [3]; (iii) redundancy analysis (RDA) for revealing the individual or set of environmental variables that significantly explained the variation in functional microbial communities; (iv) variation partitioning for RDA were used to select the minimum number of environmental variables explaining the largest amount of variation in the model [20, 49]. More details about the data analysis are described in Additional file 13.