Computational protocol: Time-scales of hydrological forcing on the geochemistry and bacterial community structure of temperate peat soils

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[…] Peat samples were taken from two sites in the Parc Naturel Regional des Marais du Cotentin et du Bessin, Normandy. The pumping station site was located in the vicinity of a groundwater well in La Bergerie (49°13'25.72″N, 1°21'50.70″W). A pristine site was chosen approximately 1.5 km downstream (49°14'11.97″N, 1°21'24.19″W) in an undisturbed area. Sixteen sediment cores were collected at each site at 50 cm below the sediment surface. The peat cores were subjected to four experimental treatments, each with four replicate cores, where peat core water saturation varied by (1) 3 days saturated – 3 days unsaturated, (2) 9 days saturated – 9 days unsaturated, (3) continuously saturated and (4) continuously unsaturated (). Peat cores from both sites were saturated using groundwater from the extraction well in the pumping station site, characterized by higher nitrate concentrations (40 mg/L) and oxidizing characteristics than peat pore water. Each saturation cycle thus represents a perturbation of the reducing conditions characteristic of the peat medium. Groundwater was chosen to saturate the peats in order to reproduce conditions observed in the field. Both sites are part of the same drainage basin and are underlain by the same aquifer. Previous work that quantified water fluxes in the study area shows that groundwater partly saturates the peat at both sites each year during the high water period and a geochemical investigation of the whole aquifer showed relatively homogeneous groundwater composition. Groundwater was collected from the pumping station before each dry-rewetting experiment, such that the water used was fresh and had geochemical composition and microbial community representative of the time of sampling.A second set of experiments (“desiccation experiment”) was carried out using peat cores from the same sites and similar experimental conditions, only peat cores were kept at the lab temperature without any water addition during 5 months before the experiment. After the 5 months, the water content was 60 w% instead of the initial 80 w%. Only geochemical analyses and biomass quantification were carried out for this experiment.At the end of each cycle period, water drained from the cores was collected for geochemical analysis, as was pore water from the middle of the core, using 0.10 μm Rhizon samplers. The water drained from the cores represents water from the interconnected pores, while water collected with the Rhizon samplers represents water from the closed and dead-end pores (). In the text, the term pore water refers to water sampled with the Rhizon samplers, while core-collected waters refers to water drained from the cores at the end of each saturation cycle. Dissolved Organic Carbon (DOC) and Dissolved Inorganic Carbon (DIC) were measured with a Total Organic Carbon analyzer (Shimadzu TOC-5050A). Major anions concentrations (Cl−, NO3−, NO2−, PO42−, SO42−) were measured by ion chromatography (Dionex DX-120), and major cation concentrations (Na+, Mg2+, K+, Ca2+, Mn2+ and FeTOTAL) were measure by ICP-MS (Agilent 4500), using indium as an internal standard. The international geostandard SLRS-4 was used to check the reproducibility of the results. Fe (II) concentration was measured by the 1.10 phenantroline colorimetric method using a UV visible spectrophotometer (AFNOR, 1997) and Fe (III) was calculated by subtracting Fe(II) from FeTOTAL. Total and active microbial biomass were measured on 4 peat samples before and at the end of the experiment. For total biomass, a modified fumigation-extraction method specific for peat was used. Active biomass was measured with the substrate-induced respiration method, by adding 18 mg/g of glucose and measuring the respiration rate (CO2 release) after 1 hour incubation by gas chromatography.For molecular analysis of bacterial communities, water was drained from the cores with a peristaltic pump from 3 of the 4 replicate cores after the first 3-day cycle, after the first 9-day cycle, and at the end of the experiment. Three replicate samples of the groundwater used to saturate the experimental cores were collected at the beginning of the experiment. A volume of 60 mL was filtered through 0.22 μm Sterivex filters (Millipore). Filters were flash frozen in liquid nitrogen and kept at −80 °C. Peat samples were collected from the sampling site, and from the center of each core at the end of the experiment. Environmental DNA was extracted using the Power Soil DNA Extraction kit (MoBio Laboratories), with a bead-beating step of 2 minutes at 30 Hz, using the Retsch MM400 mixer mill.Amplicon libraries were prepared using fusion primers that contained the typical Adaptor A or B (Lib-A protocol), a multiplex identifier (5 or 10 bases in length) and the specific primer sequence. The forward (5′-GTG CCA GCM GCC GCG GTA ATA C-3′) and reverse (5′-CCG TCA ATT CCT TTG AGT TT-3′) primers amplify a fragment spanning the V4 and V5 regions of the ribosomal 16S gene. Amplification using the AmpliTaq PCR kit (Sigma) had an initial denaturation at 94 °C for 2 min; 30 cycles of denaturation at 94 °C for 30 s, annealing at 64 °C for 30 s and extension at 72 °C for 60 s, and a final extension for 6 min. Amplicon libraries were purified using the Agencourt AMPure XP magnetic beads and quantified by fluorometry with the Quant-iT PicoGreen dsDNA kit (Invitrogen). Amplicons were multiplexed and sequenced with a Roche GS-FLX Titanium pyrosequencer at the Environmental Genomics platform of the Observatoire des Sciences de l’Univers de Rennes (University of Rennes I, France). Every sample was sequenced as two biological replicates, starting from independent amplicons.Quality screening of the pyrosequencing results removed reads that: (1) had a length shorter than 400 bp, (2) had any ambiguous base pair assignment or (3) singletons present in only 1 sample. Singletons present in at least 2 samples were kept in the analysis, but identical reads present in only one sample were excluded. Chimeric sequences were identified by uchime in Mothur and removed from the dataset. Following quality screening, 1,527,190 reads were recovered (SuppTable1).DNAclust was used for defining operational taxonomic units (OTUs) at 97% sequence identity, which were then classified by comparison with the Silva ribosomal RNA databases for Bacteria and Archaea in Mothur. Taxonomic profiles consisting of the percent composition of major bacterial phyla were generated for each sample. Rarefaction curves were calculated for each sample in Mothur.A matrix containing the total count of each OTU in each sample was generated using an in-house Perl script. OTU counts were standardized to a frequency. The following diversity indices were calculated for each sample: (1) Chao 1 species richness; (2) Shannon diversity index and (3) Shannon Equitability index. General linear models implemented in R were used to identify factors that were statistically significant among sites, treatments and sampling times. Significant differences in average abundance of phyla was calculated with Student’s t-test in R. A log(x+1) transformation was applied to all OTU relative frequencies. Ordination analyses were performed by non-metric multidimensional scaling (NMDS) using the Bray-Curtis similarity coefficient in Primer6. NMDS generates a scatter plot in which the configuration of points relates the ranks of a similarity matrix as Euclidean distances. In short, on an NMDS plot, similar samples are plotted closely together, while dissimilar samples have greater distances between them.Geochemical measurements for pore water data were log(x+1) transformed and normalized. Principal Component Analysis was used to reduce the dimensional complexity of the dataset and examine the contributions of each variable to the variance of the dataset. Associations between environmental and biological data were examined in two ways. Pore water concentrations of the most distinguishing variables identified by PCA were overlain on the NMDS plots. This allowed similarity in environment and in bacterial community structure to be examined. In addition, the BIOENV procedure implemented by Primer 6 was used to determine which combination of environmental variables resulted in the best rank correlation coefficients with respect to the sample similarity matrix for the microbial communities. 500 permutations were performed to test statistical significance of correlations between biotic and abiotic matrices. […]

Pipeline specifications

Software tools UCHIME, mothur, DNACLUST
Applications Metagenomic sequencing analysis, 16S rRNA-seq analysis
Organisms Bacteria
Diseases Pulmonary Fibrosis
Chemicals Carbon, Iron