The ISME Journal https://doi.org/10.1038/s41396-018-0334-3 ARTICLE Seasonal and ecohydrological regulation of active microbial populations involved in DOC, CO2, and CH4 fluxes in temperate rainforest soil 1,2 2,3 1,2 4 5 David J. Levy-Booth ● Ian J. W. Giesbrecht ● Colleen T. E. Kellogg ● Thierry J. Heger ● David V. D’Amore ● 6 1 1 Patrick J. Keeling ● Steven J. Hallam ● William W. Mohn Received: 9 February 2018 / Revised: 12 October 2018 / Accepted: 3 December 2018 © The Author(s) 2019. This article is published with open access Abstract The Pacific coastal temperate rainforest (PCTR) is a global hot-spot for carbon cycling and export. Yet the influence of microorganisms on carbon cycling processes in PCTR soil is poorly characterized. We developed and tested a conceptual model of seasonal microbial carbon cycling in PCTR soil through integration of geochemistry, micro-meteorology, and eukaryotic and prokaryotic ribosomal amplicon (rRNA) sequencing from 216 soil DNA and RNA libraries. Soil moisture and pH increased during the wet season, with significant correlation to net CO2 flux in peat bog and net CH4 flux in bog 1234567890();,: 1234567890();,: forest soil. Fungal succession in these sites was characterized by the apparent turnover of Archaeorhizomycetes phylotypes accounting for 41% of ITS libraries. Anaerobic prokaryotes, including Syntrophobacteraceae and Methanomicrobia increased in rRNA libraries during the wet season. Putatively active populations of these phylotypes and their biogeochemical marker genes for sulfate and CH4 cycling, respectively, were positively correlated following rRNA and metatranscriptomic network analysis. The latter phylotype was positively correlated to CH4 fluxes (r = 0.46, p < 0.0001). Phylotype functional assignments were supported by metatranscriptomic analysis. We propose that active microbial populations respond primarily to changes in hydrology, pH, and nutrient availability. The increased microbial carbon export observed over winter may have ramifications for climate–soil feedbacks in the PCTR. Introduction Soils of the Pacific coastal temperate rainforest (PCTR) of North America sequester globally important amounts of carbon (~198–900 Mg C ha−1)[1] and contribute some of Supplementary information The online version of this article (https:// the highest rates of dissolved organic carbon (DOC) export doi.org/10.1038/s41396-018-0334-3) contains supplementary − − material, which is available to authorized users. to coastal margins in the world (10.5–29.9 g C m 2 y 1) [2]. Soil CH fluxes in the PCTR range from uptake * 4 William W. Mohn (0.05–0.55 mg C m−2 h−1) in upland forests to strong [email protected] emissions (0–1.08 mg C m−2 h−1) from ombrotrophic peat 1 Department of Microbiology & Immunology, Life Sciences bogs [3]. Microbial communities regulate the flow of Institute, University of British Columbia, Vancouver, BC, Canada carbon through coastal ecosystems via decomposition 2 Hakai Institute, Tula Foundation, Heriot Bay, BC, Canada of plant biomass [4, 5], yet the controls on microbial carbon 3 School of Resource and Environmental Management, Simon cycling in hydric soils, such those in the PCTR, are little Fraser University, Burnaby, BC, Canada understood. 4 The University of Applied Sciences Western Switzerland, Nutrient limitation, low O2, and acidic soil in the PCTR CHANGINS, Delémont, Switzerland restrict organic matter degradation [6]. The carbohydrate- 5 U.S. Department of Agriculture, Forest Service, Pacific Northwest active enzymes (CAZy) database [7] can facilitate investi- Research Station, Juneau, Alaska, USA gation of carbon cycling in soil communities [8, 9], and fl 6 Department of Botany, University of British Columbia, reveal the ow of carbon and energy through peatlands Vancouver, British Columbia, Canada from biopolymer degradation to C1 metabolism [10]. D. J. Levy-Booth et al. It remains to be seen how environmental conditions in Material and Methods distinct seasons and ecohydrological classes affects micro- bial organic matter degradation and carbon cycling. Study location and site description Anaerobic metabolic pathways play a major role in the mineralization of organic carbon in waterlogged soils. The Calvert Island Field Station is located on an outer- Anaerobic degradation in these environments can overcome coast island in the Perhumid PCTR (Supplementary thermodynamic limitations through the maintenance of Figure 1), in the very wet hypermaritime coastal western low H2 concentrations by coupling fermentation by sulfate- hemlock zone (CWH vh2) zone [20]. The bog forest reducing bacteria (SRB) to the reduction CO2 to CH4 site (TSN2; N51°39′08″, W128°07′47″) is within CWH by hydrogenotrophic methanogens [11, 12]. This syntrophic vh2 site series 11. It contained shallow, nutrient poor interaction is a major component of metabolism in organic soils (80–125 cm depth) with distinct L, F, anaerobic bog soil [13], which may be stimulated by winter and H layers over Of, Om layers, and some unstructured precipitation in the PCTR. Quantifying carbon balance mineral material. The primary canopy contains Pinus due to these processes is imperative for reconciling annual contorta, Chamaecyparis nootkatensis, and Thuja plicata. terrestrial carbon budgets. The deep, ombrotrophic peat bog site (TSN3; N51°39′05″, Quantifying active microbial populations can reveal W128°07′43″) is in BEC CWH vh2 site series 32. It how communities respond to changing environmental contained deep (>2 m) peat-derived organic soil, with conditions and contribute to nutrient cycles. However, sparse P. contorta, C. nootkatensis, and T. plicata and limitations of methods of assessing active microbial groups abundant ericaceous shrubs on Sphagnum spp. lawns and must be addressed. Sufficient mRNA is difficult to extract hummocks. We use the term ecohydrological class for and purify from high-organic soils. Ribosomal RNA these sites to distinguish areas of discrete vegetation, soil (rRNA) can be recovered using high-throughput methods, depth, soil type, and hydrology. Peat bog and bog forest but cellular concentration is not well correlated with growth sites were chosen on the basis of terrestrial ecosystem rates in mixed communities [14]. Further, dormant cells mapping methods, to target ecohydrological classes that can contain detectable rRNA [15]. Yet, rRNA analysis are prevalent in the PCTR [21]. can potentially reduce bias due to dead or dormant cells [16]. As ribosome concentration indicates potential for Terrestrial sensor nodes protein synthesis and thus cellular activity, the analysis of rRNA may provide ecologically-meaningful insights Both sites contained: an HC-S3 air temperature and relative into dynamics of putatively active microbial community humidity probe and a TB4 rain-gauge controlled with a members [10, 17–19]. CR1000 datalogger (Campbell Scientific (Canada) Corp., To characterize in situ total (DNA libraries) and puta- Edmonton, Canada) and powered by solar arrays. Three tively active (rRNA libraries) microorganisms and their subplots per site each contained: three 109-L soil tem- role in DOC, CO2, and CH4 cycling and export we perature probes (Campbell Scientific (Canada) Corp.), three sequenced amplicons of archaeal and bacterial 16S rRNA, platinum soil redox potential probes with XR300 Ag/AgCl fungal ITS, and eukaryotic 18S rRNA (focused on soil Reference Electrodes (Radiometer America Inc., Brea, protists). Metatranscriptomics (mRNA) validated phylotype USA). Soil temperature and redox potential probes were functional assignment. We hypothesized that soil conditions placed at depths of 10, 25, and 40 cm in the bog forest in distinct ecohydrological classes (peat bog and bog site and 10, 20, and 30 cm in the peat bog site, with the forest) would structure total microbial communities (H1); depths corresponding to approximate median water table that putatively active microbial populations would addi- depth and the bounds of annual water table variability. tionally respond to micro-climactic variables in distinct seasons (H2); and that winter periods would increase Soil, water, and gas sampling and analysis anaerobic metabolic processes leading to increased net CH4 flux (H3). We demonstrated seasonal differences in On June 18, 2015 (early summer), July 23, 2015, July 25, the structure of putatively active microbial community 2015 (summer), October 28, 2015 (fall), February 28, 2016 members, including a response to previously uncharacter- (winter), and April 18, 2016 (spring), soil was sampled in ized increased pH and inorganic nitrogen concentrations each subplot at depths corresponding to sensor placement, in winter, resulting in enhanced net flux of both CO2 using a screw auger and peat coring device. Two cores per and CH4. Together, these data allowed us to develop subplot were pooled to give 216 total soil samples analyzed and assess a conceptual model of seasonal changes in for this work. Subsamples (5 g) for DNA and RNA microbial carbon cycling in major PCTR ecohydrological extraction were immediately frozen on dry ice. Frozen soil classes (Fig. 1a). was transferred to a V900 CryoPro Vapor Shipper (VWR Seasonal and ecohydrological regulation of active microbial populations involved in DOC,. A Ecohydrology B DOC Fluxes C Soil Microclimate -2 Bog Forest Ombrotrophic Peat Bog 3 At time of sampling 0.4 to 1.8 mg C m h CO 0 to 0.025 CH o 15 CO 0.3 to 2.1 mg C m h mg C 2 m h 10 CH -0.004 to 0 mg C m h Decomp. 1 5 Denitrifcation Monthly DOC g m 0 -1 2015-062015-072015-102016-022016-04 0 Methano-