Ko et al. Journal of Ecology and Environment (2017) 41:34 Journal of Ecology DOI 10.1186/s41610-017-0053-0 and Environment RESEARCH Open Access Bacterial and fungal community composition across the soil depth profiles in a fallow field Daegeun Ko1, Gayoung Yoo2, Seong-Taek Yun3, Seong-Chun Jun4 and Haegeun Chung1* Abstract Background: Soil microorganisms play key roles in nutrient cycling and are distributed throughout the soil profile. Currently, there is little information about the characteristics of the microbial communities along the soil depth because most studies focus on microorganisms inhabiting the soil surface. To better understand the functions and composition of microbial communities and the biogeochemical factors that shape them at different soil depths, we analyzed microbial activities and bacterial and fungal community composition in soils up to a 120 cm depth at a fallow field located in central Korea. To examine the vertical difference of microbial activities and community composition, β-1,4-glucosidase, cellobiohydrolase, β-1,4-xylosidase, β-1,4-N-acetylglucosaminidase, and acid phosphatase activities were analyzed and barcoded pyrosequencing of 16S rRNA genes (bacteria) and internal transcribed spacer region (fungi) was conducted. Results: The activity of all the soil enzymes analyzed, along with soil C concentration, declined with soil depth. For example, acid phosphatase activity was 125.9 (± 5.7 (± 1 SE)), 30.9 (± 0.9), 15.7 (± 0.6), 6.7 (± 0.9), and 3.3 (± 0.3) nmol g−1 h−1 at 0–15, 15–30, 30–60, 60–90, and 90–120 cm soil depths, respectively. Among the bacterial groups, the abundance of Proteobacteria (38.5, 23.2, 23.3, 26.1, and 17.5% at 0–15, 15–30, 30–60, 60–90, and 90–120 cm soil depths, respectively) and Firmicutes (12.8, 11.3, 8.6, 4.3, and 0.4% at 0–15, 15–30, 30–60, 60–90, and 90–120 cm soil depths, respectively) decreased with soil depth. On the other hand, the abundance of Ascomycota (51.2, 48.6, 65.7, 46.1, and 45.7% at 15, 30, 60, 90, and 120 cm depths, respectively), a dominant fungal group at this site, showed no clear trend along the soil profile. Conclusions: Our results show that soil C availability can determine soil enzyme activity at different soil depths and that bacterial communities have a clear trend along the soil depth at this study site. These metagenomics studies, along with other studies on microbial functions, are expected to enhance our understanding on the complexity of soil microbial communities and their relationship with biogeochemical factors. Keywords: Soil enzymes, Pyrosequencing, Microbial community composition, Diversity Background different soil depths is still insufficient. Majority of Soil microorganisms play an essential role in mediating studies on soil microorganisms have focused on those nutrient cycling in the terrestrial ecosystem by decom- inhabiting the topsoil (~ 25 cm of the soil surface) where position of organic matters and transformation of inor- organic matter content and the abundance of soil micro- ganic compounds (Nannipieri et al. 2003, Xu et al. 2013, organisms are high (Fierer et al. 2003, Eilers et al. 2012). Sengupta and Dick 2015). Although soil microorganisms Despite the fact that large numbers of microorganisms are present throughout the soil profile, our understand- exist at deeper soil profiles, the characteristics of these ing of the composition of microbial communities at communities and the distributions of microorganisms along the soil profiles remain less understood (Blume * Correspondence: [email protected] et al. 2002, Fierer et al. 2003, Eilers et al. 2012). 1Department of Environmental Engineering, Konkuk University, Seoul 05029, South Korea The microbial communities inhabiting subsurface soil Full list of author information is available at the end of the article layers are known to have a greater influence on the © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Ko et al. Journal of Ecology and Environment (2017) 41:34 Page 2 of 10 process of soil formation compared to those at the Soil enzyme activity topsoil (Buss et al. 2005). In addition, subsurface soil To determine microbial community metabolism, fluoro- microbial communities may play an important role in metric assays using methylumbelliferone (MUB)-linked soil C sequestration since large amounts of organic C substrates were performed. The activities of β-1,4- with long turnover times are stored in the subsurface glucosidase, cellobiohydrolase, β-1,4-xylosidase, β-1,4- horizons (Trumbore 2000, Fierer et al. 2005, Rumpel N-acetylglucosaminidase, and acid phosphatase were and Kogel-Knabner 2011). Therefore, determining the analyzed which are extracellular enzymes that de- composition and characteristics of microbial communi- grade cellulose, hemicellulose, chitin, and organic P ties at different soil depths will allow us to understand substrate in soil (Saiya-Cork et al. 2002). Two grams the important subsurface soil processes. of soil was placed in 125 ml of sodium acetate buffer, and Significant differences in microbial community com- then, the slurry was transferred to a 96-well microplate that position and function at different soil depths have been included eight analytical replicates of each enzyme assay. reported (Hansel et al. 2008, Will et al. 2010, Eilers et al. Plates for all five enzymes were incubated at the room 2012). The diversity of microorganisms typically de- temperature for 2 h. Fluorescence was measured using a creases with depth, whether diversity is determined by SynergyHTMulti-ModeMicroplateReader(BioTek phospholipid fatty acid profiles (Fierer et al. 2003), DNA Instruments Inc., Winooski, VT, USA), in which the fingerprinting (LaMontagne et al. 2003, Agnelli et al. excitation energy was set at 360 nm and emission was 2004, Goberna et al. 2005), 16S ribosomal RNA (rRNA) measured at 460 nm. Enzyme activity was expressed as − − gene sequences (Will et al. 2010), or internal transcribed nmol 4-MUB g 1 h 1. spacer (ITS) regions of the rRNA (Baldrian et al. 2012). This is due to changes in edaphic factors such as pH, DNA extraction, PCR, and pyrosequencing moisture contents, the quality and quantity of organic DNA was extracted from the soil samples using the matters, and O2 concentrations throughout the soil FastDNA SPIN Kit (MP Biomedicals, OH, USA) follow- depth profile. There is still little information on whether ing the manufacturer’s instructions. The extracted DNA certain bacterial or fungal taxa are limited to specific soil was amplified via PCR with the universal primers that depths and how that may relate to distinct microbial target V1 to V3 hypervariable regions of the 16S rRNA functions at different soil depths (Eilers et al. 2012). gene. The primers used to amplify the 16S rRNA genes Here, we analyzed the soil enzyme activities and used for bacteria were 27F (5′-GAGTTTGATCMTGGCT- barcoded pyrosequencing to understand differences in CAG-3′) and 518R (5′-WTTACCGCGGCTGCTGG-3′). the function and composition of soil bacterial and fungal For fungal amplification, barcoded primers of ITS3F communities at different soil depth in a fallow field lo- (5′-GCATCGATGAAGAACGCAGC-3′)andITS4R cated in central Korea. Our objective was to determine (5′-TCCTCCGCTTATTGATATGC-3′)wereused.PCR the differences in soil microbial community composition conditions were as follows: initial denaturation at 95 °C and function along the soil depth at this site. for 5 min, 30 cycles of denaturation at 95 °C for 30 s, primer annealing at 55 °C for 30 s, extension at 72 °C Methods for 30 s, and a final elongation at 72 °C for 5 min. The Site description and sampling PCR products were purified using the QIAquick PCR The study site is a fallow field located in Eumsung-gun, purification kit (Qiagen, Valencia, CA, USA), and the North Chungcheong Province, in the central region of non-target products were removed employing Ampure Korea (36° 57′ 36.54″ N 127° 27′ 59.80″ E). The site is beads kit (Agencourt Bioscience, MA, USA). The qual- situated on a small hillock area in the uppermost stream ity and product size were analyzed on a Bioanalyzer of Mihocheon, a primary tributary of the Geum River. 2100 (Agilent, Palo Alto, CA, USA) employing a DNA The site is a fallow dry field surrounded by rice paddies. 7500 chip. Emulsion PCR was conducted on the mixed Within this site (60 m × 20 m), eight soil samples were amplicons, and the sequencing was performed at Chunlab, collected in September 2014 to a depth of 120 cm using Inc. (Seoul, Korea) using GS Junior Sequencing a manual geoprobe. These samples were classified to five System (Roche, Branford, CT, USA) following the different depths: 0–15, 15–30, 30–60, 60–90, and 90– manufacturer’sprotocol. 120 cm. In our study, results of analyses of soils from one sampling point are reported because the soil enzyme Pyrosequencing data analysis activity at the other sampling points was too low for Pyrosequencing data were analyzed following the de- meaningful study on microbial activity and community scriptions in previous studies (Chun et al. 2010, Hur composition to be conducted. Soil physicochemical et al. 2011, Kim et al. 2012a). Reads obtained were properties including pH, total C and N concentrations, sorted according to unique barcodes of the PCR prod- and soil texture were determined. ucts.