Soil Microbial Community Structure Dynamics at Depth in Wastewater Treated Soils.

Studies investigating soil microbial community structure (SMCS) are typically limited to the top 30 cm. Here we report on SMCS and denitrification rates from samples collected to a depth of 300 cm at two municipal wastewater application sites (MWAS) in Northwest Texas. SMCS was determined using ester linked fatty acid methyl ester (EL-FAME) profiling and denitrification rates were measured from intact soil cores. Cores were collected from the Midland, TX MWAS site (MFS) (sandy loam) in October 2008 (Y1) and March 2009 (Y2) and samples from the Littlefield, TX MWAS site (LFS) (sandy clay loam) were collected in September 2008 (Y1) and January 2009 (Y2).  At each sampling event, ten cores were collected using a hydraulic drop hammer soil probe; five from the wastewater irrigated area and five from an adjacent area receiving no irrigation.  Soil cores were separated into 15 cm increments for the first 30 cm, and then one 15 cm core was collected from within each remaining 30 cm increment down to 300 cm. Denitrification rates were determined immediately using the intact core method. Immediately following incubation and gas sampling for denitrification, a subsample was collected, frozen at -80C and then freeze-dried prior to EL-FAME analysis. FAME profiles were examined using non-metric multidimensional scaling (NMS) plots after a square-root transformation. A joint plot was constructed to determine overall shifts in SMCS based on the relative proportions (mol %) of specific microbial groupings. These groups include: FAME biomarkers for Gram negative and postive bacteria, arbuscular mycorrhizal fungi (16:1ω5c), general fungal biomarkers (18:2ω6,9c, 18:1ω9c, and 18:3ω6,9,12c), and overall bacteria biomarkers (sum of Gram negative and Gram positive markers). For analysis of the NMS plots, depths were combined to create three groups: (d1) 0-60cm, (d2) 60-120cm, and (d3) 120-300cm. Prelimary analysis (of the irrigated samples only) revealed that SMCS was different between d1 and d2 and between d1 and d3 but not between d2 and d3 for the LFS site for Y1 and between the three depth groups for Y2. A joint plot overlay using biomarker groupings did not reveal any patterns for SMCS differences at depth for Y1. However, the following trends were observed for LF in Y2: d1 had relatively lower proportions of Gram negative biomarkers and overall biomarkers but d1 had the highest mol % of actinomycetes and the fungal marker 18:3ω6,9,12c. The AMF biomarker was absent in samples for group 3 and not significantly different between d1 and d2. SMCS for the MLS site in Y1 was different for each of the three depth groups. Similar to results from the LFS site in Y2, the relative abundances of actinomycetes were greater at d1 compared to d2 or d3 and the AMF biomarkers were absent at d3. However, the general fungal marker, 18:2ω6,9c was highest (mol %) at d3 compared to d2 or d1. Additionally, 18:1ω9c was absent at d3 and 18:3ω6,9,12c was absent at all three depths at the MLS in Y1. Although denitrification rates varied at the different depths, we found no correlation between overall microbial biomass as calculated as the total FAME content or with any particular microbial group (relative abundance or absoulte). Future analysis will include quantification of genes involved in the dentrification process by using quantitative PCR methods.