Integrated Assessment of Benthic Bacterial Community Physiology, Structure, and Function Across C, N, P, and S Gradients in Lake Villarrica Sediments, Chile

Benthic bacterial communities play a critical role in nutrient cycling and are highly sensitive to environmental pollution. This study aimed to investigate the physiological, compositional and functional responses of bacterial communities across a range of carbon (C), nitrogen (N), phosphorus (P), and sulfur (S) gradients in sediments from Lake Villarrica, Chile. Sediment samples were collected from 5 sites representing a gradient of nutrient pressure from the lake basin (NL < PuB < PoP < SL < VB). Nutrient forms (TC, TN, TP, TS, and OM) were chemically quantified. Community function was assessed via community-level physiological profiles (CLPPs) using Biolog^® EcoPlates (C substrates), PM3B (N substrates), and PM4A (P and S substrates). Function and composition were assessed based on total bacterial and functional nutrient-cycling gene abundances (16Sr RNA, <i>chi</i>A, <i>mcr</i>A, <i>nif</i>H, <i>amo</i>A, <i>nos</i>Z, <i>pho</i>D, <i>pqq</i>C, <i>sox</i>B, <i>dsr</i>A) using qPCR and 16S rRNA metabarcoding, respectively. In general, the CLPPs were higher for C substrates, followed by P, S, and N substrates, with metabolism of organic forms of these nutrients preferential, and P-cycling genes were the most abundant in the lake. Spatially, the most nutrient-enriched site (VB) showed a significantly (<i>p</i> ≤ 0.05) higher nutrient content (e.g., 5.4% TC, 0.54% TN, 1302.8 mg kg^-1 TP and 854.1 mg kg^-1 TS) and total bacterial abundance (2.9 × 10¹¹ gene copy g^-1 dw sediment) but displayed lower CLPPs (from 0.63 to 1.02 AWCD) and nutrient-cycling gene abundances (e.g., 9.1 × 10¹, 2.7 × 10³, 3.6 × 10³ and 4.7 × 10³ gene copy g^-1 dw sediment for <i>chia</i>A, <i>nif</i>H, <i>pho</i>D and <i>dsr</i>A, respectively) compared to the less nutrient-enriched sites (e.g., NL). The bacterial community composition shifted accordingly, with Bacillota enriched in VB and Planctomycetota occurring more frequently in less nutrient-exposed sites. Functional prediction analysis revealed enhanced methanotrophy and sulfate respiration in nutrient-rich sediments, whereas nitrification and organic P (P_o) mineralization dominated in less impacted areas. The results demonstrate that nutrient enrichment constrains bacterial functional diversity in Lake Villarrica and, so, may be useful indicators of environmental stress to be considered in pollution monitoring programmes.