DOI: 10.5061/dryad.cnp5hqcj3
title: Microbiome characterization of urbanized lakes impacted by legacy arsenic contamination

description: Bacteria and other microbes are important contributors to arsenic
 biotransformation processes, which can alter the bioavailability and
 toxicity of arsenic within a contaminated environment. Multispecies
 biofilms, known as periphyton, have been identified as a significant site
 of arsenic bioaccumulation within shallow freshwater lakes impacted by
 legacy arsenic contamination. We hypothesized that prolonged arsenic
 exposure results in the formation of distinct prokaryotic communities
 within the periphyton and other environmental compartments in
 arsenic-contaminated lakes compared to uncontaminated lakes. We also
 predicted that the periphyton prokaryotic communities would be distinct
 from, but partially overlapping with, those found in the surrounding water
 column and nearby littoral sediment. To test these hypotheses, we
 determined the taxonomic composition and modeled the assembly processes
 that yielded the bacterial communities found within three environmental
 compartments (periphyton, littoral sediment, and water column) of three
 lakes that had been differentially impacted by legacy arsenic
 contamination. We identified unique microbiomes within these environmental
 compartments and observed a clear shift in microbial community composition
 within high arsenic-contaminated periphyton. Hyperaccumulation of arsenic
 (~400 ppm) in the periphyton correlated with non-random (deterministic)
 selection for prokaryotic taxa that are more related than expected by
 chance (homogenizing selection). We also identified key prokaryotic genera
 within the arsenic-contaminated periphyton that suggest prolonged arsenic
 contamination may shift iron and methane biogeochemical cycles, which may
 regulate arsenic accumulation and mobilization. Our results imply that
 legacy arsenic contamination, by altering bacterial community composition
 and metabolic potential at the base of the food web, may influence
 biogeochemical and nutrient cycles at a larger scale within a freshwater
 lake ecosystem.
description: Lake sampling: Samples from three
 environmental compartments (littoral sediment, lake water, periphyton)
 were collected in September 2023 from three south King County, WA lakes
 that represent a gradient of arsenic contamination: Lake Killarney (high),
 Steel Lake (mid), Trout Lake (low; reference). All lakes were of similar
 depths and shared other general geomorphological features (Figure 1; Table
 1). Samples were collected from three independent sites per lake. Surface
 water was collected from a paddleboard in sterile 1L glass bottles.
 Littoral sediment was collected in sterile 50 ml tubes by hand at a water
 depth of ~0.5m. Periphyton was cultivated on acrylic plates, which were
 deployed within a plastic frame that was submerged at a depth of ~1m in
 each lake. The periphyton plates soaked in the lake for ~60 days prior to
 collection. During sample collection (n = 10 per sample type, per lake),
 sediment and periphyton were placed in sterile transport vessels and
 immediately processed in the lab. Subsamples of periphyton were 1) scraped
 off the acrylic plates using a sterile razor blade, flash frozen with
 liquid nitrogen and stored at -80°C to preserve for DNA extraction or 2)
 dried at 60°C for 72 hours to process for inductively coupled plasma mass
 spectrometry (ICP-MS) analysis. Water samples were 1) filtered using  0.2
 𝜇m sterile filters, which captured microbes and were flash frozen and
 stored at –80°C to preserve for DNA extraction or 2) filtered using 0.45μm
 pore size surfactant-free cellulose acetate vacuum filter and acidified to
 2% HNO3 to preserve for ICP-MS. Total arsenic and iron
 concentration: Samples were prepared for ICP-MS
 according to previously described methods (Hull et
 al., 2021). Briefly, the dried sediment and periphyton were
 digested in a microwave with trace metal grade HNO3 in pressurized
 digestion vessels (CEM MARS 5) following the total digestion protocol
 (modified EPA method 3015a). After digestion, sample solutions were
 diluted to 2% (v/v) HNO3 and filtered using 0.45μm pore size
 surfactant-free cellulose acetate syringe filters. Water samples did not
 undergo microwave assisted digestion. Digestion procedure efficacy was
 evaluated using certified reference material DOLT-5 (dogfish liver), which
 yielded a recovery of 97.18% (n = 5). Total arsenic concentrations in
 water, digested sediment, and periphyton samples were determined by ICP-MS
 (Agilent 7900). The lower limit of detection (LOD) was 0.25 μg/L for
 arsenic. Calibration was performed using a certified multi-element metals
 standard (Agilent Multi-Element Calibration Standard 2A) with 7 different
 dilution points. Instrument performance was assessed by checking two
 standards after every batch of 10 unknown samples. 16S
 rRNA gene amplicon sequencing for microbiome analyses:
 DNA isolation from field samples: DNA was
 extracted using the PowerSoil Pro kit (Qiagen; Hilden, Germany). Negative
 controls for the extraction process were run in parallel at all stages of
 extraction and sequencing. DNA was checked for purity and concentration on
 a Nanodrop (Thermo Scientific). DNA samples were normalized to the lowest
 concentration per sample type (sediment, periphyton, and water) and sent
 to MR DNA (Shallowater, TX) for paired-end Illumina sequencing of the 16S
 rRNA gene. The V3-V4 region of the 16S rRNA gene was amplified using 341F
 and 785R primer pairs to target high diversity and richness of samples
 (Thijs et
 al., 2017). Mock community reference samples (ATCC, n=3) were
 included as positive controls.
description: # Microbiome characterization of urbanized lakes impacted by legacy
 arsenic contamination Dataset DOI:
 [10.5061/dryad.cnp5hqcj3](10.5061/dryad.cnp5hqcj3) ## Description of the
 data and file structure This dataset includes total arsenic and total iron
 concentrations from water, littoral sediment, and periphyton samples
 collected from three urabnized shallow lakes in the Puget Sound lowlands
 of Washington, USA. 16S rRNA gene amplicon sequencing data for replicate
 samples has been deposited at ncbi.gov under BioProject PRJNA1310655
 (publicly available 10/01/25). ### Files and variables #### File:
 Buser_et_al._2025__Compartments_ICP_MS_Data.xlsx **Description of the data
 and file structure** Data are provided in .XLSX format, organized by study
 lake and sample type. This file has six tabs, and the contents of each tab
 are described below. ##### Total As H~2~O * lake - lake where samples were
 collected * sample type - all surface water  * collection date - date of
 sampling * microbiome samples - sample numbers for corresponding
 microbiome samples in BioProject PRJNA1310655 * total As (μg/L) - total
 arsenic concentration in samples as determined by acid digestion and
 ICP-MS ##### Total As Sediment * lake - lake where samples were collected
 * sample type - all littoral sediment collected by hand at a water depth
 of ~0.5 m  * collection date - date of sampling * microbiome samples -
 sample numbers for corresponding microbiome samples in BioProject
 PRJNA1310655 * total As (μg/g) - total arsenic concentration in samples as
 determined by acid digestion and ICP-MS ##### Total As Periphyton * lake -
 lake where samples were collected * sample type - all periphyton collected
 on acrylic plates after soaking for 60 days in the lake at a water depth
 of ~1 m  * collection date - date of sampling * microbiome samples -
 sample numbers for corresponding microbiome samples in BioProject
 PRJNA1310655 * total As (μg/g) - total arsenic concentration in samples as
 determined by acid digestion and ICP-MS ##### Total Fe H~2~O * lake - lake
 where samples were collected * sample type - all surface water  *
 collection date - date of sampling * microbiome samples - sample numbers
 for corresponding microbiome samples in BioProject PRJNA1310655 * total Fe
 (μg/L) - total iron concentration in samples as determined by acid
 digestion and ICP-MS ##### Total Fe Sediment * lake - lake where samples
 were collected * sample type - all littoral sediment collected by hand at
 a water depth of ~0.5 m  * collection date - date of sampling * microbiome
 samples - sample numbers for corresponding microbiome samples
 in BioProject PRJNA1310655 * total Fe (μg/g) - total iron concentration in
 samples as determined by acid digestion and ICP-MS ##### Total Fe
 Periphyton * lake - lake where samples were collected * sample type - all
 periphyton collected on acrylic plates after soaking for 60 days in the
 lake at a water depth of ~1 m  * collection date - date of sampling *
 microbiome samples - sample numbers for corresponding microbiome samples
 in BioProject PRJNA1310655 * total Fe (μg/g) - total iron concentration in
 samples as determined by acid digestion and ICP-MS