DOI: 10.57755/ch3h-7g44
title: Seasonal dynamics of bacterial communities in activated sludge and treated wastewater from a full-scale WWTP

description: <p class="MsoNormal"><span style="font-size:14px" lang="EN-US">This project contains
16S rRNA gene amplicon sequencing data from 12 environmental samples collected
from a full-scale wastewater treatment plant (WWTP) located in Poland. Samples
were obtained from activated sludge (AS) and treated wastewater (TW) during
different seasons (December, February, April, June, July, October) to
investigate temporal variations in microbial community composition. The study
aims to evaluate the seasonal dynamics of bacteria in both stages of the
treatment process, providing insights into microbiome stability and performance
in engineered ecosystems. The data include DNA and cDNA sequence reads obtained
by the 16S rRNA method, diversity of microflora composition, and metadata on
dates and sample type and information on nitrfication related genes described
as KO (KEGG Orthology) number. The dataset can be used for analysis of
microbiome dynamics in wastewater treatment processes, seasonality analysis and
comparative microbial characterization. This dataset supports a research
publication currently in preparation.</span></p>


description: <p class="TAMainText" style="line-height:normal"><span style="font-size:14px">The
total environmental DNA from the sample of interest was isolated using
Environmental DNA/RNA Extraction Kit (Eurx, Gdańsk, Poland) following the
manufacturer’s instructions. This reagent set allows for an easy and convenient
DNA isolation from material such as soil, slurry or sediment. 200 mg of sample
was weighted for processing. Samples were first disrupted by pulverization with
0.4-0.6 diameter glass beads (Sartorius AG, Göttingen, Germany) using the
TissueLyser apparatus (Qiagen, Hilden, Germany). After isolating DNA and RNA,
quality was checked by running the sample on 1% agarose gel, while template
quantity was measured by fluorimetry using Qubit 2.0 and High Sensitivity
Picogreen reagents (Thermo Fisher Scientific, Sunnyvale, CA, USA). The quality
of the isolated RNA was further improved by the use of the Clean-up RNA
Concentrator kit (A&A Biotechnology, Gdańsk, Poland), which included a
DNase-based DNA removal step. To ensure that the DNA was completely removed
from the RNA samples, a control PCR reaction was set up, with the RNA samples
serving as a template and Escherichia coli Top10 (Thermo Fisher Scientific,
Sunnyvale, CA, USA) genomic DNA serving as a positive control. The primers used
were gene-specific primers: 16S_V3-F and 16S_V4-R, which cover positions
341-357F and 785-805R, respectively, according to E. coli 16S rRNA gene
reference sequence<sup>1</sup>. PCR products were checked on 1% agarose gel.
The reverse transcription was carried out with the use of RevertAid First
Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Sunnyvale, CA, USA)
containing random hexamer primers according to the manufacturer’s instructions.
The obtained cDNA samples were stored in -20°C until further analysis.
Amplification of the conserved bacterial 16SrRNA gene fragment covering V3 and
V4 regions was done for DNA and cDNA samples in triplicate by means of the
gene-specific primers extended on 5’ end with Nextera adaptors<sup>1</sup>
(Illumina, Sand Diego, USA). </span></p>

<p class="TAMainText" style="line-height:normal"><span style="font-size:14px" lang="EN-US">The
obtained amplicons of the size c.a. 450bp were checked on 1% agarose gel and
purified by Ampure XP magnetic beads (Beckman Coulter, Brea CA, USA). Amplicon
libraries were pooled in equimolar ratio and indexed according to the Nextera
indexing strategy by PCR (Illumina, San Diego, USA). Sample indexing allowed
for pooling of amplicons for sequencing run and further extraction of the
sample sequence reads from large batch of sequencing results data. 16S
amplicons were sequenced on MiSeq sequencer in the DNA Sequencing and
Oligonuleotide Synthesis Laboratory IBB PAS in paired end mode using 600 cycle
v3 chemistry kit (Illumina, San Diego, USA).</span></p>

<p class="TAMainText" style="line-height:normal"><span style="font-size:14px" lang="EN-US">The
obtained Illumina reads were quality checked using the FastQC software
(www.bioinformatics.babraham.ac.uk/projects/fastqc<sup>2</sup>). Raw
sequencing reads were cleaned, aligned, and classified automatically by the
EzBioCloud platform using the PKSSU4.0 database<sup>3</sup> statistical
analysis and visualization were performed using R packages: phyloseq
(https://joey711.github.io/phyloseq/), microbiome
(https://microbiome.github.io/tutorials/), ggplot2
(https://ggplot2.tidyverse.org/), and Ampvis2<sup>4</sup>. </span></p>

<p class="TAMainText" style="line-height:normal"><span style="font-size:14px" lang="EN-US">Prediction
of functional abundances based on 16S marker gene sequences was performed using
Picrust2 software with default parameters<sup>5</sup>. For this purpose,
sequence reads were first processed using Qiime2 pipeline<sup>6</sup>. Paired
reads were merged and denoised using DADA2. Sequences were then taxonomically
classified using the Bayesian Naïve classifier pre-trained on the SILVA
database (“silva-138-99-nb-classifier.qza a”, https://docs.qiime2.org/2023.2
/data-resources/). Picrust2 predicted and manually selected data for KEGG
ortholog (KO) families which was further analysed using MicrobiomeAnalyst 2.0<sup>7</sup>.
Genes related to the metabolism of nitrogen compounds are listed in Table S.
The selected results were compiled and presented using Excel 2016 (MS Office)
for Windows.</span></p><p class="TAMainText" style="line-height:normal">

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