XXVI. Biochemical Congress, Budovicium, 2021

Foam is needed on draft beer, but not in wastewater treatment plants

Wastewater treatment is a complex multistep process essential in every city infrastructure that is closely monitored at every stage. The foaming represents an unpredictable and poorly understood significant problem at wastewater treatment plants (WWTP). It can lead to both reduction of the overall system performance and considerable economical losses for the WWTP. It is therefore very desirable today to study the formation of foam in WWTP, however, for early detection and prevention of foaming the responsible biochemical pathways need to be described in detail. In this process, the first step represents the identification of the microbial culprits and the proposal of an economically acceptable strategy mitigating their growth and effects.

According to this fact, we realised a retrospective pilot analysis of mesophilic anaerobic sewage sludge bacteriota stabilization under the conditions during which the formation of foam was encountered. Sludge samples were collected at wastewater treatment plant 513,000 PE, Modřice, Czech Republic. Physico-chemical parameters of the sewage sludge in situ were kindly provided by Brněnské vodárny a kanalizace, a.s. Bacteria populations in sludge samples were analyzed by V4 16S rRNA sequencing performed using Illumina’s MiniSeq with subsequent in silico analysis in DADA2 software. As a suggested canonical suspect causing foaming at WWTP we detected Microthrix parvicella and members of group Mycolata although in minuscule amounts indicating different bacteria may be at play. Besides, we observed enrichment of genera Sedimentibacter and Flexilinea capable of forming filaments, Cloacimonadales (W5 and W27) implicated in long-chain fatty acid degradation and Spirochaetaceae involved in sulphur oxidation.


 

Supplementary information

Methodology

DNA was isolated using the DNeasy PowerSoil Kit (Qiagen, Hilden, Germany), according to the manufacturer’s instructions. The V4 region of 16S rRNA was selected for amplification with unique barcoded primers. The primer sequences, consisting of an Illumina adapter (P5 or P7), an 8-nt index sequence representing the unique barcode, a 10-nt pad sequence, a 2-nt linker, and a specific sequence of the V4 region, were modified, according to Pichler et al. PCR amplification was performed according to the Earth Microbiome Project protocol using Platinum II Taq Hot-Start DNA Polymerase (Thermo Fisher Scientific, Waltham, MA, USA), as follows: initial DNA denaturation step at 94 °C for 3 min, 35 cycles of DNA denaturation at 94 °C for 45 s, annealing at 52 °C for 60 s with a 50% thermal ramp, extension at 72 °C for 90 s, and a final extension step at 72 °C for 10 min. The amplification product was purified by AMPure XP beads (Beckman Coulter, Brea, CA, USA), following the manufacturer’s instructions. The Qubit 4.0 fluorometer (Thermo Fisher Scientific, Waltham, MA, USA) and FragmentAnalyzer (Advanced Analytical Technologies, Inc., Santa Clara, CA, USA) were then used to determine the quality of the libraries. The library was sequenced using a MiniSeq system (Illumina, San Diego, CA, USA) with MiniSeq Mid Output Kit (2 × 150 bp).

Sequence analysis

Raw fastq reads were processed using the DADA2 package in R. The analysis was carried out according to the standard operating procedure. Briefly, reads were first filtered and trimmed (zero ambiguous bases, expected error threshold of 2 and the last 1 base truncated from the reverse read only). Filtered reads were then de-replicated (unique sequences were extracted) and de-noised (identified sequencing errors were removed using learned error rates and quality profiles of reads). Reads were then merged, chimaeras removed, and taxonomy was assigned by the RDP naive Bayesian classifier method against the SILVA nr99 v138.1 database. A phylogenetic tree was built using the phangorn package with the DECIPHER package used for multiple alignments. The phyloseq and vegan packages were used for subsequent phylogenetic and statistical analyses. Graphical outputs were composed in Inkscape and GraphPad Prism.