In-depth assessment of microbial communities in the full-scale vertical flow treatment wetlands fed with raw domestic wastewater

International audienceA multiphase study was proposed to examine microbial communities linked to the nitrogen cycle in the first stage of four full-scale French vertical flow treatment systems. To this end, denaturing gradient gel electrophoresis (DGGE) was performed for structural assessment and quantitative PCR (qPCR) to enumerate the abundance of ammonia-oxidizing (AOB). 16S rRNA sequencing was used to assess the taxonomic profile followed by putative assessment of functional genes. The samples were collected under different conditions, such as operational time (presence/absence of sludge layer on the surface of the filters), season (winter and summer), sampling depth (0, 15 and 30 cm) and operation cycle (rest and feed periods). A structural disparity was noted in the upper layers, whereas higher similarity at 30 cm was observed highlighting the effect of organic matter on bacterial diversity. The 7th rest day was highlighted by an apparent decline in the microbial community abundance. Additionally, qPCR indicated that the largest amount of AOB was found at 30 cm depth and during the feeding period. From the taxonomic profile, Mycobacterium, Acinetobacter, Flavobacterium, and Nitrospira were the most abundant genre found in all systems. The functional prediction results showed predicted genes linked to the denitrification process. The results suggested that operating time and season were responsible for the pattern of the microbial community behavior. This study allowed us to further understand the bacterial dynamics and to advance the idea of design modifications made in the first stage of the classical French system to improve nitrogen removal are promising

Molecular analysis of the bacterial diversity in a specialized consortium for diesel oil degradation Análise molecular da diversidade bacteriana de um consórcio degradador de óleo diesel

Diesel oil is a compound derived from petroleum, consisting primarily of hydrocarbons. Poor conditions in transportation and storage of this product can contribute significantly to accidental spills causing serious ecological problems in soil and water and affecting the diversity of the microbial environment. The cloning and sequencing of the 16S rRNA gene is one of the molecular techniques that allows estimation and comparison of the microbial diversity in different environmental samples. The aim of this work was to estimate the diversity of microorganisms from the Bacteria domain in a consortium specialized in diesel oil degradation through partial sequencing of the 16S rRNA gene. After the extraction of DNA metagenomics, the material was amplified by PCR reaction using specific oligonucleotide primers for the 16S rRNA gene. The PCR products were cloned into a pGEM-T-Easy vector (Promega), and Escherichia coli was used as the host cell for recombinant DNAs. The partial clone sequencing was obtained using universal oligonucleotide primers from the vector. The genetic library obtained generated 431 clones. All the sequenced clones presented similarity to phylum Proteobacteria, with Gammaproteobacteria the most present group (49.8 % of the clones), followed by Alphaproteobacteira (44.8 %) and Betaproteobacteria (5.4 %). The Pseudomonas genus was the most abundant in the metagenomic library, followed by the Parvibaculum and the Sphingobium genus, respectively. After partial sequencing of the 16S rRNA, the diversity of the bacterial consortium was estimated using DOTUR software. When comparing these sequences to the database from the National Center for Biotechnology Information (NCBI), a strong correlation was found between the data generated by the software used and the data deposited in NCBI.<br>O óleo diesel é um composto derivado do petróleo, constituído basicamente por hidrocarbonetos. Condições precárias no processo de transporte e armazenagem desse produto contribuem significativamente para derrames acidentais, ocasionando sérios problemas ecológicos no solo e água, alterando assim toda a diversidade microbiológica do ambiente. A estratégia de clonagem e sequenciamento do gene 16S rRNA é uma das técnicas moleculares que permitem estimar e comparar a diversidade microbiana de diferentes amostras ambientais, sejam elas impactadas ou não. O objetivo deste trabalho foi estimar a diversidade de microrganismos pertencentes ao domínio Bacteria em um consórcio degradador de óleo diesel por meio de sequenciamento parcial do gene 16S rRNA. Após extração do DNA metagenômico, o material foi amplificado por reação de PCR com oligonucleotídeos iniciadores específicos para o gene 16S rRNA. Os produtos da reação de PCR foram clonados em vetor pGEM T Easy (Promega) e transformados em células competentes de Escherichia coli. O sequenciamento parcial dos clones foi feito com oligonucleotídeos universais do vetor. A biblioteca obtida gerou 431 clones. Todos os clones mostraram similaridade com o filo Proteobacteria, onde as Gammaproteobacteria compreenderam o grupo de maior representatividade, com 49,8 % dos clones, seguida das Alphaproteobacteira, com 44,8 %, e das Betaproteobacteria, com 5,4 %. O gênero Pseudomonas destacou-se como representante com maior frequência de clones na biblioteca, seguido pelos gêneros Parvibaculum e Sphingobium. Após o sequenciamento parcial do gene 16S rRNA, a diversidade bacteriana do consórcio foi estimada utilizando-se o software DOTUR. Essas sequências, quando comparadas com as do banco do National Center for Biotechnology Information (NCBI), mostraram grande correlação entre os dados gerados pelo software utilizado e aqueles depositados no NCBI