Molecular characterization of nocardioform actinomycetes in activated sludge by 16S rRNA analysis

Abstract

The analysis of complex microbiota present in activated sludge is important for the understanding and possible control of severe separation problems in sewage treatment such as sludge bulking or sludge foaming. Previous studies have shown that nocardioform actinomycetes are responsible for these conditions, which not only affect the efficiency of sewage treatment but also represent a threat to public health due to spread of pathogens. However, isolation and identification of these filamentous, nocardioform actinomycetes is hampered by their fastidious nature. Most species are still uncultivable and their taxonomy is unresolved. To study the ecology of these micro-organisms at the molecular level, w e have established a clone library of 165 rRNA gene fragments amplified from bulk sludge DNA. A rough indication of the predominant flora in the sludge was given by sequencing randomly chosen clones, which revealed a great diversity of bacteria from different taxa. Colony hybridization with oligonucleotide probe MNPI detected 27 clones with 165 rDNA inserts from nocardioform actinomycetes and mycobacteria. The sequence data from these clones together with those from randomly chosen clones were used for comparative 165 rRNA analysis and construction of dendrograms. All sequences differed from those of previously sequenced species in the databases. Phenotypic characterization of isolates of nocardioform actinomycetes and mycobacteria cultivated in parallel from the same activated-sludge sample revealed a large discrepancy between the two approaches. Only one 165 rDNA sequence of a cultured isolate was represented in the clone library, indicating that culture conditions could select species which represent only a small fraction of the organisms in the activated sludge.

Bacterial Communities in Different Sections of a Municipal Wastewater Treatment Plant Revealed by 16S rDNA 454 Pyrosequencing

“…The microbial community, which is dominated by bacteria (Wagner et al 2002), plays an essential role in the biological treatment reactors and has been studied for several decades by both isolation (Neilson 1978) and molecular methods, such as polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (Muyzer et al 1993;Ye and Zhang 2010), terminal restriction fragment length polymorphism (Liu et al 1997), cloning (Schuppler et al 1995), and fluorescent in situ hybridization (Erhart et al 1997). The culturing methods have been a very direct and effective way to characterize the microbial community.…”

An Abundance of Escherichia coli Is Harbored by the Mucosa- Associated Bacterial Flora of Interleukin-2-Deficient Mice

“…Sequence reaction products were purified by using AutoSeq G50 columns (Amersham Pharmacia Biotech, Braunschweig, Germany) and analyzed on an ABI PRISM system 377 automated sequencer (PE Biosystems). To determine the approximate phylogenetic affiliations, the partial 16S rDNA sequences were compared to the EMBL and GenBank databases as described previously (34). The 16S rDNA sequences were added to the rDNA sequence database of the Technical University of Munich by using the ARB program package (Lehrstuhl für Mikrobiologie, Technische Universität München [http://www.mikro.biologie.tu-muenchen .de]).…”

Bacterial Communities in a Full-scale Combined A/O+BIOFOR System Treating Pharmaceutical Wastewater

“…Molecular biology techniques have been used to analyze microbial community structures in activated sludge since 1995 [8]. However, because of low throughput, the application of traditional molecular methods was limited [9].…”