Cultivation and identification of filamentous bacteria from twelve wastewater treatment plants in South Africa

Abstract

The activated sludge process is the most extensively utilized technology for domestic wastewater treatment. Due to the increasing demand for clean water supply and proper environmental management, engineers and scientists are constantly looking for ways to improve process efficiency. Since the introduction of the activated sludge process, the most prevalent operational problems reported are sludge bulking and foaming caused by over-proliferation of filamentous bacteria. Attempts have been made to resolve filamentous bulking and foaming, but short-term control strategies are often ineffective long-term. In order to gain further insight into bulking and foaming, a deeper understanding of the associated filamentous bacterial populations is required. In previous decades, the unreliability of traditional (light microscopy and culture-dependent) microbiological methods has impeded the identification of filamentous bacteria and the study of their physiology. However, knowledge gaps regarding filamentous bacteria identity and function at a molecular level still exist. Therefore, this study was aimed at identifying filamentous bacterial populations found in wastewater treatment plants in South Africa by means of both conventional (phenotypic) and molecular (phylogenetic) identification methods. Filamentous bacteria were isolated from mixed liquor samples taken from wastewater treatment plants with histories of bulking and foaming. Deoxyribonucleic acid (DNA) was extracted from the isolates and amplified via the polymerase chain reaction using universal primers, after which the amplicons were sequenced. The overall bacterial community structures in the genomic DNA extracted from the activated sludge from the selected study sites were determined using amplicon sequencing (Illumina MiSeq). Results were compared with historical results obtained using classical light-microscopy. Twelve isolates which exhibited filamentous forms were cultured. Four of eight isolates from which high-quality DNA was extracted continuously exhibited cellular morphology typical of filamentous bacteria throughout the study, while the remainder shifted from filamentous to single cell forms upon repeated sub-culturing. Sixty percent of the isolates were members of the class Gammaproteobacteria, while 40% were members of the class Bacillus, neither of which have previously been associated with Eikelboom filamentous morphotypes. Amplicon sequencing revealed that Lewinella spp, Sphaerotilus spp, Haliscomenobacter spp, Tetrasphaera spp, Fluviicola spp, Longilinea spp, Bellilinea spp, Crocinitomix spp, and Mycobacterium spp, were the most dominant filamentous bacteria that were not identified via light microscopy. Comparison of light microscopy and amplicon sequencing results for filament identification revealed notable differences and highlighted the difficulties associated with both methods. This study contributes to the body of knowledge on filament identification in the activated sludge of wastewater treatment plants from different geographical locations in South Africa. The NGS findings from this study showed that there are many filamentous bacteria dominating the activated sludge plants and may be playing crucial roles which have not yet been studied and characterised. Therefore, this study can be used as a basis to gain further knowledge on the phylogeny of the filamentous bacteria community especially in the South African context.