Profiling of selected filamentous actinomycetes isolated from activated sludge plants

Abstract

The formation of stable foams on the surface of aeration tanks has been observed worldwide in activated sludge systems. As a result of foam formation, wastewater treatment works face operational difficulties since foam seeps into effluent, adversely affecting the concentration of suspended solids, which reduces disinfection efficiency. Several efforts have been channeled towards a better comprehension of the microbial ecology of foaming. High abundance of Candidatus Microthrix parvicella and branched filaments of mycolic acid-containing actinomycetes have been reported frequently in foaming samples. The proliferation of these microorganisms has been reported from domestic to industrial treatment plants with different process configurations and throughout the different seasons. For process optimisation and troubleshooting, a greater comprehension of the structure and function of microbial community within a wastewater treatment plant is a prerequisite. Excessive growth of filamentous bacteria is promoted by the presence of a variety of physicochemical factors, and changes in process conditions. Though various factors can induce foaming, it is imperative to identify and quantify the indigenous organisms implicated in foaming to pre-empt forthcoming episodes. To date, no metagenomics data have been generated specifically from foaming samples in South African wastewater treatment works. Additionally, the fastidious nature of filamentous bacteria has limited our knowledge of pure culture studies, globally. Almost all of the available literature on pure culture studies in South Africa is more than a decade old and used conventional methods for isolation and purification. Therefore, this study aimed to profile microbial communities that are prevalent in foam samples in selected wastewater treatment works in KwaZulu Natal using a metagenomics approach. Additionally, the study explored the use of micromanipulation techniques for the isolation and cultivation of selected actinomycetes (Gordonia spp.) from foam samples. Two wastewater treatment works treating domestic and industrial wastewater were selected for the study. Microscopic examination of foam samples using wet mount technique indicated the prevalence of right-angled branched filamentous actinomycetes in both wastewater treatment works. The branched filamentous bacteria were selectively isolated using the micromanipulation technique from pre-treated mixed liquor and foam samples. Among the filamentous morphotypes that predominated in mixed liquor were Eikelboom Type 0041, Thiothrix, Gordonia spp., Eikelboom Type 021N and Eikelboom Type 0092 dominated in wastewater treatment works A and in wastewater treatment works B, Eikelboom Type 0041, Eikelboom Type 021N and Gordonia spp. dominated throughout the sampling period while Eikelboom Type 1851 and Thiothrix spp. were identified as transients. A total of forty-four isolates were obtained from the two wastewater treatment works using the micromanipulation technique. Out of these, nine isolates were further selected for physiological and molecular characterisation. The media that supported most isolates was Reasoner’s 2A agar and casitone glucose yeast agar during initial isolation stage. Upon continuous subculturing during preservation, filamentous morphology was permanently lost as the isolate from wastewater treatment works B shifted to single-celled morphology. The selected isolates were further grown in different media containing various carbon substrates such as cholesterol, benzoic acid, glucose, galactose and glycerol and were grown both aerobically and anaerobically. Aerodynamism varied amongst isolates, some displayed no growth under anaerobic conditions whilst only one isolate from wastewater treatment works B utilised all substrates aerobically and anaerobically. Isolates grew optimally at 30o C. Isolates from wastewater treatment works A mixed liquor and foam were fastidious and did not survive the process of isolation and profiling. Phylogenetic analysis of the 16S rRNA sequences indicated that the isolates were close relatives of Gordonia spp. However, the similarity index was lower than 97% indicating that the isolates may be novel or represent divergent variants to existing Gordonia spp. Additionally, quantitative polymerase chain reaction was performed to assess the dominance of selected actinomycetes in foaming samples. Gordonia spp. were successfully quantified and their abundance was related to selected plant operation parameters to establish trends that induce Gordonia spp. proliferation. The ambient temperature of the two respective plants, wastewater treatment works A and B was (24.8 ±5.5 and 28.0o C ± 5.1 respectively) observed to favour the growth of mycolic acid containing actinomycetes. The amount of Gordonia spp. in foams was significantly higher in foam samples than mixed liquor in both the plants investigated. It was also observed that Gordonia copy numbers of 3.7 X 109 ± 0.1 copies/ng were sufficient to induce foaming in these plants. Furthermore, the application of next generation sequencing provided further insight into the role of other actinomycetes in foam formation in this study. Three mixed liquor samples and three foam samples were subjected to next generation sequencing from each wastewater treatment works. Based on the next generation sequencing approach, the microbial community did not vary significantly in mixed liquor and foam, however, abundance changed significantly amongst mycolic acid containing Actinobacteria. An average increase of 17% was observed from Nocardiaceae, Mycobacteriaceae and Gordoniaceae in foams than in mixed liquor. The dominant foam formers in both the wastewater treatment were Gordonia spp., Rhodococcus spp. and Mycobacterium spp. irrespective of the influent characteristics. Gordonia amarae which has been implicated and greatly studied as a foam inducer were not found to be predominant in these wastewater treatment works highlighting the contribution of other potential Gordonia spp. (Gordonia alkanivorans, Gordonia insulae, Gordonia phthalatica, Gordonia polyisoprenivorans, Gordonia rubripertincta) in foaming. Mycobacterium doricum and Rhodococcus coprophilus were also detected from foam samples. The abundance of Mycobacterium intracellulare, Mycobacterium africanum and Mycobacterium avium in wastewater treatment works A and B raises serious health implications. M. intracellulare has been implicated in human pulmonary infections even in immunocompetent individuals. The detection of Mycobacterium tuberculosis in foams also raises health concerns considering the prevalence of HIV in South Africa and tuberculosis co-infections that emanate from immunosuppression. The prevalence of mycobacterial pathogens in foams necessitates deeper interrogation to circumvent occupational hazards. By contributing to the current knowledge base, this study has made a significant contribution towards understanding the organisms responsible for foam formation and stabilization in the subtropical region of KwaZulu-Natal.