Rapid quantification and analysis of genetic diversity among Gordonia populations in foaming activated sludge plants

Abstract

Activated sludge plants, sporadically suffers malfunction due to the proliferation of filamentous bacteria mainly Gordonia and Microthrix species. Nested Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (nested PCR-RFLP) in combination with quantitative realtime PCR (q PCR) was applied to study the distribution of Gordonia in foaming samples. Samples of mixed liquor were collected from three full-scale activated sludge plants that were experiencing filamentous biological foaming. Partial sequencing of 16S rRNA genes revealed the dominance of Gordonia amarae (60 -80%), Gordonia terrae (10%), Gordonia polyisoprenivorans (30 -40%) and an unidentified Gordonia species (20 -50%). Restriction enzyme analysis of the amplicons exhibited 87.14 to 99.6% similarity at interspecies level. The q PCR results showed that there was an average of 15.6% Gordonia 16S rRNA copies with respect to the total bacterial 16S rRNA gene in foaming sludge samples with the highest being 23.51% and lowest being 10.28%. The presence of Gordonia spp. in the foaming samples was observed throughout the year but was lower during winter and its presence was significantly higher in foaming samples, compared to Microthrix parvicella (r = 0.007, P < 5%). This approach could help to quantify and confirm the existence of genetically diverse indigenous Gordonia spp. in foaming samples and can be used as an indicator of forthcoming foaming incidents. Bulking and foaming bacteria are one of the major issues in Wastewater Treatment Works (WWTW) with varying wastewater characteristics and operating conditions [3,5,18,25,27,29,33]. Formation of thick stable foam on the surfaces of clarifier could lead to a significant reduction in the quality of effluent affecting the overall efficiency of the plant. Studies worldwide established the presence of filamentous bacteria viz., Thiothrix spp., G. amarae, Candidatus Microthrix parvi- [4,10,20,29,31,35,36]. The most frequent filamentous bacteria causing sludge foaming are Candidatus M. parvicella and G. amare [24]. However, numerous studies have also confirmed that the incident of biological foaming is highly correlated with the profusion of Mycolata including Gordonia amarae [24,26] which also includes four more genera viz., Mycobacterium, Nocardia, Rhodococcus and Tsukamurella [27]. Members of this genus Gordonia are aerobic, catalase-positive, gram-positive to gram-variable, slightly acid-fast, nonmotile and nocardioform actinomycetes. These microorganisms are able to rapidly assimilate substrates when available and then store them intra-cellularly as carbon and energy stores. These energy reserves can It is now evident that some operational parameters experienced by the activated sludge including low food to microorganism ratio, low dissolved oxygen concentration, sludge age and environmental conditions like temperature and pH are primarily responsible to create a selective environment for the proliferation of certain foam forming filamentous microorganisms [19,26,41]. Proliferation of certain f...

Development and evaluation of a TaqMan duplex real-time PCR quantification method for reliable enumeration of Candidatus Microthrix

“…Moreover, using mulitplex PCR, simultaneous detection of total Bacteria or other problematic species such as Eikelboom's types (021N, 0675, 0041, 0961, 1701, 0914 and 0092), Thiothrix eikelboomii, Nostocoida limocola, and Gordonia amarae, etc. would be possible (Dumonceaux et al, 2006;Jenkins et al, 2004;Marrengane et al, 2011;Nielsen et al, 2009;Seviour et al, 1994;Vervaeren et al, 2005). However, the development of a multiplex qPCR using the same gene is difficult due to the competition for resources.…”

Filament identification and dominance of Eikelboom Type 0092 in activated sludge from wastewater treatment facilities in Cape Town, South Africa

“…Spiers et al, 2009Spiers et al, , 2011Yoon et al, 2010); high throughput pyrosequencing of the 16S rRNA gene (e.g. Guo and Zhang, 2012;Marrengane et al, 2011); compilation of clone libraries (e.g. Yoon et al, 2010), and excision and sequencing of bands obtained from denaturing gradient gel electrophoresis (DGGE) (e.g.…”