Determination of the relationship between epiphytes and selected filamentous bacteria in activated sludge

Activated sludge (AS) flocs are paramount in biological treatment of wastewater, are comprised of microbial consortia with organic and inorganic material bound together by extra polymeric substances (EPS). The filamentous bacteria play a vital role in the floc formation process by providing the necessary structural support. Presence of epiphytic attachment on selected filamentous bacteria is a commonly occurring phenomenon in activated sludge samples. Different theories have been proposed to describe this phenomenon; however, not much research has been carried out to explore the profundity of the attachment. In this study, an attempt has been made to elucidate the intrinsic nature of the epiphytic attachment between the bacterial rods and filamentous bacteria based on microscopic (morphological and structural) analysis. Characterization of these epiphytes were performed using fluorescence in situ hybridization (FISH) at group level using Alpha, Beta and Gamma Proteo-bacterial probes. Morphological characteristics of filament hosts and the bacterial rods at the interface region was assessed using scanning electron microscopy (SEM). The SEM micrographs indicated that the attachment was facilitated by more than the EPS layer. Further ultrastructural examination using transmission electron microscopy (TEM) indicated a possible cell-to-cell interaction between epiphytes and the selected filaments. Fibrillar structures resembling amyloid-like proteins were observed within the filament cell targeted by the epiphytes. An interaction was apparent between the amyloid like proteins and the epiphytes as exhibited by the direction of fibrillar structures pointing towards the approaching epiphytes. Common bacterial appendages such as pili and fimbria were absent at the interface and further noted was the presence of cell membrane extensions on the epiphytic bacteria protruding towards the targeted filamentous cell. The sheath of host filaments however, remained intact and unpenetrated, during colonization. Amyloid-like fibrils at interface may potentially play the role of attachment sites for the attaching epiphytes, as attachment facilitating appendages were not visualized.