Bacteria and extracellular polymeric substances in activated sludge scum formation

The formation of thick viscous scum layers is a persistent problem in wastewater treatment plants (WWTPs) observed all over the world. Three factors are necessary for scum formation: (i) dispersed gas bubbles, (ii) surface-active material and (iii) hydrophobic compounds. For instance, air bubbles in the aeration tank initialize scum development. The gas-water interface is stabilized by the adsorption of surface-active molecules. The presence of hydrophobic material provides stable adhesion to solid particles promoting persistent scum layers. Surface-active substances may enter the WWTP via primary effluent or be produced by various bacteria in the presence of hydrophobic carbon sources and under nutrient deficiency. Hydrophobic material might originate from the primary effluent or be available by an abundant growth of organisms with hydrophobic cell surfaces. One objective of this study was to characterize the relevant bacteria that are involved in scum formation. The identification of these bacteria was performed by classical microscopic sludge analysis and molecular biological methods to obtain information about their taxonomic affiliation. The knowledge about the phylogeny of scum bacteria might reveal physiological properties of these bacteria, which is helpful for the understanding of the scum formation process and the application of specific control measures. A further topic of this study was to investigate whether scum formation is a passive floating mechanism of activated sludge to the water surface or an active growth process of a specific scum biocenosis close to the water surface. The elucidation of the role of these scum bacteria and extracellular polymeric substances (EPS) in the formation of stable scum layers was the third objective of this work. EPS might perform two different functions in the scum process: (i) they serve as nutrient source and promote the growth of a specific bacterial biocenosis and (ii) they directly stabilize scum layers due to their hydrophobic sites. For this purpose, scum bacteria numbers quantified by fluorescence in situ hybridization (FISH) were correlated to sludge hydrophobicity and specific EPS components extracted from activated sludge and scum. A screening of various WWTPs with scum formation revealed that in most plants examined Microthrix parvicella dominated the activated sludge and scum biocenoses. Furthermore nocardioform actinomycetes could be characterized as important scum bacteria. These microorganisms were found at very low numbers in activated sludge whereas they were clearly enriched in scum layers. The filamentous type 1863 found in several WWTPs was clearly enriched in the scum fraction but never detected as the dominant microorganism within the biocenosis. The filamentous bacterial morphotypes 1851, 0041/0675, 0092, and Nostocoida limicola were identified only in some WWTPs as the dominant organisms. However, they were often found concomitantly in M. parvicella- and nocardioform actinomycetes-dominated biocenoses. The identification of filamentous bacterial morphotypes by classical microscopy often failed because of morphological and taxonomic variations. Therefore the FISH technique using specific rRNA-targeted oligonucleotide probes was applied to reveal reliable phylogenetic information on these described morphotypes. The effectiveness of the two methods, classical sludge analysis and FISH, for detection, identification, and quantification of filamentous organisms in activated sludge and scum was compared. Classical sludge analysis and FISH corresponded well in the case of filamentous M. parvicella, which was identified as “Candidatus Microthrix parvicella”, an unclassified member of the Actinobacteria. Type 1863 was characterized as Acinetobacter spp. and type 1851 was identified as a member of the Chloroflexi subdivision 3, closely related to Roseiflexus castenholzii. Classical microscopy overlooked nocardioform actinomycetes and type 1863 single cells, which FISH detected only. Furthermore the non-branched filamentous nocardioform actinomycetes growing as short filaments or in cell clusters were clearly identified by FISH only. FISH underestimated branched filamentous nocardioform actinomycetes and morphotypes 0041/0675 or 0092 because of insufficient cell wall permeability for fluorescently labeled rRNA-targeted oligonucleotide probes, and also because of their taxonomic variability and consequently the availability of adequate probes. FISH results for N. limicola morphotypes I and III are still insufficient because of both their low in situ numbers and due to their high taxonomic variability. However, morphotype N. limicola II was frequently identified as “Candidatus Nostocoida limicola” belonging to the Actinobacteria phylum. The identification of the nocardioform actinomycetes at genus and species level was limited by classical microscopy because the different species exhibit mostly the same morphotype. The use of FISH for this purpose was restricted due to the deficit of available genera- and species-specific probes. The application of the full-cycle rRNA approach, combined with nucleic acid fingerprinting methods, restriction fragment length polymorphism (RFLP) and denaturing gradient gel electrophoresis (DGGE), proved to be a successful approach to identify unknown nocardioform actinomycete species in different scum samples. A screening of the clone libraries with Actinobacteria-specific primers followed by RFLP profiling allowed to organize clone libraries into taxonomic groups and the identification of the dominant clones for each library. The analysis of environmental DNA by DGGE and the comparison with DGGE patterns of the dominant clones indicate that the dominant clone insert represented the dominant species within the Actinobacteria phylum of the microbial scum communities analyzed. However, only the sequence analysis of clone inserts followed by probe design and the application of FISH using specific probes for Dietzia spp. and various Rhodococcus spp. (R. erythropolis and probably two new species within the Rhodococcus radiation) verified and quantified the presence of these organisms. FISH quantification revealed that these different Rhodococcus species, characterized by a typical branched filamentous morphotype (GALO), were found in high numbers in the scum fraction originating from German WWTPs. They were often accompanied by Dietzia spp. growing as non-branched short filaments or in cell clusters generally found in lower numbers. In contrast, scum samples in Australian WWTPs were dominated by Gordonia spp. and Skermania piniformis. The occurrence of the described nocardioform actinomycete species could be correlated with different growth conditions in the WWTPs concerning temperature and the availability of specific carbon sources. Physicochemical analyses such as hydrophobicity measurements and EPS characterization combined with FISH quantification of the dominant scum bacteria were performed on sludge samples originating from M. parvicella- and nocardioform actinomycetes-dominated plants with scum events. In addition, respiratory activity was monitored in activated sludge and scum to define growth strategy and ecological niche of M. parvicella and nocardioform actinomycetes. Lipophilic substances originating from the primary effluent and being adsorbed to the activated sludge EPS promoted the growth rate of M. parvicella, which was shown to be more active in activated sludge as compared to scum. The long hydrophobic M. parvicella filaments enlarge and hydrophobize the surface structure of activated sludge flocs causing stable adhesion to gas bubbles. Thus these flocs are assumed to just passively float to the water surface producing persistent scum layers. In contrast, a higher respiratory activity in the scum biocenosis as compared to the activated sludge biocenosis was determined in nocardioform actinomycetes-dominated WWTPs. Therefore nocardioform actinomycetes as was shown for several Rhodococcus species and Dietzia spp. might enhance their population density by actively growing in the floated sludge fraction. This is supported by the low numbers of nocardioform actinomycetes detected in activated sludge and the clear enrichment of these organisms in the scum fraction. The active growth process in the scum layer is genera- and species-dependent, promoted by different scum EPS compounds such as lipophilic substances or other not yet characterized substances in addition to proteins and carbohydrates. Both high numbers of Rhodococcus spp. and lipophilic substances on the water surface caused hydrophobicity and thus scum formation. The described different scum formation strategies lead to various control measures with M. parvicella to be controlled in the activated sludge fraction and nocardioform actinomycetes to be fought in the scum fraction. This study revealed that different nocardioform actinomycete species found in the scum layer require specific growth conditions. Therefore the troubleshooting measures for nocardioform actinomycetes-dominated scum should be based upon a reliable taxonomic identification at genus and species level.