Caroline Kragelund

A conceptual ecosystem model of microbial communities in enhanced biological phosphorus removal plants

The microbial populations in 25 full-scale activated sludge wastewater treatment plants with enhanced biological phosphorus removal (EBPR plants) have been intensively studied over several years. Most of the important bacterial groups involved in nitrification, denitrification, biological P removal, fermentation, and hydrolysis have been identified and quantified using quantitative culture-independent molecular methods. Surprisingly, a limited number of core species was present in all plants, constituting on average approx. 80% of the entire communities in the plants, showing that the microbial populations in EBPR plants are rather similar and not very diverse, as sometimes suggested. By focusing on these organisms it is possible to make a comprehensive ecosystem model, where many important aspects in relation to microbial ecosystems and wastewater treatment can be investigated. We have reviewed the current knowledge about these microorganisms with focus on key ecophysiological factors and combined this into a conceptual ecosystem model for EBPR plants. It includes the major pathways of carbon flow with specific organic substances, the dominant populations involved in the transformations, interspecies interactions, and the key factors controlling their presence and activity. We believe that the EBPR process is a perfect model system for studies of microbial ecology in water engineering systems and that this conceptual model can be used for proposing and testing theories based on microbial ecosystem theories, for the development of new and improved quantitative ecosystem models and is beneficial for future design and management of wastewater treatment systems.

Identity and ecophysiology of filamentous bacteria in activated sludge.

Excessive growth of filamentous bacteria in activated sludge wastewater treatment plants (WWTPs) can cause serious operational problems. With some filaments there may be the problem of bulking, where inadequate flocculation and settling of the biomass in the secondary clarifier results in a carryover of solids with the final treated liquid effluent. Their proliferation often encourages the development of stable foams on the surface of the reactors, and these foams may impact negatively on plant performance and operation. The availability of culture-independent molecular methods now allows us to identify many of the more common filamentous organisms encountered in WWTPs, which are phylogenetically diverse, affiliating to seven separate bacterial phyla. Furthermore, the extensive data published in the past decade on their in situ behaviour from the application of these culture-independent methods have not been summarized or reviewed critically. Hence, here, we attempt to discuss what we now know about their identity, ecophysiology and ecological niches and its practical value in better managing activated sludge processes. Some of this knowledge is already being applied to control and manage full-scale WWTPs better, and the hope is that this review will contribute towards further developments in this field of environmental microbiology.

Link between microbial composition and carbon substrate-uptake preferences in a PHA-storing community

The microbial community of a fermented molasses-fed sequencing batch reactor (SBR) operated under feast and famine conditions for production of polyhydroxyalkanoates (PHAs) was identified and quantified through a 16 S rRNA gene clone library and fluorescence in situ hybridization (FISH). The microbial enrichment was found to be composed of PHA-storing populations (84% of the microbial community), comprising members of the genera Azoarcus, Thauera and Paracoccus. The dominant PHA-storing populations ensured the high functional stability of the system (characterized by high PHA-storage efficiency, up to 60% PHA content). The fermented molasses contained primarily acetate, propionate, butyrate and valerate. The substrate preferences were determined by microautoradiography-FISH and differences in the substrate-uptake capabilities for the various probe-defined populations were found. The results showed that in the presence of multiple substrates, microbial populations specialized in different substrates were selected, thereby co-existing in the SBR by adapting to different niches. Azoarcus and Thauera, primarily consumed acetate and butyrate, respectively. Paracoccus consumed a broader range of substrates and had a higher cell-specific substrate uptake. The relative species composition and their substrate specialization were reflected in the substrate removal rates of different volatile fatty acids in the SBR reactor.

Identity, abundance and ecophysiology of filamentous bacteria belonging to the Bacteroidetes present in activated sludge plants

Filamentous members of the Bacteroidetes are commonly observed in activated sludge samples originating from both municipal and industrial wastewater treatment plants (WWTP), where they occasionally can cause bulking. Several oligonucleotide 16S rRNA-targeted probes were designed to target filaments with a needle-like appearance similar to Haliscomenobacter hydrossis. The design of these probes was based on an isolate and a sequence obtained from a micromanipulated filament. The abundance of filamentous Bacteroidetes was determined in 126 industrial samples applying already published and the newly developed probes. Small populations were found in 62 % of the WWTP investigated. However, only relatively few WWTP (13 %) contained large populations of filamentous Bacteroidetes potentially responsible for bulking incidences. The identity of the most abundant filamentous Bacteroidetes with H. hydrossis morphology could be detected by probes CFB719, SAP-309 and the newly designed probe HHY-654. A comprehensive study on the ecophysiology of probe-defined Bacteroidetes populations was conducted on Danish and Czech samples. The studies revealed that they were specialized bacteria involved in degradation of sugars, e.g. glucose and N-acetylglucosamine, and may participate in the conversion of lipopolysaccharides and peptidoglycan liberated by decaying cells. Many surface-associated exo-enzymes were excreted, e.g. chitinase, glucuronidase, esterase and phosphatase, supporting conversion of polysaccharides and possibly other released cell components. The role of filamentous bacteria with a H. hydrossis-like morphology in the activated sludge ecosystem is discussed.

Eikelboom's morphotype 0803 in activated sludge belongs to the genus Caldilinea in the phylum Chloroflexi

Micromanipulated filamentous bacteria from bulking and foaming activated sludge morphologically identified as Eikelboom type 0803 were shown to be affiliated to the genus Caldilinea within the phylum Chloroflexi. Specific FISH probes were designed for their in situ detection and quantification in seven Danish wastewater treatment plants with biological nutrient removal. The survey applied all species-specific probes for Chloroflexi of relevance in activated sludge treatment plants as well as the phylum-specific probes. Type 0803 filaments constituted around 20% of the total Chloroflexi population. In four of the treatment plants, type 0803 and type 0092 co-occurred and were the dominating fraction of the Chloroflexi population. In the other plants, most Chloroflexi could not be identified beyond the phylum level, suggesting a yet far larger diversity. On average, for all plants, the total Chloroflexi population constituted 12% of the entire microbial population and seems to play an important structural role in the sludge floc formation. Ecophysiological characterization of type 0803 showed their potential role in macromolecule conversion as evident by high levels of exoenzyme expression. Acetate was not consumed. Glucose was consumed with oxygen, nitrite and nitrite as electron acceptors, suggesting that type 0803 may be a denitrifier. Their surfaces were hydrophobic, explaining their occasional occurrence in foaming incidents.

Ecophysiology of the Actinobacteria in activated sludge systems

This review considers what is known about the Actinobacteria in activated sludge systems, their abundance and their functional roles there. Participation in processes leading to the microbiological removal of phosphate and in the operational problems of bulking and foaming are discussed in terms of their ecophysiological traits. We consider critically whether elucidation of their nutritional requirements and other physiological properties allow us to understand better what might affect their survival capabilities in these highly competitive systems. Furthermore, how this information might allow us to improve how these processes work is discussed.

Biodegradation of pharmaceuticals in hospital wastewater by a hybrid biofilm and activated sludge system (Hybas)

Hospital wastewater contributes a significant input of pharmaceuticals into municipal wastewater. The combination of suspended activated sludge and biofilm processes, as stand-alone or as hybrid process (hybrid biofilm and activated sludge system (Hybas™)) has been suggested as a possible solution for hospital wastewater treatment. To investigate the potential of such a hybrid system for the removal of pharmaceuticals in hospital wastewater a pilot plant consisting of a series of one activated sludge reactor, two Hybas™ reactors and one moving bed biofilm reactor (MBBR) has been established and adapted during 10 months of continuous operation. After this adaption phase batch and continuous experiments were performed for the determination of degradation of pharmaceuticals. Removal of organic matter and nitrification mainly occurred in the first reactor. Most pharmaceuticals were removed significantly. The removal of pharmaceuticals (including X-ray contrast media, β-blockers, analgesics and antibiotics) was fitted to a single first-order kinetics degradation function, giving degradation rate constants from 0 to 1.49 h(-1), from 0 to 7.78 × 10(-1)h(-1), from 0 to 7.86 × 10(-1)h(-1) and from 0 to 1.07 × 10(-1)h(-1) for first, second, third and fourth reactors respectively. Generally, the highest removal rate constants were found in the first and third reactors while the lowest were found in the second one. When the removal rate constants were normalized to biomass amount, the last reactor (biofilm only) appeared to have the most effective biomass in respect to removing pharmaceuticals. In the batch experiment, out of 26 compounds, 16 were assessed to degrade more than 20% of the respective pharmaceutical within the Hybas™ train. In the continuous flow experiments, the measured removals were similar to those estimated from the batch experiments, but the concentrations of a few pharmaceuticals appeared to increase during the first treatment step. Such increase could be attributed to de-conjugation or formation from other metabolites.

Filtration properties of activated sludge in municipal MBR wastewater treatment plants are related to microbial community structure

In the conventional activated sludge process, a number of important parameters determining the efficiency of settling and dewatering are often linked to specific groups of bacteria in the sludge--namely floc size, residual turbidity, shear sensitivity and composition of extracellular polymeric substances (EPS). In membrane bioreactors (MBRs) the nature of solids separation at the membrane has much in common with sludge dewaterability but less is known about the effect of specific microbial groups on the sludge characteristics that affect this process. In this study, six full-scale MBR plants were investigated to identify correlations between sludge filterability, sludge characteristics, and microbial community structure. The microbial community structure was described by quantitative fluorescence in situ hybridization and sludge filterability by a low-pressure filtration method. A strong correlation between the degree of flocculation (ratio between floc size and residual turbidity) and sludge filterability at low pressure was found. A good balance between EPS and cations in the sludge correlated with good flocculation, relatively large sludge flocs, and low amounts of small particles and single cells in the bulk phase (measured as residual turbidity), all leading to a good filterability. Floc properties could also be linked to the microbial community structure. Bacterial species forming strong microcolonies such as Nitrospira and Accumulibacter were present in plants with good flocculation and filtration properties, while few strong microcolonies and many filamentous bacteria in the plants correlated with poor flocculation and filtration problems. In conclusion this study extends the hitherto accepted perception that plant operation affects floc properties which affects fouling. Additionally, plant operation also affects species composition, which affects floc properties and in the end fouling propensity.

Removal of pharmaceuticals in conventionally treated wastewater by a polishing moving bed biofilm reactor (MBBR) with intermittent feeding

Previous studies have demonstrated that aerobic moving bed biofilm reactors (MBBRs) remove pharmaceuticals better than activated sludge. Thus we used a MBBR system to polish the effluent of an activated sludge wastewater treatment plant. To overcome that effluent contains insufficient organic matter to sustain enough biomass, the biofilm was intermittently fed with raw wastewater. The capacity of pharmaceutical degradation was investigated by spiking pharmaceuticals. Actual removal during treatment was assessed by sampling the inlets and outlets of reactors. The removal of the majority of pharmaceuticals was enhanced through the intermittent feeding of the MBBR. First-order rate constants for pharmaceutical removal, normalised to biomass, were significantly higher compared to other studies on activated sludge and suspended biofilms, especially for diclofenac, metoprolol and atenolol. Due to the intermittently feeding, degradation of diclofenac occurred with a half-life of only 2.1h and was thus much faster than any hitherto described wastewater bioreactor treatment.

Combination of Fluorescence In Situ Hybridization with Staining Techniques for Cell Viability and Accumulation of PHA and polyP in Microorganisms in Complex Microbial Systems

Fluorescence in situ hybridization (FISH) can be combined with a number of staining techniques to reveal the relationships between the microorganisms and their function in complex microbial systems with a single-cell resolution. In this chapter, we have focused on staining methods for intracellular storage compounds (polyhydroxyalkanoates, polyphosphate) and a measure for cell viability, reduction of the tetrazolium-based redox stain CTC. These protocols are optimized for the study of microorganisms in waste-water treatment (activated sludge and biofilms), but they may also be used with minor modifications in many other ecosystems.