Small temperature differences can improve the performance of mesophilic sludge-based digesters.

OBJECTIVE: To assess the effect of small temperature increases in mesophilic sludge-based digesters in order to develop and evaluate strategies for improving the biogas production in full-scale digesters. RESULTS: Methane production was strongly affected by small temperature differences, and this result was consistent across samples from 15 full-scale digesters. The specific methane yield varied between 42 and 97.5 ml g VS-1 after 15 days of incubation at 35 °C, and improved when increasing the digester temperature to 39 °C. Only a limited quantity of additional gas was required to balance out the cost of heating and a positive energy balance was obtained. Further increases in temperature, in some cases, negatively affected the production when operated at 42 °C compared to 39 °C. CONCLUSIONS: Small temperature increases should be applied to mesophilic sludge-based digesters to optimize the biogas production and is applicable to digesters operated in the lower mesophilic temperature range.

Control of Microthrix parvicella and sludge bulking by ozone in a full-scale WWTP.

Bulking and rising sludge are common problems in wastewater treatment plants (WWTPs) and are primarily caused by increased growth of filamentous bacteria such as Microthrix parvicella. It has a negative impact on sludge settling properties in activated sludge (AS) process, in addition to being responsible for foam formation. Different methods can be used to control sludge bulking. The aim of this study was to evaluate the dosage of on-site generated ozone in the recycled AS flow in a full-scale WWTP having problems caused by M. parvicella. The evaluation of the experiment was assessed by process data, microscopic analysis and microbial screening on the experimental and control line before, during and after the period of ozone dosage. The ozone treatment resulted in decreased abundance of M. parvicella and improved the settling properties, without impairing the overall process performance. Both chemical oxygen demand (COD)- and N-removal were unaffected and the dominant populations involved in nitrification, as analysed by fluorescent in situ hybridization, remained during the experimental period. When the ozone treatment was terminated, the problems with sludge bulking reappeared, indicating the importance of continuous evaluation of the process.

High and stable substrate specificities of microorganisms in enhanced biological phosphorus removal plants.

Microbial communities are typically characterized by conditions of nutrient limitation so the availability of the resources is likely a key factor in the niche differentiation across all species and in the regulation of the community structure. In this study we have investigated whether four species exhibit any in situ short-term changes in substrate uptake pattern when exposed to variations in substrate and growth conditions. Microautoradiography was combined with fluorescence in situ hybridization to investigate in situ cell-specific substrate uptake profiles of four probe-defined coexisting species in a wastewater treatment plant with enhanced biological phosphorus removal. These were the filamentous 'Candidatus Microthrix' and Caldilinea (type 0803), the polyphosphate-accumulating organism 'Candidatus Accumulibacter', and the denitrifying Azoarcus. The experimental conditions mimicked the conditions potentially encountered in the respective environment (starvation, high/low substrate concentration, induction with specific substrates, and single/multiple substrates). The results showed that each probe-defined species exhibited very distinct and constant substrate uptake profile in time and space, which hardly changed under any of the conditions tested. Such niche partitioning implies that a significant change in substrate composition will be reflected in a changed community structure rather than the substrate uptake response from the different species.

Impact of intermittent feeding on polishing of micropollutants by moving bed biofilm reactors (MBBR).

Moving bed biofilm reactors (MBBRs) were placed at two wastewater treatment plants, where they were constantly fed with effluent and intermittently fed with primary wastewater. Each reactor was subjected to different feast/famine periods and flow rates of primary wastewater, thus the different organic and nutrient loads (chemical oxygen demand(COD), ammonium(NH4-N)) resulted in different feast-famine conditions applied to the biomass. In batch experiments, this study investigated the effects of various feast-famine conditions on the biodegradation of micropollutants by MBBRs applied as an effluent polishing step. Rate constants of micropollutant removals were found to be positively correlated to the load of the total COD and NH4-N, indicating that higher organic loads were favourable for the growth of micropollutant degraders in these MBBRs. Rate constant of atenolol was five times higher when the biomass was fed with the highest COD and NH4-N load than it was fed with the lowest COD and NH4-N load. For diclofenac, mycophenolic acid and iohexol, their maximum rate constants were obtained with feeding of COD and NH4-N of approximately 570 mgCOD/d and 40∼60 mgNH4-N/d respectively. This also supports the concept that co-metabolism (rather competition inhibition or catabolic repression) plays an important role in micropollutants biodegradation in wastewater.

Cellulolytic and Xylanolytic Microbial Communities Associated With Lignocellulose-Rich Wheat Straw Degradation in Anaerobic Digestion.

The enzymatic hydrolysis of lignocellulosic polymers is generally considered the rate-limiting step to methane production in anaerobic digestion of lignocellulosic biomass. The present study aimed to investigate how the hydrolytic microbial communities of three different types of anaerobic digesters adapted to lignocellulose-rich wheat straw in continuous stirred tank reactors operated for 134 days. Cellulase and xylanase activities were monitored weekly using fluorescently-labeled model substrates and the enzymatic profiles were correlated with changes in microbial community compositions based on 16S rRNA gene amplicon sequencing to identify key species involved in lignocellulose degradation. The enzymatic activity profiles and microbial community changes revealed reactor-specific adaption of phylogenetically different hydrolytic communities. The enzymatic activities correlated significantly with changes in specific taxonomic groups, including representatives of Ruminiclostridium, Caldicoprobacter, Ruminofilibacter, Ruminococcaceae, Treponema, and Clostridia order MBA03, all of which have been linked to cellulolytic and xylanolytic activity in the literature. By identifying microorganisms with similar development as the cellulase and xylanase activities, the proposed correlation method constitutes a promising approach for deciphering essential cellulolytic and xylanolytic microbial groups for anaerobic digestion of lignocellulosic biomass.

Municipal wastewater treatment targeting pharmaceuticals by a pilot-scale hybrid attached biofilm and activated sludge system (Hybas™).

A hybrid wastewater treatment process with combined attached biofilm (moving bed biofilm reactor) and activated sludge, named as Hybas™, was implemented for the treatment of municipal wastewater. The system consisted of six staged reactors in series including pre-denitrification and nitrification in the Hybas™ line and post-denitrification in a pure MBBR. In addition to the significant removal of nutrients and organic matter from municipal wastewater, Hybas™ also showed removal capacity for pharmaceuticals. Of particular interest was the enhanced removal for pharmaceuticals (i.e. X-ray contrast media) compared to other biological systems. Spiking experiments showed that the maximum removal rate constants (k, h-1) for 10 out of the 21 investigated pharmaceuticals (including diclofenac) were observed to occur within the two aerobic Hybas ™ reactors, operated in a flow-shifting mode that allows even biofilm growth of nitrifying bacteria. In total, 14 out of the 21 pharmaceuticals were removed by more than 50% during continuous flow operation in the all Hybas™ line and post-denitrification MBBR. The calculated and estimated removal contributions of pharmaceuticals by each individual reactor were also assessed.

Biological removal of pharmaceuticals from hospital wastewater in a pilot-scale staged moving bed biofilm reactor (MBBR) utilising nitrifying and denitrifying processes.

Hospital wastewater contains high concentrations of pharmaceuticals, which pose risks to receiving waters. In this study, a pilot plant consisting of six moving bed biofilm reactors (MBBRs) in series (with the intention to integrate Biological Oxygen Demand (BOD) removal, nitrification and denitrification as well as prepolishing Chemical Oxygen Demand (COD) for ozonation) was built to integrate pharmaceutical removal and intermittent feeding of the latter reactors aimed for micropollutant removal. Based on the experimental resultss, nitrifying MBBRs achieved higher removal as compared to denitrifying MBBRs except for azithromycin, clarithromycin, diatrizoic acid, propranolol and trimethoprim. In the batch experiments, nitrifying MBBRs showed the ability to remove most of the analysed pharmaceuticals, with degradation rate constants ranging from 5.0 × 10-3 h-1 to 2.6 h-1. In general, the highest degradation rate constants were observed in the nitrifying MBBRs while the latter MBBRs showed lower degradation rate constant. However, when the degradation rate constants were normalised to the respective biomass, the intermittently fed reactors presented the highest specific activity. Out of the 22 compounds studied, 17 compounds were removed with more than 20%.

Ecophysiology of mycolic acid-containing Actinobacteria (Mycolata) in activated sludge foams.

Increasing incidences of activated sludge foaming have been reported in the last decade in Danish plants treating both municipal and industrial wastewaters. In most cases, foaming is caused by the presence of Actinobacteria; branched mycolic acid-containing filaments (the Mycolata) and the unbranched Candidatus'Microthix parvicella'. Surveys from wastewater treatment plants revealed that the Mycolata were the dominant filamentous bacteria in the foam. Gordonia amarae-like organisms and those with the morphology of Skermania piniformis were frequently observed, and they often coexisted. Their identity was confirmed by FISH, using a new permeabilization procedure. It was not possible to identify all abundant Mycolata using existing FISH probes, which suggests the presence of currently undetectable and potentially undescribed populations. Furthermore, some Mycolata failed to give any FISH signal, although substrate uptake experiments with microautoradiography revealed that they were physiologically active. Ecophysiological studies were performed on the Mycolata identified by their morphology or FISH in both foams and mixed liquors. Large differences were seen among the Mycolata in levels of substrate assimilation and substrate uptake abilities in the presence of different electron acceptors. These differences were ascribed mainly to the presence of currently undescribed Mycolata species and/or differences in foam age.

Identity, abundance and ecophysiology of filamentous Chloroflexi species present in activated sludge treatment plants.

Filamentous Chloroflexi species are often present in activated sludge wastewater treatment plants in relatively low numbers, although bulking incidences caused by Chloroflexi filaments have been observed. A new species-specific gene probe for FISH was designed and using phylum-, subdivision-, morphotype 1851- and species-specific gene probes, the abundance of Chloroflexi filaments were monitored in samples from 126 industrial wastewater treatment plants from five European countries. Chloroflexi filaments were present in 50% of the samples, although in low quantities. In most treatment plants the filaments could only be identified with phylum or subdivision probes, indicating the presence of great undescribed biodiversity. The ecophysiology of various Chloroflexi filaments was investigated by a suite of in situ methods. The experiments revealed that Chloroflexi constituted a specialized group of filamentous bacteria only active under aerobic conditions consuming primarily carbohydrates. Many exo-enzymes were excreted, e.g. chitinase, glucuronidase and galactosidase, suggesting growth on complex polysaccharides. The surface of Chloroflexi filaments appeared to be hydrophilic compared to other filaments present. These results are generally supported by physiological studies of two new isolates. Based on the results obtained in this study, the potential role of filamentous Chloroflexi species in activated sludge is discussed.

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.

Ecophysiology of the filamentous Alphaproteobacterium Meganema perideroedes in activated sludge.

A comprehensive study of the ecophysiology of the filamentous Meganema perideroedes affiliated to the Alphaproteobacteria, possessing a "Nostocoida limicola Type II" filamentous morphology was conducted. This morphotype often causes serious bulking problems in activated sludge wastewater treatment plants, and hardly anything is known about its physiology. The study was carried out by applying a suite of in situ methods in an industrial activated sludge treatment plant with excessive growth of this species. The experiments revealed a very versatile organism able to take up a large variety of organic substrates under aerobic conditions. It had a remarkably high storage capacity forming polyhydroxyalkanoates from most substrates tested. When nitrate was present as e-acceptor, the number of substrates to be consumed by M. perideroedes was more restricted compared to aerobic conditions. With nitrite as e-acceptor, only acetate and glucose among the substrates tested could be assimilated and used for storage and possibly growth. This indicated that M. perideroedes might be able to denitrify under certain conditions, which is unusual for filamentous bacteria in activated sludge. No substrate uptake or storage was seen under anaerobic conditions. M. perideroedes was relatively hydrophobic, compared to other filamentous bacteria and microcolonies present in the sludge, indicating the presence of a hydrophobic sheath. Several excreted surface-associated exoenzymes were detected in the sludge, but M. perideroedes never showed any activity, except once after a breakdown in the production facility. This confirmed that M. perideroedes mainly grows on soluble substrates. Based on the studies of the ecophysiology of M. perideroedes, potential control strategies are suggested.

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, ß-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.

Biodegradation of pharmaceuticals in hospital wastewater by staged Moving Bed Biofilm Reactors (MBBR).

Hospital wastewater represents a significant input of pharmaceuticals into municipal wastewater. As Moving Bed Biofilm Reactors (MBBRs) appear to remove organic micro-pollutants, hospital wastewater was treated with a pilot plant consisting of three MBBRs in series. The removal of pharmaceuticals was studied in two experiments: 1) A batch experiment where pharmaceuticals were spiked to each reactor and 2) a continuous flow experiment at native concentrations. DOC removal, nitrification as well as removal of pharmaceuticals (including X-ray contrast media, ß-blockers, analgesics and antibiotics) occurred mainly in the first reactor. In the batch experiment most of the compounds followed a single first-order kinetics degradation function, giving degradation rate constants ranged from 5.77 × 10(-3) to 4.07 h(-1), from -5.53 × 10(-3) to 9.24 × 10(-1) h(-1) and from 1.83 × 10(-3) to 2.42 × 10(-1) h(-1) for first, second and third reactor respectively. Generally, the highest removal rate constants were found in the first reactor while the lowest were found in the third one. This order was inverted for most compounds, when the removal rate constants were normalized to biomass, indicating that the last tank had the most effective biofilms. In the batch experiment, 21 out of 26 compounds were assessed to be degraded with more than 20% within the MBBR train. In the continuous flow experiment the measured removal rates were lower than those estimated from the batch experiments.

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.

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.

Population dynamics of filamentous bacteria in Danish wastewater treatment plants with nutrient removal.

Bulking and foaming are two frequently occurring operational problems in activated sludge wastewater treatment plants, and these problems are mainly associated with excessive growth of filamentous bacteria. In this study, a comprehensive investigation of the identity and population dynamics of filamentous bacteria in 28 Danish municipal treatment plants with nutrient removal has been carried out over three years. Fluorescence in situ hybridization was applied to quantify more than twenty probe-defined populations of filamentous bacteria that in total constituted a large fraction of the entire microbial community, on average 24%. Despite the majority being present within the flocs, they occasionally caused settling problems in most of the plants. A low diversity of probe-defined filamentous bacteria was found in the plants with Microthrix and various species belonging to phylum Chloroflexi (e.g., type 0803 and type 0092) as the most abundant. Few other filamentous probe-defined species were found revealing a large similarity between the filamentous populations in the plants investigated. The composition of filamentous populations was stable in each plant with only minor changes in relative abundances observed during the three-year study period. The relative composition of the different species was unique to each plant giving a characteristic "fingerprint". Comprehensive statistical analyses of the presence and abundance of the filamentous organisms did not reveal many correlations with a particular plant design or process parameter.

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.

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.

Ecophysiological analysis of microorganisms in complex microbial systems by combination of fluorescence in situ hybridization with extracellular staining techniques.

Ecophysiological analysis and functions of single cells in complex microbial systems can be examined by simple combinations of Fluorescence in situ hybridization (FISH) for identification with various staining techniques targeting functional phenotypes. In this chapter, we describe methods and protocols optimized for the study of extracellular enzymes, surface hydrophobicity and specific surface structures. Although primarily applied to the study of microbes in wastewater treatment (activated sludge and biofilms), the methods may also be used with minor modifications in several other ecosystems.

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.