Particle size as a driver of dewatering performance and its relationship to stabilization in fecal sludge.

Poor and unpredictable dewatering performance of fecal sludge is a major barrier to sanitation provision in urban areas not served by sewers. Fecal sludge comprises everything that accumulates in onsite containments, and its characteristics are distinct from wastewater sludges and from feces. There is little fundamental understanding of what causes poor dewatering in fecal sludge. For the first time, we demonstrate that particle size distribution is a driver of dewatering performance in fecal sludge, and is associated with level of stabilization. Higher concentrations of small particles (<10 µm) and smaller median aggregate size (D50) corresponded to poor dewatering performance (measured by capillary suction time (CST) and supernatant turbidity) in field samples from Kenya and Uganda and in controlled laboratory anaerobic storage experiments. More stabilized fecal sludge (higher C/N, lower VSS/TSS) had better dewatering performance, corresponding to lower concentrations of small particles. Samples with the largest aggregates (D50 > 90 µm) had higher abundance of Gammaproteobacteria Pseudomonas, and samples with the smallest aggregates (D50 ≤ 50 µm) were characterized by higher abundance of Bacteroidetes Vadin HA17 and Rikenellaceae. Contrary to common perceptions, stabilization, particle size distribution, and dewatering performance were not dependent on time intervals between emptying of onsite containments or on time in controlled anaerobic storage experiments. Our results suggest that the stabilization process in onsite containments, and hence the dewaterability of sludge arriving at treatment facilities, is not dependent on time in containment but is more likely associated with specific microbial populations and the in-situ environmental conditions which promote or discourage their growth.

Successful year-round mainstream partial nitritation anammox: Assessment of effluent quality, performance and N2O emissions.

For two decades now, partial nitritation anammox (PNA) systems were suggested to more efficiently remove nitrogen (N) from mainstream municipal wastewater. Yet to date, only a few pilot-scale systems and even fewer full-scale implementations of this technology have been described. Process instability continues to restrict the broad application of PNA. Especially problematic are insufficient anammox biomass retention, the growth of undesired aerobic nitrite-oxidizers, and nitrous oxide (N2O) emissions. In this study, a two-stage mainstream pilot-scale PNA system, consisting of three reactors (carbon pre-treatment, nitritation, anammox - 8 m3 each), was operated over a year, treating municipal wastewater. The aim was to test whether both, robust autotrophic N removal and high effluent quality, can be achieved throughout the year. A second aim was to better understand rate limiting processes, potentially affecting the overall performance of PNA systems. In this pilot study, excellent effluent quality, in terms of inorganic nitrogen, was accomplished (average effluent concentrations: 0.4 mgNH4-N/L, 0.1 mgNO2-N/L, 0.9 mgNO3-N/L) even at wastewater temperatures previously considered problematic (as low as 8 °C). N removal was limited by nitritation rates (84 ± 43 mgNH4-N/L/d), while surplus anammox activity was observed at all times (178 ± 43 mgN/L/d). Throughout the study, nitrite-oxidation was maintained at a low level (<2.5% of ammonium consumption rate). Unfortunately, high N2O emissions from the nitritation stage (1.2% of total nitrogen in the influent) were observed, and, based on natural isotope abundance measurements, could be attributed to heterotrophic denitrification. In situ batch experiments were conducted to identify the role of dissolved oxygen (DO) and organic substrate availability in N2O emission-mitigation. The addition of organic substrate, to promote complete denitrification, was not successful in decreasing N2O emission, but increasing the DO from 0.3 to 2.9 mgO2/L decreased N2O emissions by a factor of 3.4.

Successful mainstream nitritation through NOB inactivation.

The development of new wastewater treatment processes can assist in reducing the impact of wastewater treatment on the environment. The recently developed partial nitritation anammox (PNA) process, for example, consumes less energy for aeration and reduces nitrate in the effluent without requiring additional organic carbon. However, achieving stable nitritation (ammonium oxidation; NH4+ → NO2-) at mainstream conditions (T = 10-25 °C, C:N > 10, influent ammonium < 50 mgNH4-N/L and effluent < 1 mgNH4-N/L) remains challenging. This study explores the potential and mechanism of nitrite-oxidizing bacteria (NOB) suppression in a bottom-fed sequencing batch reactor (SBR). Two bench-scale (11 L) reactors and a pilot-scale reactor (8 m3) were operated for over a year and were fed with organic substrate depleted municipal wastewater. Initially, nitratation (nitrite oxidation; NO2- → NO3-) occurred occasionally until an anaerobic phase was integrated into the operating cycle. The introduction of the anaerobic phase effectively suppressed the regrowth of NOB while nitritation was stable over 300 days, down to 8 °C and at ammonium influent concentrations < 25 mgNH4-N/L. Batch experiments and process data revealed that parameters typically affecting NOB growth (e.g., dissolved oxygen, alkalinity, trace elements, lag-phase after anoxia, free nitrous acid (FNA), free ammonia (FA), pH, sulfide, or solids retention time (SRT)) could not fully explain the suppression of nitratation. Experiments in which fresh nitrifying microbial biomass was added to the nitritation system indicated that NOB inactivation explained NOB suppression better than NOB washout at high SRT. This study concludes that bottom-fed SBRs with anaerobic phases allow for stable nitritation over a broad range of operational parameters. Coupling this type of SBR to an anammox reactor can enable efficient mainstream anammox-based wastewater treatment.

Modulating medial prefrontal cortex activity using real-time fMRI neurofeedback: Effects on reality monitoring performance and associated functional connectivity.

Neuroimaging studies have found 'reality monitoring', our ability to distinguish internally generated experiences from those derived from the external world, to be associated with activity in the medial prefrontal cortex (mPFC) of the brain. Here we probe the functional underpinning of this ability using real-time fMRI neurofeedback to investigate the involvement of mPFC in recollection of the source of self-generated information. Thirty-nine healthy individuals underwent neurofeedback training in a between groups study receiving either Active feedback derived from the paracingulate region of the mPFC (21 subjects) or Sham feedback based on a similar level of randomised signal (18 subjects). Compared to those in the Sham group, participants receiving Active signal showed increased mPFC activity over the course of three real-time neurofeedback training runs undertaken in a single scanning session. Analysis of resting state functional connectivity associated with changes in reality monitoring accuracy following Active neurofeedback revealed increased connectivity between dorsolateral frontal regions of the fronto-parietal network (FPN) and the mPFC region of the default mode network (DMN), together with reduced connectivity within ventral regions of the FPN itself. However, only a trend effect was observed in the interaction of the recollection of the source of Imagined information compared with recognition memory between participants receiving Active and Sham neurofeedback, pre- and post- scanning. As such, these findings demonstrate that neurofeedback can be used to modulate mPFC activity and increase cooperation between the FPN and DMN, but the effects on reality monitoring performance are less clear.

Particulate substrate retention in plug-flow and fully-mixed conditions during operation of aerobic granular sludge systems.

Particulate substrate (XB) is the major organic substrate fraction in most municipal wastewaters. However, the impact of XB on aerobic granular sludge (AGS) systems is not fully understood. This study evaluated the physical retention of XB in AGS sequencing batch reactor (SBR) during anaerobic plug-flow and then aerobic fully-mixed conditions. The influence of different sludge types and operational variables on the extent and mechanisms of XB retention in AGS SBR were evaluated. XB mass-balancing and magnetic resonance imaging (MRI) were applied. During the anaerobic plug-flow feeding, most XB was retained in the first few cm of the settled sludge bed within the interstitial voids, where XB settled and accumulated ultimately resulting in the formation of a filter-cake. Sedimentation and surface filtration were thus the dominant XB retention mechanisms during plug-flow conditions, indicating that contact and attachment of XB to the biomass was limited. XB retention was variable and influenced by the XB influent concentration, sludge bed composition and upflow feeding velocity (vww). XB retention increased with larger XB influent concentrations and lower vww, which demonstrated the importance of sedimentation on XB retention during plug-flow conditions. Hence, large fractions of influent XB likely re-suspended during aerobic fully-mixed conditions, where XB then preferentially and rapidly attached to the flocs. During fully-mixed conditions, increasing floc fractions, longer mixing times and larger XB concentrations increased XB retention. Elevated XB retention was observed after short mixing times < 60 min when flocs were present, and the contribution of flocs towards XB retention was even more pronounced for short mixing times < 5 min. Overall, our results suggest that flocs occupy an environmental niche that results from the availability of XB during aerobic fully-mixed conditions of AGS SBR. Therefore, a complete wash-out of flocs is not desirable in AGS systems treating municipal wastewater.

Organic substrate diffusibility governs microbial community composition, nutrient removal performance and kinetics of granulation of aerobic granular sludge.

Basic understanding of formation of aerobic granular sludge (AGS) has mainly been derived from lab-scale systems with simple influents containing only highly diffusible volatile fatty acids (VFA) as organic substrate. This study compares start-up of AGS systems fed by different synthetic and municipal wastewaters (WW), characterised by increasing complexity in terms of non-diffusible organic substrate. Four AGS reactors were started with the same inoculum activated sludge and operated for one year. The development of AGS, settling characteristics, nutrient and substrate removal performance as well as microbial community composition were monitored. Our results indicate that the higher the content of diffusible organic substrate in the WW, the faster the formation of AGS. The presence of non-diffusible organic substrate in the influent WW led to the formation of small granules and to the presence of 20-40% (% of total suspended solids) of flocs in the AGS. When AGS was fed with complex influent WW, the classical phosphorus and glycogen accumulating organisms (PAO, GAO) were outcompeted by their fermentative equivalents. Substrate and nutrient removal was observed in all reactors, despite the difference in physical and settling properties of the AGS, but the levels of P and N removal depended on the influent carbon composition. Mechanistically, our results indicate that increased levels of non-diffusible organic substrate in the influent lower the potential for microbial growth deep inside the granules. Additionally, non-diffusible organic substrates give a competitive advantage to the main opponents of AGS formation - ordinary heterotrophic organisms (OHO). Both of these mechanisms are suspected to limit AGS formation. The presented study has relevant implications for both practice and research. Start-up duration of AGS systems treating high complexity WW were one order of magnitude higher than a typical lab-scale system treating VFA-rich synthetic WW, and biomass as flocs persisted as a significant fraction. Finally, the complex synthetic influent WW - composed of VFA, soluble fermentable and particulate substrate - tested here seems to be a more adequate surrogate of real municipal WW for laboratory studies than 100%-VFA WW.

Pore-Scale Hydrodynamics in a Progressively Bioclogged Three-Dimensional Porous Medium: 3-D Particle Tracking Experiments and Stochastic Transport Modeling.

Biofilms are ubiquitous bacterial communities that grow in various porous media including soils, trickling, and sand filters. In these environments, they play a central role in services ranging from degradation of pollutants to water purification. Biofilms dynamically change the pore structure of the medium through selective clogging of pores, a process known as bioclogging. This affects how solutes are transported and spread through the porous matrix, but the temporal changes to transport behavior during bioclogging are not well understood. To address this uncertainty, we experimentally study the hydrodynamic changes of a transparent 3-D porous medium as it experiences progressive bioclogging. Statistical analyses of the system's hydrodynamics at four time points of bioclogging (0, 24, 36, and 48 h in the exponential growth phase) reveal exponential increases in both average and variance of the flow velocity, as well as its correlation length. Measurements for spreading, as mean-squared displacements, are found to be non-Fickian and more intensely superdiffusive with progressive bioclogging, indicating the formation of preferential flow pathways and stagnation zones. A gamma distribution describes well the Lagrangian velocity distributions and provides parameters that quantify changes to the flow, which evolves from a parallel pore arrangement under unclogged conditions, toward a more serial arrangement with increasing clogging. Exponentially evolving hydrodynamic metrics agree with an exponential bacterial growth phase and are used to parameterize a correlated continuous time random walk model with a stochastic velocity relaxation. The model accurately reproduces transport observations and can be used to resolve transport behavior at intermediate time points within the exponential growth phase considered.

Comparing the Resistance, Resilience, and Stability of Replicate Moving Bed Biofilm and Suspended Growth Combined Nitritation-Anammox Reactors.

Combined partial nitritation-anammox (PN/A) systems are increasingly being employed for sustainable removal of nitrogen from wastewater, but process instabilities present ongoing challenges for practitioners. The goal of this study was to elucidate differences in process stability between PN/A process variations employing two distinct aggregate types: biofilm [in moving bed biofilm reactors (MBBRs)] and suspended growth biomass. Triplicate reactors for each process variation were studied under baseline conditions and in response to a series of transient perturbations. MBBRs displayed elevated NH4+ removal rates relative to those of suspended growth counterparts over six months of unperturbed baseline operation but also exhibited significantly greater variability in performance. Transient perturbations led to strikingly divergent yet reproducible behavior in biofilm versus suspended growth systems. A temperature perturbation prompted a sharp reduction in NH4+ removal rates with no accumulation of NO2- and rapid recovery in MBBRs, compared to a similar reduction in NH4+ removal rates but a high level of accumulation of NO2- in suspended growth reactors. Pulse additions of a nitrification inhibitor (allylthiourea) prompted only moderate declines in performance in suspended growth reactors compared to sharp decreases in NH4+ removal rates in MBBRs. Quantitative fluorescence in situ hybridization demonstrated a significant enrichment of anammox in MBBRs compared to suspended growth reactors, and conversely a proportionally higher AOB abundance in suspended growth reactors. Overall, MBBRs displayed significantly increased susceptibility to transient perturbations employed in this study compared to that of suspended growth counterparts (stability parameter), including significantly longer recovery times (resilience). No significant difference in the maximal impact of perturbations (resistance) was apparent. Taken together, our results suggest that aggregate architecture (biofilm vs suspended growth) in PN/A processes exerts an unexpectedly strong influence on process stability.

Biofilm increases permeate quality by organic carbon degradation in low pressure ultrafiltration.

We investigated the influence of biofouling of ultrafiltration membranes on the removal of organic model foulants and ultimately on the quality of permeate. Gravity Driven Membrane ultrafiltration (GDM) membrane systems were operated with modified river water during five weeks without control of the biofilm formation. Three GDM systems were studied: two systems with biofilms exposed to (A) variable or (B) constant load of organic foulants, and (C) one system operated without biofilm and exposed to constant foulant loading. Biodegradable dextran or non-biodegradable polystyrene sulfonate model foulants were tested. Substrate biodegradability was confirmed by Size Exclusion Chromatography (SEC) and by degradation batch tests (D). The GDM systems (A) and (B) were fed with pre-filtered river water supplemented with dextran (Dex) of 1, 150 or 2000 kDa, or polystyrene sulfonate (PSS) of 1 or 80 kDa at concentrations of 2-3.5 mgC L(-1). In exp. (C) the feed water consisted of deionized water with 25 mgC L(-1) of either PSS 1, 80 kDa or Dex 2000 kDa. The biofilm formation on UF membrane surfaces controlled the foulant permeation and thus the permeate quality. Biofilms exposed to continuous foulant loading (exp. B) degraded low molecular weight (LMW) biodegradable foulants (1 kDa Dex), which improved the permeate quality. For high molecular weight (HMW) substrates (150, 2000 kDa Dex), the improvement of the permeate quality was observed after 7 days of biofilm formation, and resulted from the foulant hydrolysis followed by degradation. For non-biodegradable foulants, an improvement of 20% of the retention was observed for the polystyrene (1, 80 kDa PSS) due to the presence of biofilms on membrane surfaces. For variable foulant loading (exp. A) the biofilms hydrolysed the large biodegradable foulants but did not degraded them fully, which resulted a deterioration of the permeate quality (except for the LMW dextran (1 kDa) that was fully degraded). Overall, the "biofilm + membrane" composite retained a larger amount of biodegradable foulant than the membrane alone, due to the activity of the biofilm. However, this resulted in an increased biofilm accumulation and reduced flux. In presence of the biofilm, the highest fluxes were observed for control (no foulant) and for small non-biodegradable foulants (PSS 1 kDa). Low fluxes were observed for the accumulating on membrane surface or degradable foulants (exp. B). But, the lowest fluxes were observed in absence of the biofilm (exp. C) due to physical accumulation of the foulants (PSS 80 kDa and Dextran 2000 kDa). Overall our study demonstrates that the presence of biofilms on membrane surfaces has some benefits: (i) biofilm helps to increase the permeate quality and (ii) biofilms protect the membrane from further fouling. Permeate flux stabilizes in the case of biofilm-membrane composite, while it continuously declines in the case of the membrane only.

Impact of coexistence of flocs and biofilm on performance of combined nitritation-anammox granular sludge reactors.

Nitrogen (N) removal from high-strength wastewater can be accomplished in single-stage combined nitritation-anammox reactors with suspended growth biomass composed of floccular sludge, granular sludge, or of any mix of these 2 different sludge fractions. To date, the influence of floccular biomass on granular sludge reactor performance and stability has not been investigated experimentally or numerically. To address this knowledge gap, two 1D multi-species models were developed in Aquasim to assess the importance of small levels of flocs in putatively granular sludge combined nitritation-anammox reactors for different bulk oxygen concentrations and organics loads. The models included the growth and decay of aerobic ammonium-oxidizing organism (AOO), nitrite-oxidizing organisms (NOO), heterotrophic organisms (OHO), and anammox organisms (AMO) in exclusively granular sludge reactors, and in granular sludge reactors with small levels (∼5% of total biomass) of flocs. While maximum N removal efficiencies were similar for both model structures, floc addition led to a lower optimal dissolved oxygen concentration (DO) as well as a narrower maximum N removal peak, suggesting that small levels of floccular material may decrease process robustness to bulk oxygen changes. For some DO levels, this led to drastic efficiency drops. Furthermore, floc addition also led to substantial segregation in activity and microbial population distribution, with AOO, NOO and OHO concentrated in flocs and AMO concentrated in granules. Increased organic loading (COD:N = 4:3) improved maximum N removal efficiency in both model structures, but yielded substantially different predictions for optimal DO setpoint and process robustness to variations in DO. Taken together, our results indicate that even small levels of floccular biomass in biofilm reactors can have profound implications for reactor performance and optimization and for segregation of linked microbial processes, and suggest that the common practice of neglecting small levels of floccular material in biofilm models and in practice may lead to erroneous predictions.

Inorganic particles increase biofilm heterogeneity and enhance permeate flux.

This study investigated the influence of inorganic particles on the hydraulic resistance of biofilm grown on membrane surface during low-pressure dead-end ultrafiltration. Gravity-driven ultrafiltration membrane systems were operated during several weeks without any flushing or cleaning. Smaller (kaolin d0.5 = 3.6 µm) or larger (kaolin with diatomaceous earth 50/50%, d0.5 = 18.1 µm) particles were added to pre-filtered creek water or to unfiltered creek water. It was demonstrated in both experiments that presence of finer particles in the feed water (kaolin) induced formation of compact and homogeneous biofilm structure. On the other hand presence of the larger particles (diatomite) helped to counterbalance the effect of fine particles due to the formation of more heterogeneous and permeable biofilm structure. The hydraulic resistance of biofilms formed with fine particles was significantly higher than the resistance of biofilm formed in (1) absence of any inorganic particles or (2) in presence of the mixed particle population. The membrane orientation (vertical or horizontal) determined which particles were accumulating at the membrane surface, with structural differences shown by Scanning Electron Microscopy (SEM). For vertical membranes, the larger particles were selectively removed due to sedimentation and did not contribute to the biofilm development. Thus the selection of smaller particles due to vertical membrane configuration negatively affected the biofilm structure and permeation rates, and such selective accumulation of fine particles should be avoided.

Systematic evaluation of biofilm models for engineering practice: components and critical assumptions.

Biofilm models are valuable tools for the design and evaluation of biofilm-based processes despite several uncertainties including the dynamics and rate of biofilm detachment, concentration gradients external to the biofilm surface, and undefined biofilm reactor model calibration protocol. The present investigation serves to (1) systematically evaluate critical biofilm model assumptions and components and (2) conduct a sensitivity analysis with the aim of identifying parameter subsets for biofilm reactor model calibration. AQUASIM was used to describe submerged-completely mixed combined carbon oxidation and nitrification IFAS and MBBR systems, and tertiary nitrification and denitrification MBBRs. The influence of uncertainties in model parameters on relevant model outputs was determined for simulated scenarios by means of a local sensitivity analysis. To obtain reasonable simulation results for partially penetrated biofilms that accumulated a substantial thickness in the modelled biofilm reactor (e.g. 1,000 microm), an appropriate biofilm discretization was applied to properly model soluble substrate concentration gradients and, consistent with the assumed mechanism for describing biofilm biomass distribution, biofilm biomass spatial variability. The MTBL thickness had a significant impact on model results for each of the modelled reactor configurations. Further research is needed to develop a mathematical description (empirical or otherwise) of the MTBL thickness that is relevant to modern biofilm reactors. No simple recommendations for a generally applicable calibration protocol are provided, but sensitivity analysis has been proven to be a powerful tool for the identification of highly sensitive parameter subsets for biofilm (reactor) model calibration.

Syntrophic acetate oxidation in two-phase (acid-methane) anaerobic digesters.

The microbial processes involved in two-phase anaerobic digestion were investigated by operating a laboratory-scale acid-phase (AP) reactor and analyzing two full-scale, two-phase anaerobic digesters operated under mesophilic (35 °C) conditions. The digesters received a blend of primary sludge and waste activated sludge (WAS). Methane levels of 20% in the laboratory-scale reactor indicated the presence of methanogenic activity in the AP. A phylogenetic analysis of an archaeal 16S rRNA gene clone library of one of the full-scale AP digesters showed that 82% and 5% of the clones were affiliated with the orders Methanobacteriales and Methanosarcinales, respectively. These results indicate that substantial levels of aceticlastic methanogens (order Methanosarcinales) were not maintained at the low solids retention times and acidic conditions (pH 5.2-5.5) of the AP, and that methanogenesis was carried out by hydrogen-utilizing methanogens of the order Methanobacteriales. Approximately 43, 31, and 9% of the archaeal clones from the methanogenic phase (MP) digester were affiliated with the orders Methanosarcinales, Methanomicrobiales, and Methanobacteriales, respectively. A phylogenetic analysis of a bacterial 16S rRNA gene clone library suggested the presence of acetate-oxidizing bacteria (close relatives of Thermacetogenium phaeum, 'Syntrophaceticus schinkii,' and Clostridium ultunense). The high abundance of hydrogen consuming methanogens and the presence of known acetate-oxidizing bacteria suggest that acetate utilization by acetate oxidizing bacteria in syntrophic interaction with hydrogen-utilizing methanogens was an important pathway in the second-stage of the two-phase digestion, which was operated at high ammonium-N concentrations (1.0 and 1.4 g/L). A modified version of the IWA Anaerobic Digestion Model No. 1 (ADM1) with extensions for syntrophic acetate oxidation and weak-acid inhibition adequately described the dynamic profiles of volatile acid production/degradation and methane generation observed in the laboratory-scale AP reactor. The model was validated with historical data from the full-scale digesters.

Mathematical modelling of biofilms and biofilm reactors for engineering design.

Mathematical models are critical to modern environmental biotechnology-both in research and in the engineering practice. Wastewater treatment plant (WWTP) simulators are used by consulting engineers and WWTP operators when planning, designing, optimizing, and evaluating the unit processes that comprise municipal and industrial WWTPs. Many WWTP simulators have been expanded to include a submerged completely-mixed biofilm reactor module that is based on the mathematical description of a one-dimensional biofilm. Leading consultants, equipment manufacturers, and WWTP modelling software developers have made meaningful contributions to advancing the use of biofilm models in engineering practice, but the bulk of the engineering community either does not use the now readily available biofilm reactor modules or utilizes them as 'black-box' design tools. The latter approach results in the mathematical biofilm models being no more useful than the empirical design criteria and formulations that have been historically applied to biofilm reactor design. The present work provides a consensus report on the state-of-the art, areas of uncertainty, and future needs for advancing the use of biofilm models in engineering design.

Evaluating operating conditions for outcompeting nitrite oxidizers and maintaining partial nitrification in biofilm systems using biofilm modeling and Monte Carlo filtering.

In practice, partial nitrification to nitrite in biofilms has been achieved with a range of different operating conditions, but mechanisms resulting in reliable partial nitrification in biofilms are not well understood. In this study, mathematical biofilm modeling combined with Monte Carlo filtering was used to evaluate operating conditions that (1) lead to outcompetition of nitrite oxidizers from the biofilm, and (2) allow to maintain partial nitrification during long-term operation. Competition for oxygen was found to be the main mechanism for displacing nitrite oxidizers from the biofilm, and preventing re-growth of nitrite oxidizers in the long-term. To maintain partial nitrification in the model, a larger oxygen affinity (i.e., smaller half saturation constant) for ammonium oxidizers compared to nitrite oxidizers was required, while the difference in maximum growth rate was not important for competition under steady state conditions. Thus, mechanisms for washout of nitrite oxidizing bacteria from biofilms are different from suspended cultures where the difference in maximum growth rate is a key mechanism. Inhibition of nitrite oxidizers by free ammonia was not required to outcompete nitrite oxidizers from the biofilm, and to maintain partial nitrification to nitrite. But inhibition by free ammonia resulted in faster washout of nitrite oxidizers.

Analyzing characteristic length scales in biofilm structures.

The quantification of biofilm structure based on image analysis requires a statistical measure like representative elemental areas (REA) to determine the necessary size of biofilm area to be imaged. In this study, REAs for biofilm structure were calculated for the descriptors Gray level and Correlation (COR) derived from a spatial gray level dependence matrix analysis (SGLDM). An important difference between these two descriptors is their response to structural features at different spatial scales. Gray level is a scale-independent descriptor, whereas COR is scale-dependent. For scale-independent descriptors, the size of the individual images is not relevant when determining REAs. This is in contrast to scale-dependent descriptors for which REAs can only be determined when the area of each image covers the range of structural variability of the biofilm. We used COR to analyze scale dependence of structural heterogeneity at different length scales. A characteristic length of 400 microm in biofilm images provides structural information relevant for mass transport phenomena in biofilms. Overall REAs for gray level and COR were on average 3.4 mm(2). The scale-dependent descriptor COR could not in all cases accurately be determined from combining individual image analysis results--even when the combined area resulted in the REA. Microscope and camera specifications define the upper and lower limit of detectable characteristic length that can be extracted from images and should therefore be considered in the experimental design when choosing an imaging technique.

Modelling the effect of the antimicrobial tylosin on the performance of an anaerobic sequencing batch reactor.

A laboratory-scale anaerobic sequencing batch reactor (ASBR) was fed a synthetic wastewater containing glucose to study the effects of the antimicrobial tylosin on treatment performance. Measurements of methane, volatile fatty acids, and COD concentrations suggested that the addition of 1.67 mg/L and 167 mg/l of tylosin to the synthetic wastewater inhibited propionate oxidizing syntrophic bacteria and aceticlastic methanogens. The latter is presumed to be an indirect effect. A modified version of the IWA Anaerobic Digestion Model No. 1 (ADM1) with extensions for microbial storage and hydrolysis of reserve carbohydrates, and tylosin liquid-solid mass transfer and inhibition adequately described the dynamic profiles observed in the ASBR.

Textural fingerprints: a comprehensive descriptor for biofilm structure development.

Automated tools to determine biofilm structure are necessary to interpret large time series of biofilm images. Image analysis based on the evaluation of Spatial Gray Level Dependence Matrices (SGLDM) enabled us to monitor biofilm structure development in response to external disturbances (i.e., periodic increases of wall shear stress) at a large scale (i.e., >1 mm). We applied our method to an experiment conducted in an annular reactor over a 10-week period. Six states of biofilm development were differentiated by their unique structure. Previous exposure to rapidly increased shear influenced the resulting biofilm structure after additional shear increases. In addition, on the scale of the biofilm images, the biofilm structure after a shear increase was spatially heterogeneous and resulted in spatially differentiated regrowth after detachment at different locations in the biofilm. SGLDM was developed further as an alternative to approaches based on image binarization as binarization leads to information loss for low-magnification and low-resolution images. During post-processing of image data, structural states of biofilm development were identified by K-means clustering and data display in Principal Component plots. Quantitatively selected representative images were used to illustrate the meaning of the clusters. Post-treatment of image data was essential for managing several thousands of raw biofilm images and therefore improved the usefulness of the image analysis.

Practical identifiability of biokinetic parameters of a model describing two-step nitrification in biofilms.

Parameter estimation and model calibration are key problems in the application of biofilm models in engineering practice, where a large number of model parameters need to be determined usually based on experimental data with only limited information content. In this article, identifiability of biokinetic parameters of a biofilm model describing two-step nitrification was evaluated based solely on bulk phase measurements of ammonium, nitrite, and nitrate. In addition to evaluating the impact of experimental conditions and available measurements, the influence of mass transport limitation within the biofilm and the initial parameter values on identifiability of biokinetic parameters was evaluated. Selection of parameters for identifiability analysis was based on global mean sensitivities while parameter identifiability was analyzed using local sensitivity functions. At most, four of the six most sensitive biokinetic parameters were identifiable from results of batch experiments at bulk phase dissolved oxygen concentrations of 0.8 or 5 mg O(2)/L. High linear dependences between the parameters of the subsets (KO2,AOB,muAOB) and (KO2,NOB,muNOB) resulted in reduced identifiability. Mass transport limitation within the biofilm did not influence the number of identifiable parameters but, in fact, decreased collinearity between parameters, especially for parameters that are otherwise correlated (e.g., muAOB) and KO2,AOB, or muNOB and KO2,NOB). The choice of the initial parameter values had a significant impact on the identifiability of two parameter subsets, both including the parameters muAOB and KO2,AOB. Parameter subsets that did not include the subsets muAOB and KO2,AOB or muNOB and KO2,NOB were clearly identifiable independently of the choice of the initial parameter values.

Comparing global sensitivity analysis for a biofilm model for two-step nitrification using the qualitative screening method of Morris or the quantitative variance-based Fourier Amplitude Sensitivity Test (FAST).

Two different methods for global sensitivity analysis were compared exemplarily for a biofilm model for two-step nitrification. Especially for biofilm models, local sensitivity analysis is not very useful as parameters can vary over a large range. Parameters that were evaluated included kinetic and stoichiometric parameters, and also biofilm parameters, such as internal and external mass transfer, the biofilm thickness, and the biomass density. Global sensitivity analyses were performed for a range of operating conditions of a biofilm reactor. The results of the qualitative screening method of Morris were compared with the results of the quantitative variance-based method FAST regarding the input parameters indicated as unimportant. Both methods resulted in similar sets of parameters with a small influence on the model output, but the screening method of Morris required a much smaller number of model evaluations to compute the sensitivity measures than the FAST method.