Activated Sludge Wastewater Treatment Systems Research Articles

Study of BOD: COD Ratio as an Indicator for Wastewater of Rubber Industry Sector

When considering the rubber industry sector, the most significant challenge is the efficient wastewater treatment before discharging it into the natural environment. The rubber industry is considered to be highly chemical and water-intensive: and involves enormous quantitative s of waste production. The ratio between Biological Oxygen Demand (BOD5) and Chemical Oxygen Demand (COD) is a widely used parameter in characterising wastewater and then the same was used in this study as an indication to measure the efficiency of wastewater treatment plant operated in the rubber industry sector. Data for the study was extracted from samples collected from 10 wastewater treatment plants of rubber industries in the Sabaragamuwa Province; two samples from each representing before and after treatment. The samples were tested in two replicated for BOD5, COD, pH, and conductivity. These selected rubber factories contain lagoon/pond type wastewater treatment systems or activated sludge wastewater treatment systems. The results showed that the treated wastewater samples were within the range of 0.1–0.4 of BOD5: COD and were below the BOD5: COD permissible level as per CEA standards. (General standards for rubber in inland surface waters; Latex-BOD 60, COD 400; Lanka rubber/crepe rubber–BOD 50, COD 400) comparing the BOD, COD, pH, and conductivity influent and effluent of the treatment process proves the effectiveness of each treatment plant of the relevant industry. Tested samples show a high BOD5: COD ratio in influent and effluent of wastewater treatment system indicate that wastewater of the industry is easily biodegradable. However, more research comparing the characteristics of different rubber manufacturing processes is needed to develop this approach further. 
 Keywords: BOD, COD, BOD: COD ratio, Wastewater treatment plant

Metabolic implications for predatory and parasitic bacterial lineages in activated sludge wastewater treatment systems

Deciphering unclear microbial interactions is key to improving biological wastewater treatment processes. Microbial predation and parasitism in wastewater treatment ecosystems are unexplored survival strategies that have long been known and have recently attracted attention because these interspecies interactions may contribute to the reduction of excess sludge. Here, microbial community profiling of 600 activated sludge samples taken from six industrial and one municipal wastewater treatment processes (WWTPs) was conducted. To identify the shared lineages in the WWTPs, the shared microbial constituents were defined as the family level taxa that had ≥ 0.1% average relative abundance and detected in all processes. The microbial community analysis assigned 106 families as the shared microbial constituents in the WWTPs. Correlation analysis showed that 98 of the 106 shared families were significantly correlated with total carbon (TC) and/or total nitrogen (TN) concentrations, suggesting that they may contribute to wastewater remediation. Most possible predatory or parasitic bacteria belonging to the phyla Bdellovibrionota, Myxococcota, and Candidatus Patescibacteria were found to be the shared families and negatively correlated with TC/TN; thus, they were frequently present in the WWTPs and could be involved in the removal of carbon/nitrogen derived from cell components. Shotgun metagenome-resolved metabolic reconstructions indicated that gene homologs associated with predation or parasitism are conserved in the Bdellovibrionota, Myxococcota, and Ca. Patescibacteria genomes (e.g., host interaction (hit) locus, Tad-like secretion complexes, and type IV pilus assembly proteins). This study provides insights into the complex microbial interactions potentially linked to the reduction of excess sludge biomass in these processes.

Negative impacts of cleaning agent DEPTAL MCL® on activated sludge wastewater treatment system.

DEPTAL MCL® is a professional cleaning agent approved by the Portuguese Food Regulatory Authority and is used in agro-food industries, namely in fish canning industries in the north of Portugal. Its extensive use during cleaning procedures results in potential significant negative impacts on the performance of the downstream municipal wastewater treatment plant (WWTP). A lab-scale extended areation activated sludge wastewater treatment system, continuously fed by influent collected at a municipal WWTP, was used to assess the impact of a range of DEPTAL MCL® concentrations during 72 h. Despite distinct activated sludge community composition (due to its dynamic nature) and variations in real influent characteristics, a relevant impact was observed. DEPTAL MCL® effect was underscored through the use of a multivariate analysis using seventeen physicochemical operational factors and nineteen quantitative image analysis (QIA) parameters. DEPTAL MCL® exerted a severe negative impact on phosphorous (P-PO4) removal, total nitrogen (TN) removal and sludge volume index (SVI). With increasing DEPTAL MCL® concentrations, both P-PO4 and TN removal were affected and diminished proportionally. Moreover, several QIA parameters indicate defloculation when DEPTAL MCL® was present, in particular for intermediate size aggregates with significant impacts. Optical density of the effluent (Ode), displayed an increase of effluent turbidity. Percentage of area covered by small aggregates (%Areasml) was also significantly higher for the intermediate and higher DEPTAL MCL® concentrations tested. Principal component analysis exhibited 3 distinct ordenations: (i) control without addition of DEPTAL MCL®; (ii) addition of 0.03% and 0.06% and of (iii) 0.13 and 0.26% (v DEPTAL MCL®/v aeration tank). Canonical correspondence analysis (CCA) was used to correlate the physicochemical data, QIA and the filamentous bacteria species prevalence to DEPTAL MCL® concentration and incubation time. A time persistent DEPTAL MCL® effect was observed, underscoring the need of a pretreatment of wastewater containing this cleaning agent.

Activated Sludge Wastewater Treatment Systems Based on Parallel Intelligence

As a typical biochemical treatment method, the activated sludge is used in various wastewater treatments. Activated wastewater treatment process is hard to control because of the complex nonlinear behavior, however, the stability of dissolved oxygen concentration (DOC) in the reaction tank is related to the quality and efficiency of processing. In this paper, a parallel wastewater treatment system based on the parallel intelligence theory is proposed to deal with the time-varying, nonlinear, and delay in the wastewater treatment process. Besides, computational experiments are designed and conducted to explore and verify the control strategy, which will guide the actual process through virtual and real interaction, so as to adapt to the complex and changeable wastewater treatment environment.

Activity-Based Cell Sorting Reveals Resistance of Functionally Degenerate Nitrospira during a Press Disturbance in Nitrifying Activated Sludge.

ABSTRACTManaging and engineering activated sludge wastewater treatment microbiomes for low-energy nitrogen removal requires process control strategies to stop the oxidation of ammonium at nitrite. Our ability to out-select nitrite-oxidizing bacteria (NOB) from activated sludge is challenged by their metabolic and physiological diversity, warranting measurements of their in situ physiology and activity under selective growth pressures. Here, we examined the stability of nitrite oxidation in activated sludge during a press disturbance induced by treating a portion of return activated sludge with a sidestream flow containing free ammonia (FA) at 200 mg NH3-N/liter. The nitrite accumulation ratio peaked at 42% by day 40 in the experimental bioreactor with the press disturbance, while it did not increase in the control bioreactor. A subsequent decrease in nitrite accumulation within the experimental bioreactor coincided with shifts in dominant Nitrospira 16S rRNA amplicon sequence variants (ASVs). We applied bioorthogonal noncanonical amino acid tagging (BONCAT) coupled with fluorescence-activated cell sorting (FACS) to investigate changes in the translational activity of NOB populations throughout batch exposure to FA. BONCAT-FACS confirmed that the single Nitrospira ASV washed out of the experimental bioreactor had reduced translational activity following exposure to FA, whereas the two Nitrospira ASVs that emerged after process acclimation were not impacted by FA. Thus, the coexistence of functionally degenerate and physiologically resistant Nitrospira populations provided resilience to the nitrite-oxidizing function during the press disturbance. These results highlight how BONCAT-FACS can resolve ecological niche differentiation within activated sludge and inform strategies to engineer and control microbiome function.IMPORTANCE Nitrogen removal from activated sludge wastewater treatment systems is an energy-intensive process due to the large aeration requirement for nitrification. This energy footprint could be minimized with engineering control strategies that wash out nitrite-oxidizing bacteria (NOB) to limit oxygen demands. However, NOB populations can have a high degree of physiological diversity, and it is currently difficult to decipher the behavior of individual taxa during applied selective pressures. Here, we utilized a new substrate analog probing approach to measure the activity of NOB at the cellular translational level in the face of a press disturbance applied to the activated sludge process. Substrate analog probing corroborated the time series reactor sampling, showing that coexisting and functionally degenerate Nitrospira populations provided resilience to the nitrite oxidation process. Taken together, these results highlight how substrate analog approaches can illuminate in situ ecophysiologies within shared niches, and can inform strategies to improve microbiome engineering and management.

Porous Polyethylene-Supported Zeolite Carriers for Improved Wastewater Deammonification

Wastewater treatment is increasingly important as anthropogenic activities continue to stress our water systems. Ammonium is one of the most common pollutants in wastewater streams and is typically oxidized to nitrate during treatment, which still results in the discharge of reactive nitrogen to the environment. Anaerobic ammonium oxidation (anammox) can completely remove reactive nitrogen, forming dinitrogen, and also decreases the cost of ammonium removal compared to conventional activated sludge wastewater treatment systems. Anammox faces challenges in mainstream implementation, however, as a result of the slow growth rates of anammox bacteria, narrow ideal growth conditions, and competition with other taxa. Addition of zeolite, such as faujasite, into low ammonium waste streams improves ammonium removal and locally concentrates ammonium ions, which in turn can improve the proliferation of anammox bacteria. Here, we report the development of a scalable approach to plastic carriers for potential use in mainstream anammox reactors that combines the processability of thermoplastics and the ammonium sequestration ability of zeolites. Carriers were prepared by melt-blending polyethylene (PE), microparticulate zeolite 13X (Z13X, a faujasite), and a sacrificial poly(ethylene oxide) (PEO) template. Removal of the PEO template by solvent etching in water exposed a percolating pore network within the PE support structure and the particulate zeolite trapped within the exterior. A pore size in the range of 10–25 μm was typical, with zeolite loadings as high as 45% by mass in the final product. The hybrid inorganic–polymer carriers were highly effective in sequestering ammonium, capable of removing >75% of ammonium from a 45 mg/L TAN (total ammonium nitrogen) aqueous environment in 24 h at less than 1% mass loading of carriers as compared to solution mass. Ammonium removal by ion exchange was confirmed by spectrophotometric methods and by energy-dispersive X-ray spectroscopy, and the kinetics of ammonium sequestration were determined to be zeroth-order with respect to ammonium and first-order with respect to zeolite. The materials described in this article are expected to find utility in future bioreactor development.

Antibiotic Resistance Profile of Bacteria Isolated from Wastewater Systems in Eastern Ethiopia.

World Health Organizations launched a global action plan on antimicrobial resistance since 2015. Along with other objectives, the plan was aimed to strengthen knowledge of the spread of antimicrobial resistance through surveillance and research. Given their high bacterial densities and that they receive antibiotics, metals, and other selective agents, wastewater systems are a logical hotspot for antibiotic resistance surveillance. The current study reports on the result of antibiotic resistance surveillance conducted in selected wastewater systems of Eastern Ethiopia from Feb. 2018 to Oct. 2019. We monitored three wastewater systems in Eastern Ethiopia, such as the activated sludge system of Dire Dawa University, waste stabilization pond of Haramaya University, and a septic tank of Hiwot Fana Specialized University Hospital for 18 months period. We collected 66 wastewater samples from 11 sampling locations and isolated 722 bacteria using selective culture media and biochemical tests. We tested their antibiotic susceptibility using the standard Kirby-Bauer disk diffusion method on the surface of the Mueller-Hinton agar and interpreted the result according to EUCAST guidelines. The result shows the highest percentage of resistance for ampicillin among isolates of hospital wastewater effluent which is 36 (94.7%), 33 (91.7%), and 32 (88.9%) for E. coli, E. faecalis, and E. faecium, respectively. A lower rate of resistance was seen for gentamicin among isolates of activated sludge wastewater treatment system which is 10 (16.4%), 8 (13.3%), 11 (18.9%), and 12 (20.3%) for E. coli, E. faecalis, E. faecium, and P. aeruginosa, respectively. Hospital wastewater exhibited higher resistance than the other two wastewater systems. The Multiple Antibiotic Resistance Index (MARI) has significantly increased in the wastewater's course treatment process, showing the proliferation of resistance in the wastewater treatment system.

Comparative life cycle assessment of municipal wastewater treatment systems: lagoon and activated sludge

Providing health conditions, prevention of water pollution and wastewater reuse are needed to select the best wastewater treatment process before designing and implementing, according to regional climatic conditions. the aim of the present study was to evaluate the lagoon and activated sludge wastewater treatment systems using life cycle assessment and ISO14040 standards within four steps. Required data in the study systems were matter and energy inputs, including NH3-N, TP, TN, CL, O2 and electricity, as well as pollutant outputs involving BOD5, COD, SS, NH3-N, TP, TN, CL, CO2, and CH4. The data were obtained from treatment systems of Ardabil (aerated lagoon) and Tabriz (activated sludge), Iran. Some of the data were obtained by calculation and the eco-invent database was used to complete the information required. Data were analyzed by Simapro 8.0.1 software. The results of the study demonstrated that the lagoon system in comparison with the other systems had 100% negative impacts in every effect classes, while the activated sludge system on global warming (6.39%) and photochemical oxidation (7.14%) had the least impact. Therefore, the lagoon system was recognized as the environmentally-harmful system, while the activated sludge as the best wastewater treatment system.

Genomic insights into the metabolism of 'Candidatus Defluviicoccus seviourii', a member of Defluviicoccus cluster III abundant in industrial activated sludge.

Filamentous cluster III Defluviicoccus (DF3) are known to proliferate and cause bulking issues in industrial wastewater treatment plants. Members of the genus Defluviicoccus are also known to exhibit the glycogen accumulating organism (GAO) phenotype, which is suggested to be detrimental to enhanced biological phosphorus removal (EBPR). Despite the reported negative impact members of the DF3 have on activated sludge wastewater treatment systems, limited research has focused on understanding the physiological traits that allow them to compete in these environments. In this study, a near complete genome of an abundant filamentous DF3 named 'Candidatus Defluviicoccus seviourii' was obtained from a full-scale sequencing batch reactor (SBR) treating winery wastewater. Annotation of the 'Ca. D. seviourii' genome revealed interesting metabolic features that help to understand the abundance of this microorganism in industrial wastewater treatment plants. Their potential for the storage of polyhydroxyalkanoates (PHA) is suggested to favour these organisms with the intermittent availability of carbon in these systems. An ability to fix nitrogen and take up urea may provide them with an additional advantage with the characteristically high carbon to nitrogen content of industrial waste. The genome and preliminary findings of this study provide a foundation for further research into these biotechnologically relevant organisms.

Composition of heterotrophic specialized sub-guilds defined by a positive RNA and polyhydroxyalkanoate correlation in activated sludge

Microbial heterotrophic guilds in activated sludge wastewater treatment systems have complex population structures and functions. A previously proposed heterotrophic-specialist model states that heterotrophs consist of sub-guilds specialized in consuming specific classes of compounds, either readily degradable substrate (RDS) or slowly degradable substrate (SDS) according to current mathematical modeling practices for wastewater treatment processes. It follows from metabolic considerations that the levels of RNA and polyhydroxyalkanoate (PHA) are correlated for strains of the same species growing in different environments; a conjecture previously tested. The proposed classification of heterotrophs into RDS or SDS consumers predicts that the same correlation would also be found across heterotrophic species in conventional activated sludge systems; this prediction was tested in the current study. The positive correlation between the RNA and PHA levels was observed in 9 conventional activated sludge plants in two independent sampling times and it was also found stable over a 6-month regular sampling period at one of these plants. Together, these results imply that the levels of RNA and PHA can be used to define heterotrophic-specialist sub-guilds. In order to gain insight in the species composition of the defined sub-guilds, flow cytometry cell sorting was used to further analyze one of the activated sludge samples. Four sorted sub-samples were obtained (high-RNA/high-PHA, low-RNA/high-PHA, high-RNA/low-PHA, and low-RNA/low-PHA), and the phylogenetic composition of each was determined using 16S rRNA gene amplicon pyrosequencing. Heterotrophic genera were identified across 12 phyla, and their representation in each sorted sub-sample showed that the high-RNA/high-PHA and low-RNA/low-PHA groups were most dissimilar. The enriched genera in these sorted sub-samples are suggested to represent the composition of heterotrophic-specialized sub-guilds defined by the kinetics of substrate consumption.

Influence of Chironomid Larvae on Activated Sludge and Nitrogen Removal in Domestic Wastewater Treatment

Chironomid larvae (CL), aquatic invertebrates in freshwater environments are the common macro‐scale organisms in activated sludge wastewater treatment systems. The effects of CL on biomass quantity, sludge characteristics, and nitrogen removal efficiency were investigated in a domestic wastewater treatment system in this study. The batch tests showed that CL induced sludge reduction and enhanced sludge settlement. A maximum sludge weight reduction rate of 42% was achieved at the CL wet‐weight density of 0.5 g CL/g total suspend solid (TSS), and the sludge volume index (SVI) decreased from 146 to 78 mL/g as the CL wet‐weight density increased from 0 to 0.5 g CL/g TSS. After 52 operation cycles, the system with the CL density of 0.5 g CL/g TSS had a lower sludge yield (0.22 g volatile suspended solid (VSS)/g removed chemical oxygen demand (CODremoved) than the control reactor without CL addition (0.34 g VSS/g CODremoved). Addition of CL enhanced the simultaneous nitrification and denitrification (SND), but partially inhibited nitrification.

Rapid adaptation of activated sludge bacteria into a glycogen accumulating biofilm enabling anaerobic BOD uptake

Glycogen accumulating organisms (GAO) are known to allow anaerobic uptake of biological oxygen demand (BOD) in activated sludge wastewater treatment systems. In this study, we report a rapid transition of suspended activated sludge biomass to a GAO dominated biofilm by selective enrichment using sequences of anaerobic loading followed by aerobic exposure of the biofilm to air. The study showed that within eight weeks, a fully operational, GAO dominated biofilm had developed, enabling complete anaerobic BOD uptake at a rate of 256mg/L/h. The oxygen uptake by the biofilm directly from the atmosphere had been calculated to provide significant energy savings. This study suggests that wastewater treatment plant operators can convert activated sludge systems readily into a “passive aeration” biofilm that avoids costly oxygen transfer to bulk wastewater solution. The described energy efficient BOD removal system provides an opportunity to be coupled with novel nitrogen removal processes such as anammox.

Integrating ultrasonic disintegration in activated sludge wastewater treatment plant modeling

AbstractThis paper presents an integrated mathematical model that is capable of predicting and assessing the impact of ultrasonic (US) treatment on the excess activated sludge production in an activated sludge wastewater treatment system. Biological processes in the reactor are simulated in Matlab®/Simulink by the ASM1 model into which two algebraic equations, which capture the US treatment, are integrated. Calibration and validation data series come from a pilot plant installed at two locations, i.e. at a communal wastewater treatment plant (Mechelen-Noord) and at an industrial food flavor production site Haasrode, both located in the Flanders region of Belgium. The results show that the built-up model is capable of correctly predicting excess sludge reduction in the treatment system (which is a sequencing batch reactor in both cases). A reduction of approximately 42% for the communal case study can be reported, while the result obtained for the industrial case study, characterized by a very high organic...

MiDAS: the field guide to the microbes of activated sludge.

The Microbial Database for Activated Sludge (MiDAS) field guide is a freely available online resource linking the identity of abundant and process critical microorganisms in activated sludge wastewater treatment systems to available data related to their functional importance. Phenotypic properties of some of these genera are described, but most are known only from sequence data. The MiDAS taxonomy is a manual curation of the SILVA taxonomy that proposes a name for all genus-level taxa observed to be abundant by large-scale 16 S rRNA gene amplicon sequencing of full-scale activated sludge communities. The taxonomy can be used to classify unknown sequences, and the online MiDAS field guide links the identity to the available information about their morphology, diversity, physiology and distribution. The use of a common taxonomy across the field will provide a solid foundation for the study of microbial ecology of the activated sludge process and related treatment processes. The online MiDAS field guide is a collaborative workspace intended to facilitate a better understanding of the ecology of activated sludge and related treatment processes—knowledge that will be an invaluable resource for the optimal design and operation of these systems.Database URL: http://www.midasfieldguide.org

ANFIS Direct Inverse Control of Substrate in an Activated Sludge Wastewater Treatment System

Nonlinearity of the substrate dynamics is the main inconveniences in its control. Low substrate concentration significantly affects the growth of the microorganisms responsible for treating the wastewater and too high substrate concentration level may lead to drop in the growth rate. Therefore, the control of substrate concentration is highly essential for effective and optimal operation of wastewater treatment plants. However, the control using conventional techniques is quite cumbersome and often impossible. This paper presents adaptive neuro fuzzy inference system (ANFIS) direct inverse control of substrate in an activated sludge system. The performances of the proposed controller are illustrated by tracking the substrate set-points. The simulation results demonstrate that the proposed controller can effectively and accurately control the substrate concentration level. The proposed inverse controller may serve as a valuable control strategy for the wastewater treatment plant.

Influence of bioselector processes on 17α-ethinylestradiol biodegradation in activated sludge wastewater treatment systems.

The removal of the potent endocrine-disrupting estrogen hormone, 17α-ethinylestradiol (EE2), in municipal wastewater treatment plant (WWTP) activated sludge (AS) processes can occur through biodegradation by heterotrophic bacteria growing on other organic wastewater substrates. Different kinetic and metabolic substrate utilization conditions created with AS bioselector processes can affect the heterotrophic population composition in AS. The primary goal of this research was to determine if these changes also affect specific EE2 biodegradation kinetics. A series of experiments were conducted with parallel bench-scale AS reactors treating municipal wastewater with estrogens at 100-300 ng/L concentrations to evaluate the effect of bioselector designs on pseudo first-order EE2 biodegradation kinetics normalized to mixed liquor volatile suspended solids (VSS). Kinetic rate coefficient (kb) values for EE2 biodegradation ranged from 5.0 to 18.9 L/g VSS/d at temperatures of 18 °C to 24 °C. EE2 kb values for aerobic biomass growth at low initial food to mass ratio feeding conditions (F/Mf) were 1.4 to 2.2 times greater than that from growth at high initial F/Mf. Anoxic/aerobic and anaerobic/aerobic metabolic bioselector reactors achieving biological nutrient removal had similar EE2 kb values, which were lower than that in aerobic AS reactors with biomass growth at low initial F/Mf. These results provide evidence that population selection with growth at low organic substrate concentrations can lead to improved EE2 biodegradation kinetics in AS treatment.

Investigation into organic matter and nutrient removal in an activated sludge wastewater treatment system: case study of Bojnurd

Investigation into organic matter and nutrient removal in an activated sludge wastewater treatment system: case study of Bojnurd

Measurement and mathematical modelling of competition between fast- and slow-growing ordinary heterotrophic organisms in low and high substrate-loaded systems

Ordinary heterotrophic organisms (OHO) of an activated sludge wastewater treatment system showed an atypical growth behaviour when they are inoculated to batch aerobic growth tests with a high substrate-loaded condition. For example, the OHO maximum specific growth rates on readily biodegradable substrates (μ H) increased with a high ratio of substrate concentration to OHO active biomass concentration (So/Xo), although they were assumed to be constant in a conventional microbial growth kinetic model with a single OHO population group. We, therefore, set a hypothesis in that the change of OHO maximum specific growth rates in the batch test condition is caused by turnover of fast-growing OHO population against slow-growing OHO population. And, a competitive microbial growth kinetic model of the fast- and slow-growing OHO population groups was developed and validated with model-data fitting analysis for the batch test results. The competitive microbial growth kinetic model of process selection, rather than that of kinetic selection, was capable of simulating microbial growth kinetics in high substrate-loaded dynamic systems (i.e., batch tests) and low substrate-loaded steady-state systems (i.e., continuously operated wastewater treatment systems), better than the conventional non-competitive growth kinetic model.

Multi-scale modelling of bioreactor–separator system for wastewater treatment with two-dimensional activated sludge floc dynamics

A simple “first generation” multi-scale computational model of the formation of activated sludge flocs at micro-scale and reactor performance at macro-scale is proposed. The model couples mass balances for substrates and biomass at reactor scale with an individual-based approach for the floc morphology, shape and micro-colony development. Among the novel model processes included are the group attachment/detachment of micro-flocs to the core structure and the clustering of nitrifiers. Simulation results qualitatively describe the formation of micro-colonies of ammonia and nitrite oxidizers and the extracellular polymeric substance produced by heterotrophic microorganisms, as typically observed in fluorescence in situ hybridization images. These results are the first step towards realistic multi-scale multispecies models of the activated sludge wastewater treatment systems and a generic modelling strategy that could be extended to other engineered biological systems.

Biomass density and filament length synergistically affect activated sludge settling: Systematic quantification and modeling

Settling of the biomass produced during biological treatment of wastewater is a critical and often problematic process. Filamentous bacteria content is the best-known factor affecting biomass settleability in activated sludge wastewater treatment systems, and varying biomass density has recently been shown to play an important role as well. The objective of this study was to systematically determine how filament content and biomass density combine to affect microbial biomass settling, with a focus on density variations over the range found in full-scale systems. A laboratory-scale bioreactor system was operated to produce biomass with a range of filamentous bacterium contents. Biomass density was systematically varied in samples from this system by addition of synthetic microspheres to allow separation of filament content and density effects on settleability. Fluorescent in-situ hybridization indicated that the culture was dominated by Sphaerotilus natans, a common contributor to poor settling in full-scale systems. A simple, image-based metric of filament content (filament length per floc area) was linearly correlated with the more commonly used filament length per dry biomass measurement. A non-linear, semi-empirical model of settleability as a function of filament content and density was developed and evaluated, providing a better understanding of how these two parameters combine to affect settleability. Filament content (length per dry biomass weight) was nearly linearly related to sludge volume index (SVI) values, with a slightly decreasing differential, and biomass density exhibited an asymptotic relationship with SVI. The filament content associated with bulking was shown to be a function of biomass density. The marginal effect of filament content on settleability increased with decreasing biomass density (low density biomass was more sensitive to changes in filament content than was high density biomass), indicating a synergistic relationship between these factors. Consideration of both biomass density and filament content, as by the methods and model described herein, should improve operation and troubleshooting of settling processes for biological solids.