Mechanistic assessment reveals the significance of HRT and MLSS concentration in balancing carbon diversion and removal in the A-stage process.

Nowadays, the shift toward energy and resource-efficient wastewater treatment plants (WWTPs) has become a necessity rather than a choice. For this purpose, there has been a restored interest in replacing the typical energy and resource-extensive activated sludge process with the two-stage Adsorption/bio-oxidation (A/B) configuration. In the A/B configuration, the role of the A-stage process is to maximize organics diversion to the solids stream and control the following B-stage's influent to allow for the attainment of tangible energy savings. Operating at very short retention times and high loading rates, the influence of the operational conditions on the A-stage process become more tangible than typical activated sludge. Nonetheless, there is very limited understanding of the influence of operational parameters on the A-stage process. Moreover, no studies in the literature have explored the influence of any operational/design parameters on the Alternating Activated Adsorption (AAA) technology which is a novel A-stage variant. Hence, this article mechanistically investigates the independent effect of different operational parameters on the AAA technology. It was inferred that solids retention time (SRT) shall remain below 1 day to allow for energy savings up to 45% and redirecting up to 46% of the influent's COD to the recovery streams. In the meantime, the hydraulic retention time (HRT) can be increased up to 4 h to remove up to 75% of the influent's COD with only 19% decline of the system's COD redirection ability. Moreover, it was observed that the high biomass concentration (above 3000 mg/L) amplified the effect of the sludge poor settleability either due to pin floc settling or high SVI30 which resulted in COD removal below 60%. Meanwhile, the concentration of the extracellular polymeric substances (EPS) was not found to be influenced or to influence process performance. The findings of this study can be employed to formulate an integrative operational approach in which different operational parameters are incorporated to better control the A-stage process and achieve complex objectives.

An alternative A-stage process - Investigating the novel alternating activated adsorption (AAA) system for carbon management under different wastewater strengths.

The interest in the A-stage of the adsorption/bio-oxidation (A/B) process has considerably increased due to its capacity of carbon redirection to the solids stream. Induced by its flexible and compact design, the Alternating Activated Adsorption (AAA) was recently implemented in full-scale as an alternative A-stage system. However, the literature on such a system is scarce. In this article for the first time, the performance of the novel AAA system is evaluated. Two lab-scale AAA systems were operated as a primary settler replacement (AAA-1) or to complement the primary settler (AAA-2). Systems were assessed in terms of process control, effluent quality and carbon diversion. As settling and aeration are performed in the same reactor, AAA maintained high MLSS (2121 ± 293 mg/L for AAA-1 and 806 ± 116 mg/L for AAA-2) compared to the literature at such a very low aerobic SRT (<6 h). Regardless wastewater strength, AAA attains low oxidation (16-17%) owing to the oxygen supply pattern and short aerobic SRT. Moreover, AAA-1 showed high COD removal efficiency for soluble (67 ± 8%) and particulates (62 ± 14%) as well as COD redirection (47 ± 7%). In addition, it is demonstrated that the simultaneous bottom feeding and top discharging regime adds unique capacity for particulates capture in AAA. On the other hand, low particulates removal and total carbon redirection were observed in the AAA-2. Yet, the overall removal efficiencies are comparable with the literature. It can be concluded that, with further optimizations, AAA system has the potential to outcompete other A-stage systems. As such, sludge settleability is found to be challenging when treating low strength wastewater.

A review of biochemical diversity and metabolic modeling of EBPR process under specific environmental conditions and carbon source availability.

Enhanced biological phosphorus removal (EBPR) is one of the most promising technologies as an economical and environmentally sustainable technique for removal of phosphorus from wastewater (WW). However, with high capacity of EBPR, insufficient P-removal is a major yet common issue of many full-scale wastewater treatment plants (WWTP), due to misinterpreted environmental and microbial disturbance. By developing a rather extensive understanding on biochemical pathways and metabolic models governing the anaerobic and aerobic/anoxic processes; the optimal operational conditions, environmental changes and microbial population interaction are efficiently predicted. Therefore, this paper critically reviews the current knowledge on biochemical pathways and metabolic models of phosphorus accumulating organisms (PAOs) and glycogen accumulating organisms (GAOs) as the most abundant microbial populations in EBPR process with an insight on the effect of available carbon source types in WW on phosphorus removal performance. Moreover, this paper critically assesses the gaps and potential future research in metabolic modeling area. With all the developments on EBPR process in the past few decades, there is still lack of knowledge in this critical sector. This paper hopes to touch on this problem by gathering the existing knowledge and to provide farther insights on the future work onto chemical transformations and metabolic strategies in different conditions to benefit the quantitative model as well as WWTP designs.

Towards mainstream deammonification: Comprehensive review on potential mainstream applications and developed sidestream technologies.

Deammonification (partial nitritation-anammox) process is a favorable and innovative process, for treatment of nitrogen-rich wastewater due to decreased oxygen and carbon requirements at very high nitrogen loadings. The bacterial groups responsible for this process are anaerobic ammonium oxidation (anammox) bacteria in symbiosis with ammonium oxidizing bacteria (AOB) which have an active role in development of nitrogen removal biotechnology in wastewater. Development and operation of sidestream deammonification processes has augmented since the initial full-scale systems, yet there are several aspects which mandate additional investigation and deliberation by the practitioners, to reach the operating perspective, set for the facility. Process technologies for treatment of streams with high ammonia concentrations continue to emerge, correspondingly, further investigation towards feasibility of applying the deammonification concept, in the mainstream treatment process is required. Mainstream deammonification can potentially improve the process of achieving more sustainable and energy-neutral municipal wastewater treatment, however feasible applications are not accessible yet. This critical review focuses on a comprehensive assessment of the worldwide lab-scale, pilot-scale and full-scale sidestream applications as well as identifying the major issues obstructing the implementation of mainstream processes, in addition to the designs, operational factors and technology advancements at both novel and/or conventional levels. This review aims to provide a novel and broad overview of the status and challenges of both sidestream and mainstream deammonification technologies and installations worldwide to assess the global perspectives on deammonification research in the recent years. The different configurations, crucial factors and overall trends in the development of deammonification research are discussed and conclusively, the future needs for feasible applications are critically reviewed.

Effect of COD on methanotrophic growth and the anaerobic digestibility of its sludge as a further step for integration in WWTPS.

Within wastewater treatment plants (WWTPs), the anaerobically produced biogas is often underutilized. Fortunately, methanotrophic based biotechnologies can be the remedy for on-site exploitation and recovery of unused biogas. In this regard, efforts have been placed on evaluating the suitably of methanotrophs to be deployed in WWTPs. Even so, the effect of chemical oxygen demand (COD) on methanotrophic activity and methanotrophic sludge digestibility have not been studied, which is the focus of the present study. A methanotrophic culture enriched from activated sludge was exposed to four different COD concentrations (0-540 mg/L) to evaluate the effect of COD on the culture activity in batch mode. It was attained that the presence of COD concentrations up to 540 mg/L has limited influence on methanotrophic activity. This finding was supported by the similar average methane uptake rate (between 2.48 and 2.53 mgCH4/hr) and consumption (61.4 ± 1.5%) observed under the different COD concentrations. On the other hand, methanotrophic sludge was digested in comparison to waste activated sludge (WAS) collected from a WWTP for more than 40 days to evaluate its digestibility. It was obtained that the methanotrophic sludge had a methane specific yield of approximately 1.72 times greater than WAS and had a higher solids destruction rate. This research is another step demonstrating the feasibility of methanotrophs integration in WWTP.

Enhancement of simultaneous nitrogen and phosphorus removal using intermittent aeration mechanism.

Biological nutrient removal grows into complicated scenario due to the microbial consortium shift and kinetic competition between phosphorus (P)-accumulating and nitrogen (N)-removing microorganisms. In this study, three sequential batch reactors with constant operational conditions except aeration patterns at 6 h cycle periods were tested. Intermittent aeration was applied to develop a robust nutrient removal system aimed to achieve high energy saving and removal efficiency. The results showed higher correspondence of P-uptake, polymeric substance synthesis and glycogen degradation in intermittent-aeration with longer interval periods compared to continuous-aeration. Increasing the intermittent-aeration duration from 25 to 50 min, resulted in higher process performance where the system exhibited approximately 30% higher nutrient removal. This study indicated that nutrient removal strongly depends on reaction phase configuration representing the importance of aeration pattern. The microbial community examined the variation in abundance of bacterial groups in suspended sludge, where the 50 min intermittent aeration, favored the growth of P-accumulating organisms and nitrogen removal microbial groups, indicating the complications related to nutrient removal systems. Successful intermittently aerated process with high capability of simple implementation to conventional systems by elemental retrofitting, is applicable for upgrading wastewater treatment plants. With aeration as a major operational cost, this process is a promising approach to potentially remove nutrients in high competence, in distinction to optimizing cost-efficacy of the system.

Development of Methane-Utilizing Mixed Cultures for the Production of Polyhydroxyalkanoates (PHAs) from Anaerobic Digester Sludge.

The fundamental components required for scaling up the production of biogas-based biopolymers can be provided through a single process, that is, anaerobic digestion (AD). In this research, the possibility of enriching methane-utilizing mixed cultures from the AD process was explored as well as their capability to accumulate polyhydroxyalkanoates (PHAs). For almost 70 days of operation in a fed-batch cyclic mode, the specific growth rate was 0.078 ± 0.005 h-1 and the biomass yield was 0.7 ± 0.08 mg-VSS/mg-CH4. Adjusting the nitrogen levels in AD centrate resulted in results comparable to those obtained with a synthetic medium. The enriched culture could accumulate up to 51 ± 2% PHB. On the other hand, when the culturing medium was supplemented with valeric acid, the enriched bacteria were able to produce polyhydroxybutyrate- co-valerate (PHBV) up to 52 ± 6% with an HV percentage of 33 ± 5%. Increasing the valeric acid concentration in the culturing medium above 100 mg/L decreased the overall amount of PHBV by 60%, whereas the number of HV units incorporated was not affected. Changing the methane-to-oxygen ratio (M/O) from 1:1 to 4:1 caused an almost 80% decline in PHB accumulation. In addition, M/O had a significant effect on the fraction composition of PHBV at different valeric acid concentrations.

Influence of biomass density and food to microorganisms ratio on the mixed culture type I methanotrophs enriched from activated sludge.

Methanotrophic based process can be the remedy to offset the wastewater treatment facilities increasing energy requirements due to methanotroph's unique ability to integrate methane assimilation with multiple biotechnological applications like biological nitrogen removal and methanol production. Regardless of the methanotrophic process end product, the challenge to maintain stable microbial growth in the methanotrophs cultivation bioreactor at higher cell densities is one of the major obstacles facing the process upscaling. Therefore, a series of consecutive batch tests were performed to attentively investigate the biomass density influence on type I methanotrophs bacterial growth. In addition, food to microorganisms (F/M), carbon to nitrogen (C/N) and nitrogen to microorganisms (N/M) ratio effect on the microbial activity was studied for the first time. It was clarified that the F/M ratio is the most influencing factor on the microbial growth at higher biomass densities rather than the biomass density increase, whereas C/N and N/M ratio change, while using nitrate as the nitrogen source, does not influence methanotrophs microbial growth. These study results would facilitate the scaling up of methanotrophic based biotechnology by identifying that F/M ratio as the key parameter that influences methanotrophs cultivation at high biomass densities.

Development of long-term dynamic BioWin® model simulation for ANAMMOX UASB micro-granular process.

Three different innovative mathematical models were established to assess the volumetric nitrogen conversion rates of a lab-scale ANAMMOX upflow anaerobic sludge blanket reactor. Despite the vast technological and economical advantages of ANAMMOX, major challenges in process implementation call for mathematic simulations of the process, optimization of operating conditions, and kinetic/statistical analysis of the entire process. In this study, all developed mathematical models implemented via BioWin®, were calibrated and validated, with adequate representations of a bench-scale micro-granular ANAMMOX process, to understand the potential setbacks of ANAMMOX process start-up and stabilization. Fundamental calculations of the kinetic and stoichiometric constants were integrated in the BioWin® software, and the adjusted parameters based on experimental analysis were applied for the assessments. Based on the results from the statistical approach, one of the models (Model III) exhibited a precise prognosis of the effluent data for the entire operational phases with a mean relative error (MRE) of approximately 1.96, 4.36 and 2.54% for nitrogen removal efficiency, removal rate and loading rate, respectively. Evaluating alkalinity and pH during the operation, led to identifying an acceptable fit between the experiment and Model III results, with a MRE of -7.19 and -0.35%, correspondingly. This study confirms the reliability of ANAMMOX-based process modeling and high predictive ability with BioWin®. The presented simulation constants and modeling outline, can be further employed in full-scale applications design and development.

Evaluation of PAO adaptability to oxygen concentration change: Development of stable EBPR under stepwise low-aeration adaptation.

Recent research has shown the adaptability of Biological Nutrient Removal (BNR) systems to very low level dissolved oxygen (DO) concentration, mainly focusing in the nitrification ability that maintains the nitrogen oxidation process even at very low DO levels. Although step-wise aeration decrease on Enhanced Biological Phosphorus Removal (EBPR) is not fully comprehended. This study investigated the effect of reaching micro-aeration with adaptation strategies on EBPR performance. A step-wise oxygen concentration decrease, arriving at an average aeration level of 0.4 mgO2/L was evaluated, with an outcome of approximately 90 % phosphorus removal efficiency. Compared with different aeration modes, the highest phosphorus (P)-removal efficiency, P-release and lowest effluent phosphorus was achieved in gradual DO decrease strategy. On the other hand, an instant decrease in aeration from stable EBPR process from 2 mgO2/L to 0.4 mgO2/L adversely impacted P-removal by decreasing the efficiency to average 60 % and deteriorating the phosphorus removing microbial consortium. Comparison of results between instant and gradual DO-decrease, indicated the sensitivity of microorganisms to aeration. Microbial adaptation to decreased oxygen availability is crucial to reach high process performance. This study proposes, a potential aeration mode, which contributes in reduction of energy consumption in BNR systems through wastewater treatment.

Production of poly-hydroxy-butyrate using nitrogen removing methanotrophic mixed culture bioreactor.

Methanotrophic biotechnologies for methane mitigation and nitrogen removal are becoming more apparent. However, the sludge produced during these processes is often underutilized and instead can be applied for resources recovery. Fortunately, methanotrophic bacteria can utilize methane while also producing poly-hydroxy-butyrate (PHB), bioplastics, under nutrients deficient conditions. Bioplastics are increasing in popularity and can be produced from unexploited resources, such as methane and carbon dioxide, within wastewater facilities. This research demonstrates that methanotrophic sludge generated during a methanotrophic-based nitrogen removal process, which has been recently suggested, can be directly utilized for PHB production. It was found that the PHB storage response of the methanotrophic driven mixed culture was greatest when methane and oxygen were supplied in equal volume to volume ratios. In addition, the PHB response due to imposing feast-like conditions along with nitrogen or phosphorus deprivation were assessed. The highest PHB storage achieved was 21 ± 1.31% after one cycle under methane sufficient and nitrogen limited conditions. Whereas, only applying feast-like conditions demonstrated a PHB storage of 15 ± 0.67% while simultaneously removing nitrate. Finally, further optimization and continued feast- and famine-like cycles can lead to a greater PHB storage response by the culture.

Biomedical Applications of Biomolecules Isolated from Methanotrophic Bacteria in Wastewater Treatment Systems.

Wastewater treatment plants and other remediation facilities serve important roles, both in public health, but also as dynamic research platforms for acquiring useful resources and biomolecules for various applications. An example of this is methanotrophic bacteria within anaerobic digestion processes in wastewater treatment plants. These bacteria are an important microbial source of many products including ectoine, polyhydroxyalkanoates, and methanobactins, which are invaluable to the fields of biotechnology and biomedicine. Here we provide an overview of the methanotrophs' unique metabolism and the biochemical pathways involved in biomolecule formation. We also discuss the potential biomedical applications of these biomolecules through creation of beneficial biocompatible products including vaccines, prosthetics, electronic devices, drug carriers, and heart stents. We highlight the links between molecular biology, public health, and environmental science in the advancement of biomedical research and industrial applications using methanotrophic bacteria in wastewater treatment systems.

Holistic insights into extracellular polymeric substance (EPS) in anammosx bacterial matrix and the potential sustainable biopolymer recovery: A review.

Anaerobic ammonia oxidation (anammox) process has been proven to be a favorable and innovative process, for treatment of nitrogen-rich wastewater due to decreased oxygen and carbon requirements at very high nitrogen loading rates. Anammox process is mainly operated through biofilm or granular sludge structures, as for such slow-growing microorganisms, elevated settling velocity of granules allows for adequate biomass retention and lowered potential risk of washouts. Stability of granular sludge biomass is extremely critical, yet the formation mechanism is poorly understood. There are number of important functions linked to Extracellular Polymeric Substance (EPS) in anammox bacterial matrix, such as; structural stability, aggregation promotion, maintenance of physical structure in the granules, water preserving and protective cell barrier. There is an increasing demand to introduce accurate methods for proper EPS extraction and characterization, to expand the perception of anammox granule stability and potential resource recovery. Analyzing EPS with a focus on various (mechanical and physical) properties can lead to biopolymer production from granular sludge. Biopolymers such as EPS are attractive alternatives substituting the conventional chemical polymers furthermore their recovery from the waste sludge and the potential applications in industrial sectors, leads to a radical enhancement of both environmental and economical sustainability, accelerating the circular economy advancements. Here, this study aims to overview the newest understanding on the structure of anammox sludge EPS, obtained recently and to assess the potential challenges and prospects to identify the knowledge gaps towards constructing an inclusive anammox EPS recovery and characterization procedure.

Screening for Methane Utilizing Mixed Communities with High Polyhydroxybutyrate (PHB) Production Capacity Using Different Design Approaches.

With the adverse environmental ramifications of the use of petroleum-based plastic outweighing the challenges facing the industrialization of bioplastics, polyhydroxyalkanoate (PHA) biopolymer has gained broad interest in recent years. Thus, an efficient approach for maximizing polyhydroxybutyrate (PHB) polymer production in methanotrophic bacteria has been developed using the methane gas produced in the anaerobic digestion process in wastewater treatment plants (WWTPS) as a carbon substrate and an electron donor. A comparison study was conducted between two experimental setups using two different recycling strategies, namely new and conventional setups. The former setup aims to recycle PHB producers into the system after the PHB accumulation phase, while the latter recycles the biomass back into the system after the exponential phase of growth or the growth phase. The goal of this study was to compare both setups in terms of PHB production and other operational parameters such as growth rate, methane uptake rate, and biomass yield using two different nitrogen sources, namely nitrate and ammonia. The newly proposed setup is aimed at stimulating PHB accumulating type II methanotroph growth whilst enabling other PHB accumulators to grow simultaneously. The success of the proposed method was confirmed as it achieved highest recorded PHB accumulation percentages for a mixed culture community in both ammonia- and nitrate-enriched media of 59.4% and 54.3%, respectively, compared to 37.8% and 9.1% for the conventional setup. Finally, the sequencing of microbial samples showed a significant increase in the abundance of type II methanotrophs along with other PHB producers, confirming the success of the newly proposed technique in screening for PHB producers and achieving higher PHB accumulation.

Enhancement of the cultivation process conditions of mixed culture methanotrophic Proteobacteria phylum enriched from waste activated sludge as the first step for value added recovery process.

Methanotrophs are of great interest due to their distinguish ability of recovering value-added commodities such as methanol and lipids while mitigating methane. The enhancement of methanotrophs cultivation process conditions is a pivotal step to develop a feasible methanotrophic bioreactor. In this study, multiple batch tests have been performed to evaluate the aqueous growth medium elements including nitrogen, copper, and biomass density and the gaseous headspace composition influence on methanotrophs activity and the associated microbial community. It was found that increasing copper concentration to 20 µM and nitrate concentration to 40 mM result in higher growth rate and yield. In contrast, increasing the biomass density resulted in a declination in the growth rate and yield. The 16S rRNA gene sequencing shows that the used culture was dominated by type I Methylomonas genus at relative abundance of 60.9% compare to 1% of the enriched population for type II and type III methanotrophs. Thereafter, the specific nitrate uptake rate has been determined to be ranging from 0.05 to 0.62 mgN-NO3/mgTSS/day based on the cultures OD600. The attained results would facilitate the continuous cultivation of type I methanotrophs dominated culture as a first step for any methanotrophic based biotechnology.

Long-term dynamic and pseudo-state modeling of complete partial nitrification process at high nitrogen loading rates in a sequential batch reactor (SBR).

Recently, partial nitrification has been adopted widely either for the nitrite shunt process or intermediate nitrite generation step for the Anammox process. However, partial nitrification has been hindered by the complexity of maintaining stable nitrite accumulation at high nitrogen loading rates (NLR) which affect the feasibility of the process for high nitrogen content wastewater. Thus, the operational data of a lab scale SBR performing complete partial nitrification as a first step of nitrite shunt process at NLRs of 0.3-1.2kg/(m3d) have been used to calibrate and validate a process model developed using BioWin® in order to describe the long-term dynamic behavior of the SBR. Moreover, an identifiability analysis step has been introduced to the calibration protocol to eliminate the needs of the respirometric analysis for SBR models. The calibrated model was able to predict accurately the daily effluent ammonia, nitrate, nitrite, alkalinity concentrations and pH during all different operational conditions.

Development of partial nitrification as a first step of nitrite shunt process in a Sequential Batch Reactor (SBR) using Ammonium Oxidizing Bacteria (AOB) controlled by mixing regime.

Shortcut biological nitrogen removal is a non-conventional way of removing nitrogen from wastewater using two processes either nitrite shunt or deammonification. In the nitrite shunt process, the ammonia oxidation step stops at the nitrite stage, which is known as partial nitrification, then nitrite is directly reduced to nitrogen gas. Effective partial nitrification could be achieved by accumulating Ammonia Oxidizing Bacteria (AOB) and inhibiting Nitrite Oxidizing Bacteria (NOB). In this research, a novel control strategy has been developed to control the DO using the variable mixing regime in a suspended growth system using a Sequential Batch Reactor (SBR) in order to achieve a stable ammonia removal efficiency (ARE) and nitrite accumulation rate (NAR) at a high nitrogen loading rate (NLR). The new controlled SBR system has been successfully running at NLR up to 1.2kg/(m3.day) and achieved an ARE of 98.6±2.8% and NAR of 93.0±0.7%.

Mitigation of nitrous oxide (N2O) emissions from denitrifying fluidized bed bioreactors (DFBBRs) using calcium.

Nitrous oxide (N2O) is a significant anthropogenic greenhouse gases (AnGHGs) emitted from biological nutrient removal (BNR) processes. In this study, N2O production from denitrifying fluidized bed bioreactors (DFBBR) was reduced using calcium (Ca2+) dosage. The DFBBRs were operated on a synthetic municipal wastewater at four different calcium concentrations ranging from the typical municipal wastewater Ca2+ concentration (60 mg Ca2+/L) to 240 mg Ca2+/L at two different COD/N ratios. N2O emission rates, extracellular polymeric substances (EPS), water quality parameters, and microscopic images were monitored regularly in both phases. Calcium concentrations played a significant role in biofilm morphology with the detachment rates for R120Ca, R180Ca, and R240Ca 75% lower than for R60Ca, respectively. The N2O conversion rate at the typical municipal wastewater Ca2+ concentration (R60Ca) was about 0.53% of the influent nitrogen loading as compared with 0.34%, 0.42%, and 0.41% for R120Ca, R180Ca, and R240Ca, respectively corresponding to 21-36% reduction.

Influence of biofilm thickness on nitrous oxide (N2O) emissions from denitrifying fluidized bed bioreactors (DFBBRs).

Nitrous oxide (N2O) is a significant anthropogenic greenhouse gas emitted from biological nutrient removal (BNR) processes. This study tries to get a deeper insight into N2O emissions from denitrifying fluidized bed bioreactors (DFBBRs) and its relationship to the biofilm thickness, diffusivity, and reaction rates. The DFBBR was operated at two different organic and nitrogen loading rates of 5.9­7 kg COD/(m3 d) and 1.2­2 kg N/(m3 d), respectively. Results showed that the N2O conversion rate from the DFBBR at a biofilm thickness of 680 µm was 0.53% of the total influent nitrogen loading while at the limited COD and a biofilm thickness of 230 µm, the N2O conversion rate increased by 196­1.57% of the influent nitrogen loading concomitant with a sevenfold increase in liquid nitrite concentration. Comparing the N2O emissions at different biofilm thickness showed that the N2O emission decreased exponentially with biofilm thickness due to the retention of slow growth denitrifiers and the limited diffusivity of N2O.

Development of a calibration protocol and identification of the most sensitive parameters for the particulate biofilm models used in biological wastewater treatment.

Biofilm models are valuable tools for process engineers to simulate biological wastewater treatment. In order to enhance the use of biofilm models implemented in contemporary simulation software, model calibration is both necessary and helpful. The aim of this work was to develop a calibration protocol of the particulate biofilm model with a help of the sensitivity analysis of the most important parameters in the biofilm model implemented in BioWin® and verify the predictability of the calibration protocol. A case study of a circulating fluidized bed bioreactor (CFBBR) system used for biological nutrient removal (BNR) with a fluidized bed respirometric study of the biofilm stoichiometry and kinetics was used to verify and validate the proposed calibration protocol. Applying the five stages of the biofilm calibration procedures enhanced the applicability of BioWin®, which was capable of predicting most of the performance parameters with an average percentage error (APE) of 0-20%.