Impact of calcium on biofilm morphology, structure, detachment and performance in denitrifying fluidized bed bioreactors (DFBBRs) Ahmed Eldyasti a , George Nakhla a,b,⇑ , Jesse Zhu b a Department of Civil and Environmental Engineering, The University of Western Ontario, London, Ontario N6A 5B9, Canada b Department of Chemical and Biochemical Engineering, The University of Western Ontario, London, Ontario N6A 5B9, Canada highlights  Impact of Ca 2+ on biofilm morphology and stability was evaluated in CFBBR at low C/N.  Role of calcium in EPS formation and biofilm attached was evaluated.  Correlations between denitrification performance and biofilm were established.  Optimization of biofilm was achieved at 120 mg Ca 2+ /L in the wastewater influent.  Calcium fate closure in biofilm processes was presented and evaluated. article info Article history: Received 24 January 2013 Received in revised form 20 July 2013 Accepted 23 July 2013 Available online 31 July 2013 Keywords: Calcium Fluidized bed Extracellular polymeric substances (EPS) Denitrification Biofilm morphology abstract The impact of calcium concentrations on the biofilm morphology, structure, detachment and denitrifica- tion efficiency in denitrifying fluidized bed bioreactors (DFBBRs) was investigated. The DFBBRs were operated on a synthetic municipal wastewater at five different calcium concentrations ranging from the typical Ca 2+ concentration of the tap water (20 mg Ca 2+ /L) to 240 mg Ca 2+ /L at two different C/N ratios of 5 and 3.5 in phases I and II, respectively for a period of 200 days. Extracellular polymeric substances (EPS), Ca 2+ concentration, water quality parameters, and microscopic images were monitored regularly in both phases. Calcium concentrations played a significant role in biofilm morphology with the detach- ment rates for R 120Ca (bioreactor with a Ca 2+ concentration of 120 mg/L), R 180Ca , and R 240Ca 90% and 70% lower than for R 20Ca and R 60Ca , respectively. The optimum influent calcium concentration at both organic and nitrogen loading rates was 120 mg Ca 2+ /L, with higher concentrations exhibiting fractured and weak biofilms. Specific denitrification rates did not change with changing the C/N ratio at elevated Ca 2+ concentration bioreactors while with lower Ca 2+ concentrations, the specific denitrification rates dropped by 20–40%. Nutrients and Ca 2+ mass balances were closed with reasonable accuracy. Ó 2013 Elsevier B.V. All rights reserved. 1. Introduction Among the biological processes for the municipal and industrial wastewater, the fluidized bed bioreactor (FBBR) system is a prom- ising technology for biological nutrient removal (BNR) and proves to be economic and efficient [1–4]. Recently, several FBBRs have been used and investigated for the carbon oxidation, nitrification, denitrification, and anaerobic treatment of the municipal and industrial wastewater [1–3]. Due to the large surface area of the media, which ranges from 2000 m 2 /m 3 to 4000 m 2 /m 3 , the denitri- fying fluidized bed bioreactor (DFFBR) can maintain very high bio- mass (biofilm) concentrations of up to 40,000 mg VSS/L [4,5]. Biofilm accumulation is a dynamic process that is the net result of growth and the detachment processes and affected in the FBBR by several external factors, including wastewater composition and concentration, liquid velocity, concentration of particles, particle– particle collisions, and particle–wall collisions [6]. Divalent cations, such as magnesium (Mg 2+ ) and calcium (Ca 2+ ), are a component of municipal wastewater that have been proven to influence acti- vated sludge bioflocs and enhance the density and settling proper- ties [7–10]. Additionally, divalent cations have been shown to change the biofilm structure and detachment rate [10–12], due to electrostatic interaction and bridging of negatively charged moi- eties of extracellular polymeric substances (EPS), as postulated by the divalent cations bridging theory (DCB) [13,14]. Simultaneously, divalent cations have an indirect role in attachment processes by acting as important cellular cations and enzyme cofactors [13–17]. While most of the studies of divalent cations have focused on suspended growth i.e. activated sludge, there have also been investigations on biofilm systems that indicate the importance of 1385-8947/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.cej.2013.07.084 ⇑ Corresponding author at: Department of Chemical and Biochemical Engineer- ing, The University of Western Ontario, London, Ontario N6A 5B9, Canada. Tel.: +1 519 661 2111x85470; fax: +1 519 850 2921. E-mail address: gnakhla@eng.uwo.ca (G. Nakhla). Chemical Engineering Journal 232 (2013) 183–195 Contents lists available at ScienceDirect Chemical Engineering Journal journal homepage: www.elsevier.com/locate/cej