Water Research 37 (2003) 2732–2738 Study on the use of NADH fluorescence measurements for monitoring wastewater treatment systems G. Farabegoli a,b , C. Hellinga a,1 , J.J. Heijnen a , M.C.M. van Loosdrecht a, * a Department of Biochemical Engineering, Delft University of Technology, Julianalaan 67, Delft 2628 BC, The Netherlands b Department of Hydraulics, Transportations and Roads, Via Eudossiana 18, 00184 Rome, Italy Received 14 August 2001; accepted 21 January 2003 Abstract Fluorescence measurement of intracellular nicotinamide adenine dinucleotide (NADH) provides information about the physiological response of microbes towards changing conditions in their environment and has been suggested to be useful for the control of wastewater treatment plants. In this study, the practical usefulness of such measurements was evaluated from batch experiments with a commercially available NADH sensor in a bench scale reactor. The sensor was linear in the NADH concentration, robust, almost maintenance free, and hardly sensitive to floc size distribution. Measured fluorescence intensity proved to depend strongly on the concentration of active heterotrophic biomass. The NADH level was supposed to be dependent on the ratio of electron donor/electron acceptor availability inside the cells; however, neither acetate nor ammonium addition was reflected by the measurement signal. A jump wise NADH signal change was observed at complete oxygen or nitrate depletion as also reflected by bends in the redox curve. In the near zero concentration ranges of oxygen and nitrate (0.1–0.5 mg/l) the signal changes only slightly in the opposite direction to the redox trend. r 2003 Elsevier Science Ltd. All rights reserved. Keywords: Fluorescence measurements; Monitoring; NADH; Redox; Wastewater 1. Introduction Robust and almost maintenance-free fluorescence measurement devices are available nowadays to be used for monitoring wastewater treatment plants. These measurements are related to microbial activity, as was discovered in 1957 by Duysens and Amesz [1]. They showed that bakers yeast suspension exhibit fluorescence when exposed to UV light and that the fluorescence spectrum resembles that of NADH. Harrison and Chance [2] showed that when a culture is exposed to fluorescence light at a wavelength of 340 nm, fluores- cence measured at 460 nm is proportional to its NAD(P)H content. NADH/NAD + plays a key role in the electron transfer from electron donor to electron acceptor inside living cells. It is generally assumed that the NADH/ NAD + ratio reflects the balance between the cell internal electron donor and electron acceptor supply with respect to the catabolic redox reactions [3].A surplus of electron donor (or shortage of electron acceptor) leads to a high NADH/NAD + ratio, whereas a relative shortage of electron donor (or a surplus of electron acceptor) is reflected in a low NADH/ NAD + ratio. The normal situation under substrate limitation is thus a low NADH/NAD + ratio [4]. At short time scales the summed amount of NAD + and NADH is constant so that a changing NADH/NAD + ratio is reflected by a change in the NADH concentra- tion which is expected to give a variation in the fluorescence measurement. *Corresponding author. Fax: +31-152-782355. E-mail addresses: geneve.farabegoli@uniromal.it (G. Farabegoli), chris@halotec.com (C. Hellinga), mark.vanloosdrecht@tnw.tudelft.nl (M.C.M. van Loosdrecht). 1 Current address: Halotec Delft B.V., Delftechpark 26, Delft 2628 XH, The Netherlands. 0043-1354/03/$ - see front matter r 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0043-1354(03)00064-2