Available at www.sciencedirect.com journal homepage: www.elsevier.com/locate/watres Sensitive and meaningful measures of bacterial metabolic activity using NADH fluorescence Melissa Wos, Peter Pollard à Centre for Riverine Landscapes, School of Environmental Engineering, Griffith University, Queensland 4111, Australia article info Article history: Received 18 May 2005 Received in revised form 23 March 2006 Accepted 26 March 2006 Keywords: Microbial Bacterial metabolic activity NADH Fluorimetry Activated sludge ABSTRACT Successful biological wastewater treatment depends on bacterial metabolic activity. Commercial fluorimeters are designed to monitor this activity using the native fluorescence of Nicotinamide Adenine Dinucleotide [NADH]. However, fluorescence measurements in wastewater treatment plants remain scarce due to difficulties with interpreting fluores- cence data. This paper shows that fluorescence probe measurements taken from waste- water do not represent bacterial cell metabolic activity because intracellular NADH is likely swamped by the stable extracellular NADH fraction. Thus, a simple filtration/extraction/ centrifugation method was developed to collect the bacterial cells, extract the intracellular NADH using heat treatment in Tris buffer and collect the purified intracellular NADH fraction. NADH standards were used to quantify NADH from the unknown wastewater samples where limits of detection were between 1 nmol mL 1 and 0.35 mmol mL 1 . Fluorescence of [NADH] greater than 0.35 mmol mL 1 was self-quenched. At high pH’s NADH was stable outside the cell. NADH was stable at neutral and basic pH ranges of pH 7 to 11, but declined proportionally below a pH of 7. Since commercially available fluorescence probes used for measuring NADH are more likely detecting extracellular NADH, separating bacterial cells from water samples followed by NADH extraction was essential to distinguish intracellular and extracellular [NADH]. Here we have proposed three simple steps to meaningful measures of bacterial metabolic activity based on the autofluorescence of NADH. The three simple steps to getting it right are (1) filter the sample, (2) extract the bacteria collected on filter into a hot Tris buffer and (3) measure autofluorescence of the extract against a set of NADH standards. Future development of an on-line monitoring system based on these three steps is achievable with a little ingenuity. & 2006 Elsevier Ltd. All rights reserved. 1. Introduction Biological wastewater treatment processes are used globally to protect aquatic environments. The importance of bacterial cells in these engineered environments is related to their ability to metabolise substrates to generate energy and minimal biomass (Low et al., 2000). There are more than bacteria in biological treatment processes, fungi, rotifers, and protozoans are also residents of activated sludge. However, in terms of metabolic activity the most dominating influence is bacterial. Bacteria constitute the majority of microorganisms present in activated sludge using organic compounds for their ARTICLE IN PRESS 0043-1354/$ - see front matter & 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.watres.2006.03.020 à Corresponding author. Tel.: +61 7 3875 6597; fax: +61 7 3875 5479. E-mail address: p.pollard@griffith.edu.au (P. Pollard). WATER RESEARCH 40 (2006) 2084– 2092