Water Research 38 (2004) 2865–2873 Indicators of biofilm development and activity in constructed wetlands microcosms S.R. Ragusa a, *, D. McNevin a , S. Qasem a , C. Mitchell b a Department of Chemical Engineering, University of Sydney, NSW 2006, Australia b Institute for Sustainable Futures, University of Technology, Sydney, P.O. Box 123, Broadway, NSW 2007, Australia Received 28 January 2003; received in revised form 30 January 2004; accepted 26 March 2004 Abstract Methods to measure protein, exopolysaccharide, viable cell number and INT reduction activity were tested on biofilm growing in a wastewater batch reactor. They were shown to be meaningful indicators of biofilm growth and correlated well with each other. Protein, exopolysaccharide, viable cells and INT reduction rates increased linearly over time. Viable cell number exhibited strong linear correlations with protein (R 2 =0.98) and exopolysaccharide (R 2 =0.99) while INT reduction rate was somewhat less well correlated (R 2 =0.90). Our results indicate production rates of 0.91  10 7 mg EPS per viable cell and 1.0  10 7 mg protein per viable cell. Protein and polysaccharide specific INT reduction rates decreased by approximately 50%, whereas viable cell specific INT reduction rates decreased by 65% and the protein to polysaccharide ratio stayed relatively constant at between 1.1 and 1.2 as the biofilm developed. Measurement of protein, polysaccharide, viable cells and INT reduction rate at depth within the bioreactor showed that they were concentrated in the top 1cm of the influent end of the reactor and each decreased to a base level within 4.5cm of the inlet. Protein to polysaccharide ratios increased with depth in the reactor and the specific INT reduction rates were maximal at 4.5 cm depth. The results indicate that the biomass can take upwards of 100 days to stabilize during batch (fill and draw) operation of subsurface wetlands and that the relative ratios of biomass components remain relatively constant during biofilm growth. Also, it appears that filtration of suspended solids results in biomass concentration at the inlet to the wetland. r 2004 Elsevier Ltd. All rights reserved. Keywords: Biofilm; Constructed wetlands; Batch; INT reduction; Protein; Polysaccharide 1. Introduction Constructed wetlands have proved to be an effective low cost treatment system which utilizes the interactions of emergent plants and microorganisms in the removal of wastewater pollutants [1,2]. The primary mechanism for removal of carbon is via filtration of suspended solids, followed by biological degradation [3,4]. Con- structed wetlands may be classified as free water surface (FWS), subsurface flow (SSF) or batch systems. Recently it has been shown that batch systems can enhance aerobic degradation processes [5]. Urban population pressures have placed restrictions on the land available for wastewater treatment facilities. Vertical subsurface flow wetlands operated in batch mode enhance aeration and nitrification of ammonia [5]. In this study, we have adapted methods used to study microbial communities in sediments and within biofilms to study the development and activity of biofilms within ARTICLE IN PRESS *Corresponding author. E-mail address: sragusa@chem.eng.usyd.edu.au (S.R. Ragusa). 0043-1354/$-see front matter r 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.watres.2004.03.039