Research Article Received: 2 July 2012 Revised: 17 September 2012 Accepted: 19 September 2012 Published online in Wiley Online Library: (wileyonlinelibrary.com) DOI 10.1002/jctb.3975 In situ imaging of biopolymers and extracellular enzymes in activated sludge flocs of a municipal wastewater treatment plant Szabolcs Szilveszter, Botond R ´ aduly, * Be´ ata ´ Abrah ´ am and Szabolcs L ´ anyi Abstract BACKGROUND: The aim of this study was to investigate biopolymers and extracellular enzymes in whole activated sludge flocs originating from a full-scale wastewater treatment plant, by means of confocal laser scanning microscopy (CLSM) techniques. Both sectioned and whole activated sludge floc samples have been stained using specific fluorochromes and immunostains in order to visualize the structural and functional characteristics of these bioaggregates. Samples were stained for visualization of lipids, sugars, total cells, esterase enzyme producing bacteria, and for β -glycosidase (EC3.2.1.21), alkaline phosphatase (EC3.1.3.1) and trypsin (EC3.4.21.4) enzymes. Simultaneous staining schemes were applied and immunostaining specificity tests were performed. RESULTS: By the CLSM imaging and the 3-D reconstruction of the stained flocs the distributions of the targeted floc components were successfully assessed. The immunostain specificity controls gave satisfactory results in each case. The reflected total cells-to-enzymes ratio was repeatedly higher for the sectioned samples. CONCLUSIONS: The CLSM imaging of whole sludge flocs delivers valuable information on the spatial distribution of the floc build-up materials, with a satisfactory visualization accuracy of the individual components. The images of whole and sectioned samples showed similar distributions of the floc components, but the consistent differences revealed in cells-to-enzymes ratios call for further research. c  2012 Society of Chemical Industry Supporting information may be found in the online version of this article. Keywords: activated sludge floc; CLSM imaging; biopolymers; extracellular enzymes; immunostaining; fluorochromes INTRODUCTION The activated sludge process is an environmental biotechnology widely used for the treatment of municipal wastewater. It is based on the growth of a mixed microbial community that will consume organic matter found in the wastewater through its biochemical metabolism. The microorganisms and other particles present in the wastewater aggregate and adhere through the bioflocculation process, forming so called activated sludge flocs. The formed flocs have relatively good settling properties, and therefore are normally separated from the treated effluent by gravity settling, to be then recycled in the bioreactor, for the removal of further amounts of organic pollutant. Besides the formation of microbial colonies by cell division, physico-chemical adhesion of cells and other particles is the main factor affecting bioflocculation. 1 Microbial aggregates are kept together by means of biopolymers of microbial origin, called extracellular polymeric substances (EPSs). EPS is used as a comprehensive term for different classes of macromolecules, such as polysaccharides, proteins, nucleic acids, (phospho-) lipids and other polymeric compounds that have been found to occur in the extracellular space of microbial aggregates. 2 EPSs are responsible for the cohesive forces that keep these aggregates together, forming biofilms, granules and sludge flocs. 3 These biopolymers fill and form the space between the cells, determine the architecture and morphology of the matrix in which the cells live, and can be considered as the ‘house’ of the microorganisms onto which organic fibers, particles and various colloids can adsorb. 4 The EPS matrix is three-dimensional, highly hidrated, often charged, and constitutes the main fraction of the activated sludge flocs, while bacteria make up only a minor fraction (approximately 5–20%) of the floc. 5 The different EPS components are synthesized and degraded in a manner to support the life of the microorganisms in microbial aggregates, hence the EPS matrix is heterogeneous in composition, architecture, and over time it behaves as a dynamic system. 4 The important contributions of the different EPS components to the structural stability of aerobic granules were described in recent studies. 6 – 9 These studies were conducted on granules of laboratory provenience (acetate-, phenol-, glucose- and pepton-fed granules), aiming to reveal structural differences ∗ Correspondence to: B. R´ aduly, Department of Bioengineering, Sapientia Hungarian University of Transylvania, Pta. Libertatii 1, 530104 Miercurea-Ciuc, Jud. HR, Romania. E-mail: radulybotond@sapientia.siculorum.ro Department of Bioengineering, Sapientia Hungarian University of Transylvania, 530104 Miercurea Ciuc, Romania J Chem Technol Biotechnol (2012) www.soci.org c  2012 Society of Chemical Industry