Bacterial community structure and enzyme activities in a membrane bioreactor (MBR) using pure oxygen as an aeration source Kadiya Calderón a , Alejandro González-Martínez b , Camino Montero-Puente a , Patricia Reboleiro-Rivas a , José M. Poyatos b , Belén Juárez-Jiménez a , María Victoria Martínez-Toledo a , Belén Rodelas a,⇑ a Department of Microbiology, University of Granada, Spain b Department of Civil Engineering, University of Granada, Spain article info Article history: Received 31 July 2011 Received in revised form 27 September 2011 Accepted 29 September 2011 Available online 10 October 2011 Keywords: Membrane bioreactor MBR Enzyme activities TGGE Pure oxygen abstract A pilot-scale membrane bioreactor was used to treat urban wastewater using pure oxygen instead of air as a source of aeration, to study its influence on bacterial diversity and levels of enzyme activities (acid and alkaline phosphatases, glucosidase, protease, and esterase) in the sludge. The experimental work was developed in two stages influenced by seasonal temperature. Operational parameters (temperature, pH, BOD 5 , COD, total and volatile suspended solids) were daily monitored, and enzyme activities measured twice a week. Redundancy analysis (RDA) was used to reveal relationships between the level of enzyme activities and the variation of operational parameters, demonstrating a significant effect of temperature and volatile suspended solids. Bacterial diversity was analyzed by temperature-gradient gel electropho- resis of PCR-amplified partial 16S rRNA genes. Significant differences in community structure were observed between both stages. Sequence analysis revealed that the prevalent Bacteria populations were evolutively close to Alphaproteobacteria (44%), Betaproteobacteria (25%) and Firmicutes (17%). Ó 2011 Elsevier Ltd. All rights reserved. 1. Introduction Since the development of the activated sludge process in the early 20th century, aeration is required in wastewater treatment plants (WWTPs) to transfer oxygen to the biologically active mass of microorganisms, in order to achieve an adequate removal of or- ganic matter. Different types of aeration are employed, depending on specific treatment requirements (Mueller et al., 2002). Aeration systems involve an important contribution to wastewater plant construction and operation cost (Mueller et al., 2002; Germain and Stephenson, 2007). The use of pure oxygen as a substitute for air in the activated sludge process was proposed in the 1940s and put into commercial use in 1970 (Shammas and Wang, 2009). The main advantages found when pure oxygen is used instead of air in conventional activated sludge processes are: increased oxygen mass transfer, smaller installations due to a lower required tank volume, reduced power requirements, decreased sludge production, reduced bulking and foaming problems, ability to treat high-strength wastewaters, im- proved biokinetics, and faster treatment rates at high suspended so- lid concentrations (Brindle et al., 1998; Mueller et al., 2002; Shammas and Wang, 2009; Zupanc ˇic ˇ and Roš, 2008). These advanta- ges balance out the significant cost of oxygen generation equipment or the purchase of oxygen tanks. Membrane bioreactor (MBR) systems for wastewater treatment are based on the combination of the activated sludge process and membrane technology to separate the particulate material from water, avoiding the requirement of the secondary clarifier (Chang et al., 2011). For the last 40 years, MBR technology is one of the most used to treat industrial, domestic and municipal wastewater for the numerous advantages offered such as excellent effluent quality, low sludge production, small configuration, and flexibility for future expansion and upgrade (Miura et al., 2007; Gómez-Silván et al., 2010). In aerobic MBRs, aeration is used not only to administrate dis- solved oxygen to the microbial biomass, but also to help keeping sol- ids in suspension and minimize membrane fouling (Germain and Stephenson, 2007; Calderón et al., 2011). Use of pure oxygen as a source of aeration in MBRs allows the achievement of high removal rates of organic matter (over 90% reduction of COD and BOD 5 )(Rodríguez et al., 2010, 2011). How- ever, the effect of pure oxygen aeration on the biology of bacterial communities in MBR sludge remains poorly characterized. During the formation of the activated sludge, microorganisms use their enzymes to hydrolyze and degrade the organic matter, mostly composed by carbohydrates and proteins (Burgess and Pletschke, 2008). Evaluating microbial diversity and enzyme activities through the operating phase of MBRs is essential to achieve the 0960-8524/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2011.09.133 ⇑ Corresponding author. Address. Departamento de Microbiología, Facultad de Farmacia, Universidad de Granada, 18071 Granada, Spain. Tel.: +34 958241755; fax: +34 958246235. E-mail address: mrodelas@ugr.es (B. Rodelas). Bioresource Technology 103 (2012) 87–94 Contents lists available at SciVerse ScienceDirect Bioresource Technology journal homepage: www.elsevier.com/locate/biortech