Long-term storage and subsequent reactivation of aerobic granules Xinhua Wang, Hanmin Zhang * , Fenglin Yang, Yongfei Wang, Mingming Gao Key Laboratory of Industrial Ecology and Environmental Engineering, MOE, School of Environmental and Biological Science and Technology, Dalian University of Technology, Linggong Road 2, Dalian, Liaoning 116024, PR China article info Article history: Received 29 November 2007 Received in revised form 29 February 2008 Accepted 1 March 2008 Available online 28 April 2008 Keywords: Aerobic granules Storage Reactivation Extracellular polymeric substances (EPS) Fluorescence in situ hybridization (FISH) abstract This study investigated a seven month storage and the subsequent reactivation of aerobic granules. The granule size and structure integrity were remained during storage, whereas some cavities and pleats appeared on the surface and further deteriorated the settleability. Along with the reactivation, the phys- ical characteristics and microbial activities of aerobic granules were gradually improved. Activities of heterotrophs and nitrifiers can be fully recovered within 16 days and 11 days, respectively. Nitrifiers decayed slower during storage and reinstated rapider during reactivation than heterotrophs. In fresh aer- obic granules, the dominated ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) were Nitrosomonas and Nitrospira, respectively. During storage, the initially dominated populations decayed rapider than the initially less dominated ones. Extracellular polymeric substances (EPS) signifi- cantly decreased within the first month, and then gradually accumulated during the last six months stor- age. Accumulation of EPS was an effective strategy for maintaining structural integrity of aerobic granules during long-term storage. Ó 2008 Elsevier Ltd. All rights reserved. 1. Introduction Aerobic granulation technology has been extensively investi- gated (Morgenroth et al., 1997; Peng et al., 1999; Tay et al., 2001; Wang et al., 2006a). Aerobic granules have many advantages over conventional bioflocs, such as strong structure, excellent sett- leability, high biomass retention, and resistance to inhibitory and toxic compounds. It is believed that aerobic granulation would be a novel and promising biotechnology for wastewater treatment. From environment engineering viewpoint, the storage stability, physicochemical characteristics, and reactivation efficiency of aer- obic granules are crucial for their practical application. Several studies on storage of aerobic granules for less than four months showed that granules had good storage stability and bioactivity reactivation ability (Zhu and Wilderer, 2003; Liu et al., 2005; Zeng et al., 2007). However, it is unknown about the differences in decay and reactivation processes taking place in different microbial pop- ulations within aerobic granules. In addition, longer storage of aer- obic granules needs to be investigated to ensure the feasibility of aerobic granules commercial production. Extracellular polymeric substances (EPS) is biopolymers con- sisting of polysaccharides (PS), proteins (PN), nucleic acid, etc. They contribute to the structure stability and integrity of aerobic gran- ules, and serve as maintenance energy source during starvation (Zhang and Bishop, 2003; Wang et al., 2005, 2006b, 2007b). During storage of aerobic granules, there are two distinct demands for EPS: they ought to be utilized for energy maintenance, while they also needed to be reserved to keep structure integrity. Investiga- tion on the evolution of EPS would enrich the current knowledge on aerobic granules storage. This study investigated a long-term storage up to seven months as well as the subsequent reactivation of aerobic granules. Special attention was focused on the decay and reactivation processes for different microbial populations and the role of EPS on storage. It is expected that this research could provide useful information for the application of aerobic granulation technology in biological wastewater treatment and further commercial production of aero- bic granules. 2. Methods 2.1. Cultivation of aerobic granules Aerobic granules were cultivated in a internal-circulate sequencing batch reactor (SBR; working volume of 2.5 l; 100 cm in height and 8 cm in diameter for down-comer; 70 cm in height and 4 cm in diameter for riser) at room temperature 20–30 °C. The reactor was operated sequentially in 6 h cycles, with 2 min of substrate filling, 355 min of aeration, 2 min of settling and 1 min of effluent withdraw. Effluent was discharged at the middle port of the reactor. Fine air bubbles for aeration were supplied through a fine-bubble diffuser at the reactor bottom with an air- flow rate of 1.8 cm s 1 . Synthetic wastewater mainly consists of 0960-8524/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2008.03.024 * Corresponding author. Tel.: +86 411 84706172; fax: +86 411 84708083. E-mail address: zhhanmin@126.com (H. Zhang). Bioresource Technology 99 (2008) 8304–8309 Contents lists available at ScienceDirect Bioresource Technology journal homepage: www.elsevier.com/locate/biortech