Abundance of amoA genes of ammonia-oxidizing archaea and bacteria in activated sludge of full-scale wastewater treatment plants Tawan Limpiyakorn a,b,⇑ , Puntipar Sonthiphand b,c , Chaiwat Rongsayamanont b,c , Chongrak Polprasert d a Department of Environmental Engineering, Faculty of Engineering, Chulalongkorn University, Bangkok, Thailand b National Center of Excellence for Environmental and Hazardous Waste Management, Chulalongkorn University, Bangkok, Thailand c International Postgraduate Programs in Environmental Management, Graduate School, Chulalongkorn University, Bangkok, Thailand d School of Bio-Chemical Engineering and Technology, Sirindhorn International Institute of Technology, Thammasat University, Pathumthani, Thailand article info Article history: Received 8 September 2010 Received in revised form 18 November 2010 Accepted 19 November 2010 Available online 25 November 2010 Keywords: Ammonia-oxidizing archaea Ammonia-oxidizing bacteria amoA gene Activated sludge Wastewater treatment plant abstract In this study, the abundance and sequences of amoA genes of ammonia-oxidizing archaea (AOA) and bacteria (AOB) were determined in seven wastewater treatment plants (WWTPs) whose ammonium concentrations in influent and effluent wastewaters varied considerably (5.6–422.3 mgN l À1 and 0.2– 29.2 mgN l À1 , respectively). Quantitative real-time PCR showed that the comparative abundance of AOA and AOB amoA genes differed among the WWTPs. In all three industrial WWTPs, where the influent and effluent contained the higher levels of ammonium (36.1–422.3 mgN l À1 and 5.3–29.2 mgN l À1 , respectively), more than four orders of magnitude higher numbers of AOB amoA genes than AOA amoA genes arose (with less than the limit of detection of AOA amoA genes). In contrast, significant numbers of AOA amoA genes occurred in all municipal WWTPs (with ammonium levels in the influent and effluent of 5.6–11.0 mgN l À1 and 0.2–3.0 mgN l À1 , respectively). Statistical analysis suggested that compared to other plants’ parameters, the ammonium levels in the plants’ effluent showed correlation with the high- est p value to the abundance of AOA amoA genes. Ó 2010 Elsevier Ltd. All rights reserved. 1. Introduction For a few decades, it had been believed that autotrophic ammo- nia oxidation only occurred in members of the domain Bacteria (e.g., aerobic ammonia-oxidizing bacteria, AOB and anaerobic ammonia-oxidizing bacteria, Anammox). This understanding has been changed by recent molecular and culture-dependent evi- dence showing that autotrophic ammonia oxidation also occurs in the domain Archaea. The use of molecular tools led to the discov- ery of an archaeal ammonia monooxygenase (amo) gene that en- coded ammonia monooxygenase (AMO), a key enzyme for ammonia oxidation, in marine and terrestrial system environ- ments (Venter et al., 2004; Treusch et al., 2005). The first and the only available ammonia-oxidizing archaea (AOA) isolate, ‘‘Candida- tus Nitrosopumilus maritimus’’, was obtained from a marine aquarium tank (Konneke et al., 2005). This AOA isolate acquires en- ergy for growth by aerobically oxidizing ammonia to nitrite. Like AOB, it contains putative genes for all three subunits of AMO (i.e., amoA, amoB, and amoC). Later, thermophilic and moderately thermophilic enriched AOA cultures, ‘‘Candidatus Nitrosocaldus yellowstonii’’ and ‘‘Candidatus Nitososphaera gargensis,’’ were cul- tivated from hot spring systems (De La Torre et al., 2008; Hatzenp- ichler et al., 2008). Quantitative real-time PCR analysis has been applied to several environmental samples to reveal the compara- tive abundance of AOA and AOB. In many cases, such as in seawa- ter, estuarine sediments, and soil, the archaeal amoA gene outnumbered that of AOB (Wuchter et al., 2006; Caffrey et al., 2007). In Wastewater treatment plants (WWTPs), AOA was found for the first time to occur in five out of nine activated sludge bioreac- tors in the US (Park et al., 2006) and, later, in two WWTPs in Hong Kong (Zhang et al., 2009). This suggests the widespread presence of AOA in WWTPs and their potential role in removing nitrogen from wastewater. Nonetheless, the comparative abundance of AOA and AOB had not been investigated in both studies. Erguder et al. (2009) reviewed and provided a conclusive discussion on the fac- tors influencing the presence or/and dominance of AOA in different environments. AOA is proposed to be the possible major ammonia oxidizer in environments low in ammonium (Erguder et al., 2009). Evidence from culture-dependent studies corresponds to this hypothesis (Hatzenpichler et al., 2008; Martens-Habbena et al., 2009). It must be noted that, in WWTPs, the ammonium concentra- 0960-8524/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2010.11.085 ⇑ Corresponding author. Address: Department of Environmental Engineering, Faculty of Engineering, Chulalongkorn University, Phayathai Road, Pathumwan, Bangkok 10330, Thailand. Tel.: +66 2 218 6668; fax: +66 2 218 6667. E-mail addresses: tawan.l@chula.ac.th, miketawan@yahoo.com (T. Limpiyakorn). Bioresource Technology 102 (2011) 3694–3701 Contents lists available at ScienceDirect Bioresource Technology journal homepage: www.elsevier.com/locate/biortech