Ecological Engineering 105 (2017) 66–77 Contents lists available at ScienceDirect Ecological Engineering jo ur nal home p ag e: www.elsevier.com/locate/ecoleng Methanogenic activity in the biomass from horizontal subsurface flow constructed wetlands treating domestic wastewater Mario Sepúlveda-Mardones, Daniela López, Gladys Vidal ∗ Engineering and Environmental Biotechnology Group, Environmental Science Faculty & Center EULA–Chile, University of Concepción, P.O. Box 160-C, Concepción, Chile a r t i c l e i n f o Article history: Received 26 January 2017 Received in revised form 19 April 2017 Accepted 20 April 2017 Available online 9 May 2017 Keywords: Subsurface flow constructed wetland Methane production Methanogenic activity assay Wastewater Accumulated solids seasonality a b s t r a c t The aim of this study was to evaluate the methanogenic activity of horizontal subsurface flow (HSSF) constructed wetlands treating domestic wastewater. The analysis was carried out in four 4.5 m 2 pilot-scale HSSF systems, two planted with Phragmites australis and two planted with Schoenoplectus californicus. A specific methanogenic activity (SMA) assay was carried out with the microbial biomass attached to the gravel of the HSSF systems to account for the different seasons. Accumulated solids throughout the entire operational time were also assessed. Results showed that biochemical oxygen demand (BOD 5 ) removal efficiencies averaged 67.6 ± 9.9% with organic loading rates (OLR) of4.4–5.8 g BOD 5 m -2 d -1 . Total suspended solids (TSS) removals were 92.9 ± 3.4%. Solids accumulation rates ranged between0.7 ± 0.3 and 1.5 ± 0.6 kg TSS m -2 year -1 , respectively. Microbial biomass extracted from the HSSF presented a SMA regarding volatile suspended solids (VSS) of 0.018–1.220 g COD CH4 g -1 VSS d -1 , corresponding to methane productions between 176 and 15227 mg CH 4 m -2 d -1 . Methanogenic activity after 550d were significantly lower (73.6%) than after 1200d of operation. The influent inlet zone of the HSSF systems showed 14–39% higher methanogenic activity than the middle and exit zones in the first 550d of operation. However, after 1100d of operation, the middle and exit zones presented 18–55% higher methanogenic activity than the inlet zone. The plant species did not affected the methanogenic activity of the biofilm from the HSSF system. The results of the present study showed that microbial biomass development through operation time, seasonality and the applied OLR influence methane production in HSSF systems. © 2017 Elsevier B.V. All rights reserved. 1. Introduction Horizontal subsurface flow (HSSF) constructed wetlands have been a widely used technology for the removal of organic mat- ter and suspended solids from domestic wastewater (Puigagut et al., 2007; Vera et al., 2011; López et al., 2015). Organic mat- ter removal efficiencies measured as biochemical oxygen demand (BOD 5 ) and chemical oxygen demand (COD) in HSSF systems have been described in the ranges73–97 and 54–91%, respectively, accounting for average organic removal rates of 14.9 g COD m -2 d -1 (Vymazal and Kröpfelová, 2009). Suspended solids removal effi- ciencies have been documented in the ranges of 85–92% in terms of total suspended solids (TSS) (Caselles-Osorio et al., 2007). In this manner, HSSF effluent organic matter and suspended solids con- ∗ Corresponding author. E-mail address: glvidal@udec.cl (G. Vidal). centrations are usually in the ranges of 9–55 and 10–70 mg L -1 of BOD 5 and TSS, respectively (Vera et al., 2011). Specifically, particulate organic matter and suspended solids are mainly removed by such physical mechanisms as filtration and sed- imentation (Kadlec and Wallace, 2009). It has been shown that almost 90% of the particles in an HSSF system are removed in the first quarter of the length of the system, reducing the influ- ent organic matter by 50% (García et al., 2004). On the other hand, the dissolved organic matter is removed by biochemical reactions performed by the microbial biofilm attached to the gravel and macrophytes root system (García et al., 2010). This biofilm is mostly composed of anaerobic bacteria and archaea since HSSF deep sys- tems (>0.4m) usually operate at oxidation-reduction potentials (ORP) between -200 and +100 mV and dissolved oxygen (DO) levels < 1mg L -1 (Baptista et al., 2003; García et al., 2003). As a consequence, methane is generated as the final product of the anaerobic digestion of organic matter within an HSSF system. Methane emissions have been assessed in several HSSF systems showing broad ranges, with values of 19.2–2208 mg CH 4 m -2 d -1 http://dx.doi.org/10.1016/j.ecoleng.2017.04.039 0925-8574/© 2017 Elsevier B.V. All rights reserved.