Seasonal shifts of the microbial community structure in a winery waste-impacted wetland soil Lukas M. Rohr 1 , Nthabiseng Mashaphu 1 , Craig Sheridan 2 , Marla Tuffin MRSSAf 1 , Stephanie G. Burton MRSSAf 2 & Don A. Cowan FRSSAf 1 * 1 Institute for Microbial Biotechnology and Metagenomics (IMBM), University of the Western Cape, Bellville, Cape Town, 7530 South Africa *Author for correspondence: e-mail: dcowan@uwc.ac.za 2 Biocatalysis and Technical Biology Unit, Cape Peninsula University of Technology, South Africa Wetlands have been identified as viable systems for treating agroindustrial and other effluents. However, the microbial contribution to the bioremediation processes in wetlands is poorly understood. We have investigated the diversity and dynamics of microbial communities in a natural wetland system used to bioremediate winery waste in the Western Cape region, South Africa. Denaturing gradient gel electrophoresis (DGGE) profiles of 16S rRNA gene fragments amplified from surface wetland soil samples (0–5 cm depth) collected between February 2005 and February 2006 indicated that major qualitative and quantitative changes occurred in the structure of the bacterial, total archaeal and methanogenic communities over the course of the year. Conversely, no significant differences were detected in samples recovered over the same period at ca. 25 cm depth. The DGGE patterns from surface samples grouped into two distinct seasonal clusters, which correlated well with considerable differences in effluent chemistry and hence may be related to harvesting activities at the wine estate. The analysis of temporal intensity variations of specific DGGE bands, which reflect the population dynamics of the respective phylotypes, was used to identify taxa potentially involved in the degradation of winery waste compounds. We propose that this is a valid approach to the identification of dominant and temporally responsive microorganisms in passive bioremediation studies. Keywords: anaerobic degradation, DGGE, microbial community structure, wetland, winery waste. INTRODUCTION Agroindustrial wastewaters often contain higher concentra- tions of organic matter and nutrients than processed municipal effluents and therefore need to be treated before being dis- charged in the environment (Ibekwe et al., 2003). Artificial and natural wetlands have long been recognised as potential treat- ment options for wastewater streams, especially in rural areas where land is more available and capital and technical resources are limited (Office of Energy Management and Conservation, 2001). Removal of pollutants in wetlands is mainly achieved by physicochemical processes such as sedimentation, filtration, precipitation and adsorption as well as through bacterial trans- formations, but higher plants have also been found to contribute significantly to the bioremediation process (Brix, 1997; Gagnon et al., 2007; Arienzoa et al., 2009). The Cape Winelands, situated in the Western Cape Province of South Africa, is one of the world’s premium wine-growing regions, producing the bulk of the country’s more than 900 million litres of wine per annum (National Department of Agriculture, 2007). The volume of wastewater generated in wine production is dependent on factors such as production capacity of the winery, type of wine and vat size and has been estimated to 0.2–3 l per litre of wine produced (Shepherd et al., 2001a and references therein). Winery wastewater primarily originates from washing operations during the crushing and pressing of grapes, as well as from rinsing of fermentation tanks, barrels and other equipment (Petruccioli et al., 2000). Such wastewater contains residues of grape pulp, skin and seeds and the different compounds used in the filtration, pre- cipitation and cleaning processes (Bustamante et al., 2005). Winery effluents typically have a pH of 3–5 with ethanol (2.5–8 g l –1 ) as the primary contributor to the chemical oxygen demand (COD); sugars, glycerol, organic acids and phenolic substances have been reported as minor components (Malandra et al., 2003; Colin et al., 2005; Mulidzi, 2007). The release of wastewater, and thus the COD, varies considerably seasonally, with the highest levels occurring during the harvest period and lesser peaks later in the year when the wine is racked (Kalyuzhnyi et al., 2001; Grismer et al., 2003). Since the intermittently high flow rate and organic load of winery effluents can cause the failure of activated sludges in municipal wastewater treatment plants (Beck et al., 2005), on-site COD reduction is often preferable. The main technologies described to achieve this include natural evaporation in ponds (Bories et al., 2005), aerobic biological treatment (Petruccioli et al., 2000, 2002; Malandra et al., 2003; Eusébio et al., 2004), and a variety of methods based on anaerobic digestion. Conventional plants, however, often require large investment, are difficult to control and, in case of aerobic systems, have a high energy consumption and produce large amounts of sludge that needs to be disposed of (Daffonchio et al., 1998; Bustamante et al., 2005). There is therefore a need for efficient and sustainable treatment methods for winery effluents (Ruíz et al., 2002). The performance of constructed wetlands in winery waste treatment has been the subject of a series of studies (Grismer et al., 2001; Shepherd et al., 2001a,b; Grismer et al., 2003; Mulidzi, 2007; Arienzoa et al., 2009; Christen et al., 2010). A pilot-scale subsurface-flow system combined with an upflow sand Transactions of the Royal Society of South Africa Vol. 66(1), February 2011, 41–53 ISSN 0035-919X Print / 2154-0098 Online © 2011 Royal Society of South Africa DOI: 10.1080/0035919X.2011.572428 http://www.informaworld.com/ttrs