Complementary independent molecular, radioisotopic and fluorogenic techniques to assess biofilm communities in two wastewater wetlands

Abstract

Optimisation of nutrient removal processes requires a detailed understanding of the microbial physiologies actually occurring within the biomass of different treatment zones, and also knowledge of how these communities respond to environmental factors. This paper describes a suite of four independent and complementary, microbiological techniques, utilised to obtain detailed assessments of wastewater bacterial biomass. Examples of these techniques are shown as applied to biofilms from two surface-flow wetlands used for treating sewage effluent. Intact biofilms were prepared via freezing, followed by cryosectioning and direct microscopic observation. This approach was combined with prior, in situ incubation of biofilm with radiolabelled thymidine to assess in situ bacterial growth rates. Individual bacterial cells were assessed microscopically for in situ respiratory activity and phylogenetic identity, respectively through use of an inducibly-fluorescent redox stain (CTC), and fluorescent in situ hybridisation (FISH) of ribosomal RNA (rRNA). These complementary techniques enabled assessment of individual biofilm clusters according to their in situ status (growth, respiration, spatial location and phylogenetic affiliation). This approach detected significant spatial variations in biofilm; limited the bias inherent in data obtained using any one technique, and provided greater detail of subpopulations within the microbial community.

Characterization of bacterial communities of a constructed wetland in cold conditions

“…3). These percentages, like the b/g ratio variation, were similar to those reported by Flood et al (1999) in a FWS biofilm, showing a decrease in b (62 to 41%) and an increase in g (11 to 34%) from the inlet to the outlet of a wetland, respectively. In our system, the b/g ratio evolved from a 3 : 1 value in the influent to 7 : 1 in the SP effluent, decreasing to the initial proportion in the final effluent.…”

“…Previous works on wetlands have focused on the biofilm communities during summer with different results. Flood et al (1999) found 12 and 4% NEU in the inlet and outlet parts, respectively, of a low loaded wetland. Silyn-Roberts and Lewis (2001) studied a wetland as a tertiary treatment of a dairy effluent and the percentage of NEU in the biofilm fluctuated from 0 to 1.5%.…”

“…Juretschko et al, 1998) and trickling filters (see e.g. Okabe et al, 1999) almost no evidence on its application to low-cost systems as constructed wetlands is found in the literature (Flood et al, 1999;Silyn-Roberts and Lewis, 2001). Moreover, studies on these systems are usually carried out during the growing season and no data is usually available on the winter period, which is the time of year for which low-cost treatment systems are designed as they have to maintain their removal efficiency even in harsh conditions.…”

“…The specific probe/DAPI ratio was selected for a better comparison with the few studies on FISH in constructed wetlands (Flood et al, 1999;Silyn-Roberts and Lewis, 2001). In the present study the bacterial community was generally dominated by the Betaproteobacteria (average % of DAPI: 38.3%Ϯ19.1) (Fig.…”

Evaluation of the Redox Dye 5-Cyano-2,3-Tolyl-Tetrazolium Chloride for Activity Studies by Simultaneous Use of Microautoradiography and Fluorescence In Situ Hybridization

“…An easy and often used method is the reduction of CTC by the respiratory electron transport chain to insoluble, fluorescent formazan crystals (CTF). This method is commonly applied to determine respiratory activity and viability of bacteria in many different complex microbial systems (4,6,13). However, several studies indicate that this method is unable to detect all active cells and thus underestimate the real number if it is compared to the other two methods (e.g., references 6 and 14).…”