Microthrix parvicella foam control using a rapid method for assessing chlorine effect in activated sludge Ramírez G.W.*, Alonso J. L.*, Basiero J.A.**, Bernácer I.** and Morenilla J.J.** *Instituto de Hidrología y Medio Natural, Universidad Politécnica, Camino de Vera 14, Valencia, Spain (E- mail: **Entidad de Saneamiento de Aguas, Generalidad Valenciana, Valencia, Spain. The filamentous bacterium Microthrix parvicella is one of the bacteria causing the main dysfunctions of activated sludge systems, particularly foaming problems. Chlorine has been used for filamentous microorganisms foaming control. Addition of chlorine is effective when the applied dosage is enough to kill the filaments extending from the activated sludge floc, but not so high as to inhibit significantly the floc-forming microorganisms. Introduction The authors would like to express appreciation for the support of the sponsors: the Entidad de Saneamiento de Aguas de la Comunidad Valenciana. Results & Discussion Chlorine applications were done while the floc was alive (Figure 3c) and they were stopped when the floc was injured. The filamentous microorganisms were successfully controlled without any increases in effluent suspended solids (TSS) (Fig. 4) and effluent COD (Figure 5). The scum diminishes due to effect of chlorine. Exposure to chlorine has been shown to damage cell membranes and to alter associated function. The filamentous microorganisms were successfully controlled with the chlorine dosage applied (6 and 8 g Cl 2 /kg VSS.d). Dosages lower than 6 g Cl 2 /kg VSS.d were not effective. The effect of chlorine-induced damage on Microthrix parvicella was assessed using suitable fluorescent intracellular stains (BL). A major advantage of the nucleic acid dye technology, used in this study, is the potential for monitoring the in situ resistance to chlorine of filamentous bacteria in bulking sludge, as well as to assess rapidly the effect of toxicant additions on bacteria flocs during the period in which treatments are made. Conclusions Figure 2: Gandia plant, A) aeration tank and B) secondary clarifier High numbers of filamentous bacteria were detected. From the microscopic observation and fluorescent in situ hybridisation (Figures 3a and 3b) results it was evident that the Microthrix parvicella were the predominant filamentous group in the activated sludge. By using FISH it was possible to determine accurately the identity of the bacterium, allowing remedial actions for a precise bacterial species. Figure 5: Relationship between chlorine dosage (L) and effluent COD (mg.L -1 ) in chlorinated activated sludge A major advantage of the nucleic acid dye technology, used in this study, is the potential for monitoring the in situ resistance to chlorine of filamentous bacteria in foaming sludge, as well as to assess rapidly the effect of toxicant additions on bacteria flocs during the period in which treatments are made Figure 1: Scanning electron micrograph of Microthrix (x2,000). Figure 4: Relationship between chlorine dosage (L) and effluent TSS (mg.L -1 ) in chlorinated activated sludge AB Foaming chlorination and wastewater treatment plant The Gandia plant receives wastewater from civil area of Gandia (Valencia, Spain), 128,000 inhabitant equivalent, and treats 60,000 m 3 /d of sewage. The process uses mechanical treatments, equalization, biological treatment, secondary clarification, sludge treatment, anaerobic digestion and chlorine disinfection. Chlorine was added in surface and the dosages used were 6 and 8 g Cl 2 /kg VSS.d. Live/Dead (L/D) counts Samples (1 ml) were mixed with 3 µl L/D stain mixture of A (syto 9) and B (propidium iodide) (1:1) nucleic acid stains (Molecular Probes Inc., 1998), and incubated in the dark for 15 min at room temperature. Filamentous bacteria were evaluated as live, injured and dead following the technique of Ramírez et al. (2000). Material & Methods Recently, a new fluorescent technique LIVE/DEAD  BacLight TM Viability kit (Molecular Probes Inc., Eugene, Oregon) (BL) has facilitated the rapid assessment of the extent and site of disinfection damage in filamentous and floc bacteria. Activated sludge foaming by filamentous bacteria Microthrix parvicella (Figure 1) was detected in an urban wastewater treatment plant (Figures 2a and 1b). The massive proliferation of Microthrix parvicella was easily reduced by chlorination. In this study the effect of chlorine induced damage on Microthrix parvicella and floc bacteria was assessed employing the BL method. A B Figure 3: A) Direct ilumination light; B) Identification of Microthrix parvicella filaments using Mpa645 probe (x1,250) C) BL, floc alive (green) and M. parvicella dead (red) C