The activated sludge seed failed to maintain an adequate level of biomass within reactor R1 and could not acclimate quickly enough to allow phenol-degrading microorganisms to multiply and remove the phenol. As a consequence, phenol rapidly accumulated in the reactor and the biomass was completely washed out of R1 within four days after startup. On the other hand, the use of acetate-fed granules as a starting seed resulted in the development of stable phenol-degrading granules with good settling ability, good biomass retention and good metabolic activity, as evidenced by the low SVI values, stable biomass concentrations and nearly complete phenol removal. Although there was a slight lag in the ability of the acetate-fed granules to degrade phenol initially, the compact structure of the acetate-fed granules likely provided the microorganisms with adequate protection against phenol toxicity and minimized sludge washout, thus allowing the buildup of a critical population of phenol-degrading microorganisms such as the filamentous bacteria observed in Fig. 10.12. The different DGGE banding patterns in the steady-state phenol-degrading granules compared to the acetate-fed granule seed indicated that some form of community restructuring had taken place. The granules quickly acclimated to the phenol load and achieved complete phenol removal three days after start-up. The granules stabilized within two weeks after start-up, with little change in biomass concentration, phenol removal, and specific mineralization activity.
Exposure of the granules to phenol triggered a two-fold increase in ECP content two weeks into the reactor operation. This was associated with an increase in PN production and the proliferation of sheath bacteria on the granule surface. ECPs are the construction materials for microbial aggregates and are responsible for their structural integrity. They also serve a protective function and are known to form a shield against the adverse influences of the external environment by acting as a diffusion limitation barrier to delay or prevent toxicants from reaching the microorganisms (Wingender et al., 1999). PS and PN play different roles within the ECP matrix, the stability of which depends on the interactions between PS and PN and the other macromolecules present (Flemming and Wingender, 2001; Sutherland, 2001). A similar preferential production of PN over PS in ECPs has also been observed in other biofilms and granules exposed to high phenol concentrations (Fang et al., 2002; Jiang et al., 2004a).
The propagation of filamentous bacteria is generally thought to be favored by low nutrient or low oxygen conditions (Jenkins et al., 1993). According to the kinetic selection theory, filamentous bacteria are considered to be slow-growing microorganisms with maximum growth rates (^max) and affinity constants (Ks) lower than floc-forming bacteria (Martins et al., 2004). In systems where the substrate concentration is high, like in plug-flow reactors and the SBR system used in the current study, the filamentous bacteria should be suppressed since their growth rate is expected to be lower than that for the floc-forming bacteria. Therefore, the emergence and eventual dominance of filamentous bacteria in the granules in R2 was an interesting and unexpected development. Even under the high concentrations of phenol substrate in R2, filamentous bacteria were the dominant bacterial morphotype residing on the granule surface. Although stresses such as substrate overloads are known to induce the proliferation of filamentous bacteria, this is thought to be the result of the oxygen shortage induced by the transient substrate overload rather than the massive substrate input itself (Pernelle et al., 2001). However, oxygen deficiency is not expected to be a problem in the current study because of the high aeration rates employed in R2. The precise reasons for the dominance of the filamentous bacteria in the phenol-degrading granules must be linked to their ability to compete in a highly toxic environment. The tolerance to phenol is probably due to the presence of a sheath that is composed of proteins, polysaccharides, and lipids, which would serve as a protective barrier against phenol toxicity. This notion is corroborated by surveys of aquatic biofilms in highly polluted rivers where the dominance of filamentous bacteria was associated with their ability to tolerate high concentrations of pollutants and metals in the rivers (Brummer et al., 2003). Chlorine decay assays also lend support to this idea, as sheathed Sphaerotilus natans are known to be several-fold more resistant to chlorination than the floc-forming but sheathless Acinetobacter anitratus (Caravelli et al., 2003).
Activated sludge-derived granules were a more appropriate inoculum than activated sludge for the development of phenol-degrading granules. The use of activated sludge resulted in system failure. On the other hand, the compact structure of the granules afforded sufficient protection against phenol toxicity and minimized sludge washout, thus facilitating a rapid microbial acclimation towards phenol biodegradation. This strategy of using granules as a microbial inoculum has practical implications for starting up aerobic granulation systems treating wastewaters containing high concentrations of toxic chemicals.
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