While overexposure to phenol is usually associated with decreases or complete loss in specific growth rate, specific oxygen utilization and enzyme activity, microorganisms in the aerobic granules should be capable of a variety of adaptive physiological responses to tolerate phenol toxic-ity. To investigate how phenol loading affected the structure, activity and metabolism of aerobic granules, four SBRs were fed with phenol as sole carbon and energy source at loading rates of 1.0, 1.5, 2.0, and 2.5 g phenol l-1 d-1 (Jiang et al., 2004a). After about two months of operation, all four reactors reached a steady state, as evidenced by stable biomass concentrations and constant phenol removal efficiencies. Compact granules with good settling ability were maintained at loadings up to 2.0 g phenol l-1 d-1, but structurally weakened granules with enhanced production of extracellular polymers and proteins and significantly lower hydrophobicities were observed at the highest loading of 2.5 g phenol l-1 d-1. Specific oxygen uptake rate, catechol 2,3-dioxygenase (C23O) and catechol 1,2-dioxygenase (C12O) activities peaked at a loading of 2.0 g phenol l-1 d-1, and declined thereafter. The granules degraded phenol completely in all four reactors, mainly through the meta-cleavage pathway as C23O activities were significantly higher than C12O activities.
At the highest loading applied, the anabolism and catabolism of microorganisms were regulated such that phenol degradation proceeded exclusively via the meta-pathway, apparently to produce more energy for overstimulation of protein production as additional protection against phenol toxicity. Microorganisms are known to regulate synthesis of extracellular polymers (ECPs) and modify ECP properties as a microbial response against the effect of antimicrobial agents. ECPs can form a protective shield for the cells against the adverse influences of the external environment, and delay or prevent toxicants from reaching microbes by acting as a diffusion limitation barrier. Such preferential production of proteins over polysaccharides in the ECPs in the aerobic granules has also been observed in other biofilms exposed to phenol (Fang et al., 2002). Possible explanations for the elevated production of proteins include induction of heat shock-like proteins as a defense mechanism against high phenol concentrations, and induction of special proteins that could be involved in the catalytic degradation of phenol and other potentially toxic compounds (Benndorf et al., 2001).
Degradation of phenol may proceed via either the ortho (C12O) or the meta (C23O) cleavage pathway, which are often found to occur simultaneously in the same strain (Kiesel and Muller, 2002). With aerobic granules, phenol biodegradation proceeded mainly via the meta-pathway, as C23O
activities were significantly higher than C12O activities (Jiang et al., 2004a). Previous studies have shown that the ortho-pathway dominated the meta-pathway at low growth rates due to affinity reasons, whereas the meta-pathway attained the highest growth rates (Filonov et al., 1997). Half-saturation constants (Ks) are also usually higher for the meta-pathway than for the ortho-pathway (Muller and Babel, 1996). Thus high-affinity/ low-rate properties are found at low substrate concentrations in contrast to low-affinity/high-rate properties in situations with increased levels of substrate. From a kinetics point of view, the high phenol concentrations employed might help explain the observed predominance of the meta over the ortho-cleavage pathway in the aerobic phenol-degrading granules.
It should be noted that the choice of cleavage pathways is also mediated by metabolic factors. For kinetics reasons, the shorter route for energy production through the meta-pathway corresponds to a higher overall growth rate (Kiesel and Muller, 2002). This may be considered a selective advantage when alternative metabolic routes have to compete successfully for a common carbon/energy source whenever there is excess substrate, but the rate increase is obtained at the expense of a lower efficiency of carbon conversion into biomass. It is very likely that the selection pressure exerted by high phenol loads can drive the microbial community to regulate its metabolic pathways so as to maintain a balance with the external pressure by consuming non-growth-associated energy to counteract the toxicity-related inhibition of cellular activity and deterioration in granule structure. Part of non-growth-associated energy produced by metabolism might be used to maintain the integrity of cell membranes, since energy expended for this purpose would be expected to be higher at higher phenol concentrations, and part of the energy was directed towards the production of ECPs as shown earlier (Jiang et al., 2004a).
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