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Studied was the activity of the enzymes involved in the bacterial assimilation of toluene by carbon source limited Acinetobacter sp. grown in titrostat under strictly controlled conditions with specific growth rates ranking from 0.015 to 0.109 h^-1 corresponding to metabolic rates (g toluene assimilated by 1 g cells per hour) from 0.038 to 0.144 h^-1. The study confirms the supposed catabolic pathway of toluene by Acinetobacter sp. via benzoate, benzaldehyde and catechol. The activities of two enzymes of this pathway were found to increase with the rise of microbial growth rate: benzaldehyde dehydrogenase and catechol-2,3-dioxygenase (C23O). Observed was a sharp increase of the specific C23O-activity with the rise of microbial metabolism from nearly maintenance rate, 0.022 h^-1, to 0.046 h^-1 which could be explained with the generally accepted view that the meta pathway catalyzed by C23O provides considerably more energy needed for the activated constructive metabolism of the organism.

Low-temperature (<20°C) anaerobic digestion is an emerging, cost-efficient technology, which has been successfully applied to a variety of high-strength hazardous and non-hazardous waste-streams. Operated at low-temperature, expanded granular sludge bed (EGSB) reactors favour the development of a methanogenic consortium adapted to high biodegradation rates. Reactor operating parameters, such as wastewater characteristics and organic loading rate, affect microbial consortium physiology and therefore could shift bacterial 16S rRNA ratios, for example by changing growth rates and/or physiological activity. Thus, as a screening tool, temporal DGGE-profiling, comparing DNA and cDNA, was employed to highlight the potentially key organisms directly involved in psychrophilic (12°C) toluene methanogenesis in a laboratory-scale EGSB bioreactor. Biomass samples were taken and analysed over a 5-day period, immediately following an imposed increase in toluene loading rate. Functionally active potential toluene-degraders were successfully characterized. The results suggested that a putatively psychrophilic Geobacter-like organism was involved in the process along with hydrogenotrophic Methanospirillum-like and acetotrophic Methanosaeta-like Archaea. The presence of additional mesophilic and thermophilic putative toluene-degraders suggested, however, that reactor operating temperature may not have been the main factor for the development of this well-established and active microbial methanogenic consortium.