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The colour removal achieved under anaerobic conditions is also called reductive decolourisation, which is composed of a biological part, i.e. the reducing equivalents are biologically generated, anda chemical part, i.e. the formed electrons reducechemically the dyes. The overall objective of this research was to explore different strategies to increase the reduction of dyes in bioreactors by using thermophilic anaerobic granular sludge and redox mediators. Our results clearly show that thermophilic treatment with anaerobic granular sludge does accelerate the reduction of azo dyes compared to the performance achieved under mesophilic conditions. In Chapter 3, batch assays showed increases on the decolourisation rates of azo dyes in sixfold and twofold, in the absence and presence of the redox mediator anthraquinone-2,6-disulphonate (AQDS), respectively, between thermophilic (55ºC) and mesophilic (30ºC) conditions by using the same sludge source. Therefore, most likely the transfer of reducing equivalents at 30ºC was the rate-limiting step. In Chapter 4, thermophilic EGSB (expanded granular sludge blanket) bioreactors were extremely efficient in treating the recalcitrant azo dye ReactiveRed 2 (RR2), as well as having a high stability when high loading rates of RR2 were applied(up to 2.7 gRR2 l -1 day -1 ). Long-term experiments revealed that the AQDS-free reactor achieved efficiencies of around 91% in comparison with the efficiencies around 95% for the AQDS-supplemented reactor. In Chapter 5, we demonstrated that the normal rate limiting step, the transfer of reducing equivalents to the azo dye, was accelerated under thermophilic conditions. Both biotic and abiotic mechanisms involved in the biochemistry of reductive decolourisation were enhanced at 55ºC. The faster biological reduction of the redox mediator, AQDS, achieved by sludge incubations at 55°C in comparison with 30°C evidences the biological contribution in enhancing the rate of electron transfer. For instance, about 1 mM of AQDS was completely reduced at 55ºC after 0.7 days of incubation, whereas mesophilic reduction after 0.7 days was just 12.9% of this value. Furthermore, no lag-phase was found at 55ºC. The abiotic chemical reduction of RR2 by sulphide, as expected, followed the Arrhenius equation, and the decolourisation rates were accordingly accelerated by the temperature increase. Furthermore, the AQS-supplemented incubations presented a lower Ea requirement. The calculated Ea values are27.9 kJ/moland 22.9 kJ/mol for the AQS-free and AQS-supplemented incubations, respectively. Therefore, the activation energy was decreased 1.2-fold due to the addition of 0.012mM of AQS.In Chapter 6, the significant enhancement of the electron transfer capacity and subsequent increase on the reductive decolourisation of azo dyes, simply by applying high temperature, was demonstrated. For instance, at a hydraulic retention time (HRT) of 2.5 h and in the absence of AQDS, the colour removal was 5.3-fold higher at 55ºC in comparison with the efficiency of bioreactors achieved at 30°C. Furthermore, the catalytic effect of AQDS on the thermophilic reductive decolourisation, i.e. the impact of AQDS on the decolourisation rates, was even decreased 3.2-fold compared to experiments carried out at 30ºC. Very likely, this indifference to the presence of AQDS was the consequence of the high efficiency reached at 55°C, which masked the impact of AQDS on reductive decolourisation of dyes. The similar degree of COD (chemical oxygen demand) removal achieved in all reactors of about 75% indicated that the reducing equivalents were generated at a similar rate in the mesophilic and thermophilic bioreactors. Apparently, the difference in the decolourisation rates was not related to the difference in the production rates of reducing equivalents. Consequently, we concluded that the higher degree of colour removal was attributed to the impact of temperature and AQDS on electron shuttling. Results described in Chapter 7 with reductive decolourisation of anthraquinone dyes, demonstrated that in comparison with incubations at 30ºC, incubations at 55ºC present distinctly higher decolourisation rates not only with real wastewater, but also with the model compound Reactive Blue 5 (RB5). The k-value of RB5 at 55ºC is enhanced 11.1-fold in the presence of anthraquinone-2-sulphonate (AQS), and sixfold in the absence of AQS, upon comparison with mesophilic incubations at 30ºC. However, the anthraquinone dye Reactive Blue 19 (RB19) exhibited a very strong toxic effect on volatile fatty acids (VFA) degradation and methanogenesis at both 30ºC and 55ºC. Further experiments at both temperatures revealed that RB19 was mainly toxic to methanogens, because the glucose oxidizers including acetogens, propionate-forming, butyrate-forming and ethanol-forming microorganisms are not affected by the dye toxicity. Finally in Chapter 8, we studied the contribution of acidogenic bacteria and methanogenic archaea to azo dye reduction by a thermophilic anaerobic consortium, as well as the competition for reducing equivalents between methanogenesis and azo dye reduction. Our results indicated that acidogenic bacteria and methanogenic archaea play important roles in this reductive process. Experiments with the thermophilic methanogens Methanothermobacter thermoautotrophicusDH and a Methanothermobacter -related strain NJ1 revealed that these strains were unable to reduce the dye in the absence of the redox mediator, riboflavin. This suggested that anaerobic dye reduction is not a universal property among methanogenic archaea and that redox mediators may play an important role for allowing some microbial groups, commonly found in wastewater treatment systems, to participate more effectively in reductive decolourisation of dyes.