Thesis for Doctor of Philosophy
Luísa Teresa Pires Grancho Caetano Davies
“Biodegradation of an azo dye in a vertical flow constructed wetland”
Ph. D. Biotechnology, IST, 2006
Supervisors: S. Martins-Dias; J.M. Novais
Abstract
This thesis summarizes the study of the degradation of an azo dye, acid orange 7 (AO7) in a Vertical flow Constructed Wetland (VFCW), at pilot scale, emphasising the hydrological aspects of those systems and highlighting the Phragmites australis (P.australis) role in its elimination. AO7 and subsequent aromatic amines degradation and mineralization were found to be carried out in the VFCW aerobically (+230 mV). This contradicts the general idea that azo bond cleavage only occur in anaerobic conditions.
The VFCW was operated in a continuous feeding mode, being applied mass loads (MLs) ranging from 9.3 to 41.6 mMAO7 m-2 d-1, being effectively removed 7.1 up to 30.5 mMAO7 m-2 d-1 respectively. With the goal of enhancing the oxygen transfer rates, an intermittent feeding mode was selected and a simulation of an accidental discharge was carried out (120 l m-2 d-1 at 700 mgAO7 l-1 applied during 8 cycles a day). Thus, for an inlet ML of 208 mMAO7 m-2 d-1 the system efficiently removed 145 mMAO7 m-2 d-1. The VFCW was modelled by analogy with a combination of ideal reactors (2 CSTRs in series and 1 in parallel). This model parameters helped to understand, the experimental data concerning the hydrological and kinetic processes occurring in the VFCW being possible to establish that the overall degradation kinetics is of first order. Another feature of the developed model was the possibility to predict the correct dimensioning of a VFCW capable of dealing with most extreme concentrations (e.g. accidental discharge) in order to fulfil the legislation requirements.
Concerning the direct contribution of P. australis to the AO7 degradation within a CW, it was observed in vitro and in vivo that the peroxidases in the roots, stems and leaves of P. australis were able to use AO7 as an organic substrate and concomitantly reduce the H2O2. This is a reactive oxygen specie that plays a role in the establishment of oxidative stress conditions. Finally, since information on the P. australis genes responsible for its tolerance to xenobiotics and signal transduction is scarce, despite its widespread use in CW, a methodology was developed to design specific primers to perform a transcriptomic analysis. The transcriptomic analysis was carried, leading to gene sequencing encoding reactive oxygen species scavenging enzymes (catalase and glutathione peroxidase) responsible for the elimination of H2O2 and lipidic hydroperoxides, whose expression is enhanced in the oxidative stress invoked by AO7.
Keywords: Azo Dye; Acid Orange 7; Catalase; Enzymes; Genomic; Glutathione peroxidase; Mechanistic model; Phragmites australis; Peroxidases; Superoxide dismutase; Transcriptomic analysesVertical flow constructed wetland