Studies of decolorization of azo dyes by ascomycete yeasts

Poster apresentado no Micro'2001, Póvoa de Varzim, 2001.Azo dyes are the most widely used colored materials in textile industries and its biodegradability is, therefore, an important issue in the biological treatment of dye-containing wastewater. However, these treatments are not totally effective in removing color of textile dye wastewater since dyes are typically resistant to oxidative degradation. Most biodegradation studies on azo dyes involve bacterial species, and anaerobic or microaerophylic conditions are usually refered to as being favorable to a reduction step producing colorless amines. Our group has succeeded in isolating a number of yeast species, which revealed an interesting potential in the color removal of azo dyes in aerated culture media. Three of these species (UM2, UM41 and UM45), where used in the present work. The tested dyes included several sulphonic and carboxylic monoazo dyes, and several reactive textile dyes. The results which will be described refer to (i) the conditions and time-course of the decolorization process, (ii) the optimization of the decolorization medium, aiming at the reduction in yeast extract concentration, (iii) the identification of dye reduction products formed in the course of the decolorization process and (iv) the effect of those reduction products on yeast specific growth rates.BIOEFTEX Project

Azoreductase activity in intact cells of a non-conventional yeast strain

Poster apresentado no Annual Workshop COST 847 Textile Quality and Biotechnology, 2, Villa Olmo, Como, Itália, 10-11 Outubro 2002.Several non-conventional ascomycete yeast strains, isolated from dye-contaminated environments, display azo-reductive capabilities. Those strains can therefore decolorize azo dyes, which are widely used in the textile industries. The azoreductase activity has been described and investigated in a wide variety of anaerobic bacterial species, particularly from the intestinal microflora, and also, although more rarely, in aerobic bacteria. The present work describes some characteristics of the azoreductase present in one of the isolated yeast strains. This enzyme activity seems to be constitutive in the microorganism, as the decolourization process is not affected by pre-adaptation of the cells to the tested dyes. The azo reduction activity, measured at constant OD (optical density), has a maximum at the end of the exponential growth phase. For cells collected in this phase, the decolourization profile for the two dyes derived from N,N-dimethylaniline (I and II) presents a plateau between pH 3 and 5, while the analogue dyes derived from beta-naphtol (III and IV) have a maximum at pH 3. The azoreductase activity in resting cells shows a typical saturation kinetics in relation to the concentration of the dye. For dye II, at pH 4.0, a Km of 0,42 mM and a vmáx of 3,4 mmol*h-1*gDW-1 were obtained. Despite the low specificity of this yeast azoreductase, both the initial decolourization rate and the extent of the process are dependent on the dye structure.BIOEFTEX Project

Characterization of the azo reductase activity in a novel ascomycete yeast strain

Poster apresentado no XIII Congresso Nacional de Bioquímica, Lisboa, 5-7 Dezembro 2002.An ascomycete yeast strain, UM41 , was isolated from a textile wastewater treatment facility, and selected on the basis of its azo dye decolourising activity. Growing yeast cultures in liquid media containing glucose and 0.2mM dye removed more than 80% of the colour in 15 h, and total decolourisation of the tested dyes occured in 24 h. Colour loss is due to a constitutive azoreductase activity, expressed in intact cells, but not detectable in supernatant samples or cell-free extracts. This activity suffers thermal inactivation at temperatures higher than 50ºC and its dependence on dye concentration is satisfactorily described by the Michaelis-Menten model. Specific decolourising activities of resting cells have a maximum during the late exponential growth phase and are enhanced by up to 3-fold by the redox mediator AQS. The effect of pH on the decolourisation rates is structure-dependent. UM41 growing on glucose-containing medium initially exhibits a predominantly fermentative metabolism and produces ethanol, while oxygen is quicky depleted from the culture medium. However the same batch of cells retained decolourising activity for up to five successive cycles of dye removal, without any further nutrient addition. The microorganism growth and dye decolourisation observed after glucose exhaustion probably occur at the expense of ethanol, suggesting that decoulorisation is also compatible with a respiratory metabolism.BIOEFTEX

Sida e a mulher

Os padrões epidemiológicos da pandemia do VIH/SIDA estão a mudar, fazendo prever que, nos próximos anos, o ratio homem/mulher seja de 1:1. Isto é tanto mais verdade em localizações geográficas onde a transmissão do vírus por via heterossexual é a predominante, com particular relevância para os países em vias de desenvolvimento. De entre estes, África merece destaque, já que aí as cifras assustadoras relativas a esta doença traduzem, de facto, a precariedade da condição de mulheres e crianças. As mulheres são, no presente, o grupo mais vulnerável à SIDA, não só pelas suas condições anatomofisiológicas, mas também pela sua situação social, económica e cultural. O seu papel na família, como cuidadora, parceira sexual e, eventualmente, mãe coloca-as face aos desafios de uma doença que, apesar de crónica, tem, ainda hoje, um desfecho fatal. Simultaneamente vítima e portadora/mensageira da doença, a mulher é confrontada com os seus comportamentos passados ("vítima culpada") ou acaba por se ver presa numa teia de contaminações sucessivas. Com frequência, começa por ser contaminada pelo seu parceiro sexual (muitas vezes, como verdadeira "vítima inocente", já que desconhece condutas e passado do homem com quem vive), tornando-se, depois, ela própria, transmissora da doença aos filhos que, provavelmente, tanto quis proteger

Decolourisation of a synthetic textile effluent using a bacterial consortium

In the present study we examined the performance of a thermoalkalophilic bacterial consortium, where the predominant strain was Bacillus sp. SF, in the degradation of Reactive Black 5 (RB5). We used a reactor working in continuous mode and investigated the effects of pH, hydraulic retention time (HRT) and several added salts on colour and chemical oxygen demand (COD) reductions. For the chosen operational conditions (pH 9, 55ºC and HRT of 12 h) the efficiencies achieved were 91.2 ± 0.8 % for colour removal and 81.2% for COD removal. The system tolerated, with no significant decrease in colour removal efficiency, 30 g/L Na2SO4, Na2CO3 or NaCl. The latter two salts, however, led to a reduction in COD removal of 30% and 50%, respectively. The system proved to be very effective in the decolourisation of C.I. RB5 under alkaline conditions and at a comparatively high temperature.(undefined

Reductive biological treatment of textile effluents

Poster apresentado no "Simpósio Corantes e Pigmentos Orgânicos", na Universidade de Trás-os-Montes e Alto Douro, em Vila Real, Portugal, em Novembro de 2004.Our group is undertaking an investigation on the potential application of ascomycete yeasts to the decolourisation of azo dyes. Two of these strains, Candida zeylanoides (UM2) and Issatchenkia occidentalis (UM41), were isolated from contaminated soil and have been shown to mediate dye decolourisation through reductive cleavage of the azo bond. The rates of colour loss in the presence of yeast cells are independent of their previous exposure to the dye, suggesting that the decolourising activity, under the conditions tested, is constitutive. The process requires intact cells and an external carbon and energy source and depends on pH, temperature and dissolved oxygen. Interestingly, anaerobic conditions do not allow decolourisation. The kinetic study of the cells decolourising activity demonstrated that such activity has a maximum in the late exponential growth phase. Although glucose is the standard carbon and energy source we have also observed decolourisation by cells growing at the expense of ethanol. Decolourisation rates are considerably dependent on the dye structure. Of considerable practical interest is the observation that some of the amines produced by azo dye reduction can be used as carbon and nitrogen sources by the yeast. In order to get further insight on the yeast decolourising activity we have prepared some mutants of a laboratory strain of Saccharomyces cerevisiae and performed inhibition studies. The experimental evidence suggests that major part of the decolourising activity of intact yeast cells is due to a very well characterized plasma membrane redox system.BIOEFTEX Project

Role of the component Fre1p of the plasma membrane ferric reductase on the azo reductase activity of intact Saccharomyces cerevisiae cells

In Press. Aceite em 2005 para publicação na revista Applied and Environmental Microbiology.Unspecific bacterial reduction of azo dyes is a widely studied process in correlation with the biological treatment of coloured waste waters but the enzyme system associated to this bacterial capability has never been positively identified. Several ascomycete yeast strains display similar decolourising activities. The yeast-mediated process requires an alternative carbon and energy source and is independent of previous exposure to the dyes. When substrate dyes are polar their reduction is extracellular, strongly suggesting the involvement of an externally-directed plasma membrane redox system. The present work demonstrates that, in Saccharomyces cerevisiae, the ferric reductase system participates in the extracellular reduction of azo dyes. The Saccharomyces cerevisiae mutant strains Δfre1 and Δfre1Δfre2, but not Δfre2, showed a much reduced decolourising capability, suggesting that, under the conditions tested, Fre1p is a major component of the azo reductase activity.European BIOEFTEX Project

Is Saccharomyces cerevisiae azoreductase the plasma membrane ferric reductase?

Poster apresentado na International Conference on Plasma Membrane Redox Systems and their Role in Biological Stress and Disease, 7, Asilomar, Califórnia, Estados Unidos da América, 14-18 Abril 2004.Unspecific bacterial reduction of azo dyes is a widely studied process in correlation with the biological treatment of coloured waste waters but the enzyme system associated to this bacterial capability has never been positively identified. Several ascomycete yeast strains, isolated by our group from contaminated soils, display similar decolourising activities. A study of the yeast-mediated process in batch culture demonstrated that colour loss was due to a reductase activity, expressed constitutively, which could transform azo dyes into colourless amines. In order to get a better understanding of the azoreductase activity of yeast cells we selected a Saccharomyces cerevisiae strain (BLC0276) with a high decolourising capacity. A likely candidate might be the plasma membrane ferric reductase system. Both azoreductase and ferric reductase have peak activities in the late exponential growth phase, and their substrates -ferricyanide and soluble azo dyes - are impermeant to the cell plasma membrane. This hypothesis was confirmed through two different lines of evidence: the use of different inhibitors and the construction of mutants defective in the iron reductase system. Inhibitors like carbonylcyanide m-chlorophenylhydrazone (CCCP), diphenylene iodonium (DPI), p-chloromercuribenzoate (pCMB) and chloroquine (CQ), were tested. In most cases the percentage inhibition of both activities was similar. The genes Fre1 and Fre2 were deleted and the effect on the decolourising activity was analysed in the mutant strains. The effect of Fre2 deletion was negligible, but fre1 and fre1fre2 strains showed a much reduced decolourising capability. These results demonstrate the involvement of the ferric reductase system in the azo decolourising capacity of S. cerevisiae. However the capacity was not completely removed indicating that cells have an alternative reducing system.BIOEFTEX Project

Improved conditions for the aerobic reductive decolourisation of azo dyes by Candida zeylanoides

A number of anaerobic and aerobic bacterial species are known to decolourise azo dyes through the reduction of the azo bonds, forming the corresponding amines. In this work, we describe improved decolourisation conditions for model azo dyes by the ascomycete yeast Candida zeylanoides. The dyes were derived from the diazonium salts of metanilic and sulfanilic acids and N,N-dimethylaniline or 2-naphthol as coupling components. Total decolourisation times observed in culture media supplemented with 0.2mM dye ranged from 40 go 60 hours. The initial decolourisation rates were 14-52µmol.(g dry cell)-¹.h-¹, depending on dye structure. In the course of decolourisation either metanilic acid or sulfanilic acid were detected in the supernatant fluid, showing that decolourization by this yeast strain is due to azo bond reduction. None of those aminobenzenesulphonates supported microorganism growth as carbon and energy source but both could be used, to a limited extent, as nitrogen sources. The azo reductase activity is not significantly affected by pre-adaptation of the microorganism to the dyes.Junta Nacional de Investigação Científica e Tecnológica - PraxisXXI/2/2.2/QUI/44/94. Erasmus programme

A contribution for the identification of the azo reductase activity in intact yeast cells

Poster apresentado no International Congress on Yeasts (ICY) -Yeasts in Science and Biotechnology : The Quest for Sustainable Development", 11, Rio de Janeiro, Brasil, Agosto 2004.Azo dyes are synthetic organic colorants which are extensively used in textile, food and cosmetic industries. A large fraction of these dyes is released into the environment even after conventional wastewater treatments. This is a worldwide problem and particularly a problem in regions where textile industries release large quantities of coloured wastewater to water courses. In an attempt to develop a biological treatment for colour removal we isolated several ascomycete yeast strains from contaminated soil based on its capacity to decolourize soluble azo dyes. We studied the process in batch cultures and have demonstrated that the colour loss was due to a reductase activity, expressed constitutively, which could transform azo dyes into colourless amines [1]. In order to understand the process involved in this reduction we selected a Saccharomyces cerevisiae strain (BLC0276) with this capacity. This strain reduced model azo dyes in 8-16 h. We decided to further investigate which enzyme(s) could be involved in this reduction. It was found that in S. cerevisiae there is a membrane redox system with an externally directed reductase – the ferric reductase system, responsible for the extracellular reduction of ferric to ferrous iron previous to uptake, and involving the genes fre1 and fre2. Several know inhibitors of this system were investigated like excess of iron in the medium, CCCP, DPI, chloroquine and p-chloromercurybenzoic acid. Several of these compounds managed to inhibit the decolourizing activity. We also deleted the genes fre1 and fre2 in our strain and analysed the effect on decolourizing activity. Once again the process was retarded in fre1 and fre1fre2 strains. The effect of fre2 deletion was negligible. The deletion of fre1 retarded the decolourizing capability showing the involvement of this system. However the capacity was not completely removed indicating that cells have an alternative reducing system. [1] Ramalho, P.A., Scholze, H., Cardoso, M.H., Ramalho, M.T., Oliveira-Campos, A.M. 2002 Improved conditions for the aerobic reductive decolourization of azo dyes by Candida zeylanoides. Enz Microbiol Technol 31: 848-854.BIOEFTEX Project