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Anaerobic azo dye reduction
Zee, F.P. van der - \ 2002
Wageningen University. Promotor(en): G. Lettinga; J.A. Field. - S.l. : S.n. - ISBN 9789058086105 - 142
anaërobe behandeling - azoverbindingen - kleurstoffen (dyes) - redoxreacties - antrachinonen - anaerobic treatment - azo compounds - dyes - redox reactions - anthraquinones
Azo dyes, aromatic moieties linked together by azo (-N=N-) chromophores, represent the largest class of dyes used in textile-processing and other industries. The release of these compounds into the environment is undesirable, not only because of their colour, but also because many azo dyes and their breakdown products are toxic and/or mutagenic to life. To remove azo dyes from wastewater, a biological treatment strategy based on anaerobic reduction of the azo dyes, followed by aerobic transformation of the formed aromatic amines, holds promise. However, the first stage of the process, anaerobic azo dye reduction, proceeds relatively slow. Therefore, this thesis research aimed at optimising anaerobic azo dye reduction, by studying the reaction mechanism and by consequently applying the obtained insights.
In this thesis it is shown that non-adapted anaerobic granular sludge has the capacity to non-specifically reduce azo dyes. As there was no correlation between a dye's reduction rate and its molecular characteristics (i.e. its size and its number of sulphonate groups and other polar substituents), it is unlikely that the mechanism of azo dye reduction involves cell wall penetration. Moreover, the presence of bacteria is not a prerequisite: azo dyes can also be reduced by sulphide in a purely chemical reaction. As dye containing wastewater usually contains sulphate and other sulphur species that will be biologically reduced to sulphide during treatment in anaerobic bioreactors, azo dye reduction will be a combination of biotic and abiotic processes. However, it was demonstrated that under normal conditions in high-rate anaerobic bioreactors (high sludge content, moderate sulphide levels), chemical azo dye reduction by sulphide hardly contributes to the overall reaction. Anaerobic azo dye reduction is therefore mainly a biological process, either a direct enzymatically catalysed reaction involving non-specific enzymes or a reaction with enzymatically reduced electron carriers. Azo dye reduction by sludge that had not earlier been exposed to dyes was found to relate to the oxidation of endogenous substrate and, especially, to the oxidation of hydrogen when present in bulk concentrations. Enrichment was required for the utilisation of electrons from volatile fatty acids for dye reduction.
Examination of the reduction of twenty chemically distinct azo dyes by anaerobic granular sludge revealed a large variation in the reaction rates. Especially reactive azo dyes with triazyl reactive groups were slowly reduced. For these common occurring reactive dyes, long contact times may be necessary to reach a satisfying extent of decolourisation. Consequently, they pose a serious problem for applying high-rate anaerobic treatment as the first stage in the biological degradation of azo dyes. However, this problem can be overcome by using redox mediators, compounds that speed up the reaction rate by shuttling electrons from the biological oxidation of primary electron donors or from bulk electron donors to the electron-accepting azo dyes.
It was observed that one of the constituent aromatic amines of the azo dye Acid Orange 7 had an autocatalytic effect on the dye's reduction, probably by acting as a redox mediator. Other compounds, e.g. the artificial redox mediator anthraquinone-2,6-disulphonate (AQDS), a compound that is known to catalyse the reductive transfer of several pollutants, and the commonly occurring flavin enzyme cofactor riboflavin, were found to be extremely powerful catalysts, capable of raising the pseudo first-order reaction rate constants by orders of magnitude. Moreover, a large stimulatory effect was found for autoclaved sludge, presumably due to the release of internal electron carriers, e.g. enzyme cofactors like riboflavin, during autoclaving.
AQDS was successfully applied to improve the continuous reduction of Reactive Red 2 (a reactive azo dye with a triazyl reactive group) in a lab-scale anaerobic bioreactor that was operated under moderate hydraulic loading conditions. Without AQDS, the reactor's dye removal efficiency was very low, which gave rise to severe dye toxicity towards the biological activity. Addition of catalytic concentrations of AQDS to the reactor influent caused an immediate increase of the dye removal efficiency and recovery of the methane production. Eventually, almost complete RR2 colour removal could be reached.
Though effective AQDS dosage levels are low, continuous dosing has disadvantages with respect to the costs and the discharge of this biologically recalcitrant compound. Therefore, the feasibility of activated carbon (AC), which is known to contain quinone groups at its surface, to act alternatively as an insoluble/immobilised redox mediator was explored. Incorporation of AC in the sludge of lab-scale anaerobic bioreactors that treated Reactive Red 2 in synthetic wastewater containing volatile fatty acid as primary electron donor resulted in enhanced continuous dye reduction as compared to the control reactors without AC. The effect of AC was in large excess of its dye adsorption capacity. In addition, it was shown that bacteria could utilise AC as terminal electron acceptor in the oxidation of acetate. Moreover, AC catalysis of chemical azo dye reduction by sulphide was demonstrated. These results clearly suggest that AC accepts electrons from the microbial oxidation of organic acids and transfers the electrons to azo dyes, thereby accelerating their biological reduction.
The research presented in this thesis makes clear that the reduction of azo dyes can be optimised by utilising redox mediators, i.e. either by continuous dosing of soluble quinones or by incorporation of AC in the sludge blanket. The potential of using redox mediators is probably not limited to enhancing azo dye reduction but may be extrapolated to other non-specific reductive (bio)transformations, e.g. reduction of halogenated or nitroaromatic compounds. The potential of using redox mediators is furthermore probably not limited to wastewater treatment but may also apply to bioremediation of soils polluted with e.g. polychlorinated solvents or nitroaromatic pesticides.
Red, redder, madder : analysis and isolation of anthraquinones from madder roots (Rubia tinctorum)
Derksen, G.C.H. - \ 2001
Wageningen University. Promotor(en): Æ. de Groot; A. Capelle; T.A. van Beek. - S.l. : S.n. - ISBN 9789058084620 - 150
antrachinonen - rubia tinctorum - analyse - plantextracten - anthraquinones - rubia tinctorum - analysis - plant extracts
The roots of Rubia tinctorum L. (madder) are the source of a natural dye. The dye components are anthraquinones with alizarin being the main dye component. Alizarin as such is present in madder root in only small quantities, most of the alizarin is present as its glycoside ruberythric acid. The sugar in this disaccharide is primeverose. Madder roots have been used to dye textiles in many parts of the world since ancient times. From 1600-1900 there was a heavy trade in madder throughout Europe. Madder root was an important export product for Holland. In 1868 Graebe and Liebermann discovered how to prepare alizarin synthetically. At the end of the 19 thcentury the madder culture rapidly declined due to the cheaper production of synthetic alizarin. Production of synthetic alizarin gives polluting side products. Nowadays the use and production of natural dyes becomes more popular due to the growing awareness for the environment and the need for alternative crops. An important element in the revitalisation of madder as an industrial crop is that the dye preparation from madder should be able to compete in quality and price with synthetic alizarin.Due to this renewed interest this research was initiated with this thesis as result.
For the simultaneous identification of the anthraquinone glycosides and aglycones in extracts of madder root a high-pressure liquid chromatography method (HPLC) was developed. The anthraquinones were separated on an end-capped C 18 -RP column with a water-acetonitrile gradient as eluent and measured with ultra violet (UV) detection at 250 nm. For the identification of anthraquinones on-line a mass spectrometer (MS) and a diode-array detector were used.
The main anthraquinones in an ethanol-water extract of madder root are the glycosides lucidin primeveroside and ruberythric acid and the anthraquinones pseudopurpurin and munjistin, which contain a carboxylic acid moiety. Beside these compounds also small amounts of the aglycones alizarin and purpurin could be detected and sometimes also lucidin was present.
For the production of a commercially useful dye preparation from madder, the glycoside ruberythric acid should be hydrolysed to the aglycone alizarin, which is the main dye component. An intrinsic problem of the hydrolysis of ruberythric acid in madder root is the simultaneous conversion of lucidin primeveroside to the unwanted mutagenic aglycone lucidin. Madder root was treated with strong acid, strong base or enzymes to convert ruberythric acid into alizarin. The anthraquinone composition of the suspensions was analysed with HPLC-UV, HPLC-DAD and HPLC-MS.
Stirring of dried madder root in water at room temperature for 90 min gave a suspension with pseudopurpurin, munjistin, alizarin and nordamnacanthal. Nordamnacanthal originates from lucidin primeveroside, which is hydrolysed to lucidin and subsequently oxidised to the corresponding aldehyde nordamnacanthal by an endogenous hydrolase and oxidase respectively. Nordamnacanthal is not mutagenic. During this conversion oxygen is obligatory and can be added by stirring the suspension. This stirring is an easy method for simultaneously hydrolysing ruberythric acid and to getting rid of the mutagenic lucidin.
Different madder root cultivars were screened for their anthraquinone composition and amount of the main anthraquinones. The concentration of alizarin varied from 6.1 to11.8 mg/g root. If madder root was cultivated for three instead of two years the amount of alizarin increased from 6.7 mg/g to 8.7 mg/g.
A number of different methods were compared for their capacity to isolate alizarin from the rest of the plant material. To make a first selection attention was mainly paid to the yield of alizarin. Three routes were selected as most promising for an industrial application. These three methods were tested at a larger scale. The first method consisted of the following steps: conversion of madder root (250 g) by endogenous enzymes, extraction of madder root with refluxing ethanol-water, hot filtration, evaporation to half of the original volume and precipitation at 4°C. An extract of 14.7 g was obtained which consisted for 35 % of anthraquinones. Of the total amount of alizarin available in the starting material 78% was extracted. The second method consisted of the following steps: extraction of madder root (250 g) with refluxing water, hot filtration, conversion of the glycosides in the filtrate by a madder root enzyme extract and precipitation at 4°C. An extract of 3.2 g was obtained of which 38 % were anthraquinones. Of the total amount of alizarin available in the starting material 19% was extracted. The third method consisted of the following steps: extraction of madder root with an aqueous surfactant solution, twice C 18 chromatography for extracting alizarin, elution of alizarin with methanol and evaporation. An extract of 17.1 g was obtained of which 11 % were anthraquinones. Of the total amount of alizarin available in the starting material 98% was extracted.
For the development of an economically feasible route these three methods have to be further optimised. After optimisation the three routes have to be compared in terms of amount of extract obtained, alizarin content, dyeing capacity, costs and industrial applicability of the procedure.