Biodegradation of Grape Cluster Stems and Ligninolytic Enzyme Production by Phanerochaete chrysosporium during Semi‐Solid‐State Cultivation

The degradation undergone by grape cluster stems (woody component of vine bagasse), an agroindustrial waste, was investigated during the semi‐solid‐state cultivation of Phanerochaete chrysosporium BKM‐F‐1767 (ATCC 24725). For this, the content of lignin, cellulose and hemicellulose in grape cluster stems was determined before and after the enzymatic process. It was found that about 20% of Klason lignin, 48% of hemicellulose and 5% of cellulose were degraded during the process, being the ligninolytic enzymes (manganese‐dependent peroxidase and lignin peroxidase) produced by such cultures responsible for the degradation of grape cluster stems. In parallel, semi‐solid‐state cultures of P. chrysosporium grown on an inert support (cubes of nylon sponge), which is not susceptible to undergoing degradation during the enzymatic process, were used as reference cultures. In addition, the in vivo decolourisation of a model dye, the polymeric dye Poly R‐478, by both grape cluster stem and nylon cultures was studied in order to assess their degradative ability. A percentage of biological decolourisation higher than 90% after four days of dye addition was obtained using nylon sponge cultures, whereas grape cluster stem cultures led to a decolourisation of around 70% after eight days of dye incubation. The lower percentage of dye degradation achieved by the cultures grown on grape cluster stems was due to the enzymes produced, which were not only employed in the decolourisation of the dye but also in the degradation of the support, as indicated by the data mentioned above.     

Different bioreactor configurations for the decolourisation of the azo dye reactive black 5 by Geotrichum sp. CCMI 1019

Decolourisation of the azo dye Reactive Black 5 by Geotrichum sp. CCMI 1019 was studied using stirred tank reactors (STR) and two types of bubble columns (porous plate (PP) bubble column and aeration tube (AT) bubble column). For the bubble columns, the k_La increased with the gas fractional hold-up (ε_G) and the aeration rate. A linear relationship between ε_G and superficial gas velocity was obtained for all reactors. At same aeration rates, the PP bubble columns showed higher k_La and hold-up values than the AT bubble column. In the STRs, large and dense aggregates were formed which adhered to surfaces whereas bubble columns gave smaller and less compact pellets.

Manganese peroxidase and laccase were detected in the extracellular media in all reactors. However, laccase was only detected after the onset of decolourisation, suggesting that additional enzymes may be involved. Mn peroxidase activity was detected (about 46 U/ml) in both the STRs and AT bubble columns but higher values (110 U/ml) were obtained with the PP bubble columns.

Out of the three reactor systems studied, the AT bubble columns gave the most favourable results for Reactive Black 5 decolourisation. Rapid and complete colour removal was obtained throughout the visible spectrum. Bubble columns are simple in design as well as operation and may be useful for the bioremediation of textile wastewater.     

The role of riboflavin in decolourisation of Congo red and bioelectricity production using <Emphasis Type="Italic">Shewanella oneidensis</Emphasis>-MR1 under MFC and non-MFC conditions

Dissimilatory metal reducing bacteria can exchange electrons extracellularly and hold great promise for their use in simultaneous wastewater treatment and electricity production. This study investigated the role of riboflavin, an electron carrier, in the decolourisation of Congo red in microbial fuel cells (MFCs) using Shewanella oneidensis MR-1 as a model organism. The contribution of the membrane-bound protein MtrC to the decolourisation process was also investigated. Within the range of riboflavin concentrations tested, 20 µM was found to be the best with >95% of the dye (initial concentration 200 mg/L) decolourised in MFCs within 50 h compared to 90% in the case where no riboflavin was added. The corresponding maximum power density was 45 mW/m². There was no significant difference in the overall decolourisation efficiencies of Shewanela oneidensis MR-1 ΔMtrC mutants compared to the wild type. However, in terms of power production the mutant produced more power (P_max 76 mW/m²) compared to the wild type (P_max 46 mW/m²) which was attributed to higher levels of riboflavin secreted in solution. Decolourisation efficiencies in non-MFC systems (anaerobic bottles) were similar to those under MFC systems indicating that electricity generation in MFCs does not impair dye decolourisation efficiencies. The results suggest that riboflavin enhances both decolourisation of dyes and simultaneous electricity production in MFCs.     

Effect of redox mediator, AQDS, on the decolourisation of a reactive azo dye containing triazine group in a thermophilic anaerobic EGSB reactor

The feasibility of thermophilic (55 °C) anaerobic treatment applied to colour removal of a triazine contained reactive azo dye was investigated in two 0.53 l expanded granular sludge blanket (EGSB) reactors in parallel at a hydraulic retention time (HRT) of 10 h. Generally, this group of azo dyes shows the lowest decolourisation rates during mesophilic anaerobic treatment. The impact of the redox mediator addition on colour removal rates was also evaluated. Reactive Red 2 (RR2) and anthraquinone-2,6-disulfonate (AQDS) were selected as model compounds for azo dye and redox mediator, respectively. The reactors achieved excellent colour removal efficiencies with a high stability, even when high loading rates of RR2 were applied (2.7 g RR2 l⁻¹ per day). Although AQDS addition at catalytic concentrations improved the decolourisation rates, the impact of AQDS on colour removal was less apparent than expected. Results show that the AQDS-free reactor R2 achieved excellent colour removal rates with efficiencies around 91%, compared with the efficiencies around 95% for the AQDS-supplied reactor R1. Batch experiments confirmed that the decolourisation rates were co-substrate dependent, in which the volatile fatty acids (VFA) mixture was the least efficient co-substrate. The highest decolourisation rate was achieved in the presence of either hydrogen or formate, although the presence of glucose had a significant impact on the colour removal rates.     

Biological sulphate reduction and redox mediator effects on azo dye decolourisation in anaerobic–aerobic sequencing batch reactors

In this work, the anaerobic period of an anaerobic–aerobic sequencing batch reactor was found to allow the reductive decolourisation of azo dyes. 1-l reactors were operated in 24-h cycles comprising anaerobic and aerobic reaction phases, fed with a simulated textile effluent including a reactive type (Remazol Brilliant Violet 5R) or an acid type (Acid Orange 7) azo dye. The aim was to assess the role of different redox phenomena in the anaerobic decolourisation process. Selective inhibition of sulphate reducing bacteria was carried out in the sulphate-containing, reactive dye fed reactor, resulting in nearly complete, though reversible and inhibition of decolourisation. The acid dye fed reactor's supplementation with sulphate, though resulting in sulphate reduction, did not improve decolourisation. Other redox mediators, namely quinones, were more effective in promoting electron transfer to the azo bond. Bio-augmentation of the acid dye fed reactor with a pure sulphate reducer strain known to decolourise azo dyes, Desulfovibrio alaskensis, was also carried out. Decolourisation was improved, but apparently as a result of the carbon source change required to support D. alaskensis growth. A chemically mediated reduction of the azo bond coupled to biological sulphate reduction, thus seemed to account for the high decolourisation yields of both dyes.     

Enzymatic reactors for the removal of recalcitrant compounds in wastewater

Abstract

Enzymatic treatments based on oxidative enzymes, such as peroxidases, laccases and tyrosinases, have been proposed as an alternative to conventional methods to remove a broad range of contaminants present in wastewater. The aim of this study is to discuss existing technologies for the removal of pollutants based on the use of oxidative enzymes, including a discussion on the most important factors affecting the efficiency of the proposed systems. Factors involved in the catalytic cycle of the enzyme (biocatalyst, substrates and mediators), the addition of certain components to the reaction medium (additives, surfactants or solvents) as well as operational parameters (temperature, pH or agitation) will be discussed. Finally, two types of reactors: one-stage and two-stage enzymatic membrane reactors, especially designed for the treatment of micropollutants present in secondary effluents, will be described in detail.     

Performance of staged and non-staged up-flow anaerobic sludge bed (USSB and UASB) reactors treating low strength complex wastewater

The use of anaerobic processes to treat low-strength wastewater has been increasing in recent years due to their favourable performance-costs balance. For optimal results, it is necessary to identify reactor configurations that are best suited for this kind of application. This paper reports on the comparative study carried out with two high-rate anaerobic reactor systems with the objective of evaluating their performances when used for the treatment of low-strength, complex wastewater. One of the systems is the commonly used up-flow anaerobic sludge blanket (UASB) reactor. The other is the up-flow staged sludge bed (USSB) system in which the reactor was divided longitudinally into 3, 5 and 7 compartments by the use of baffles. The reactors (9 l) were fed with a synthetic, soluble and colloidal waste (chemical oxygen demand (COD) < 1000 mg/l) and operated at 28°C and 24 h hydraulic retention time. Intermediate flow hydraulics, between plug-flow and completely-mixed, in the UASB and 7 stages USSB reactors allowed efficient degradation of substrates with minimum effluent concentrations. Low number of compartments in the USSB reactors increased the levels of short-circuiting thus reducing substrate removal efficiencies. All reactors showed high COD removal efficiencies (93–98%) and thus can be regarded as suitable for the treatment of low strength, complex wastewater. Staged anaerobic reactors can be a good alternative for this kind of application provided they are fitted with a large enough (≥7) number of compartments to fully take advantage of their strengths. Scale factors seem to have influenced importantly on the comparison between one and multi staged sludge-bed reactors and, therefore, observations made here could change at larger reactor volumes.     

Decolourisation of the textile dye Astrazon Red FBL by Funalia trogii pellets.

The effects of various conditions such as initial pH, dye concentrations, amount of pellet, temperature and agitation on decolourising activity of Funalia trogii were investigated. These, except initial pH, were all found to be important for dye decolourising activity of F. trogii. The decolourisation of the dye involved adsorption of the dye compound by fungal pellets at the initial stage, followed by the decolourisation through microbial metabolism. Heat-killed pellets were also tested for their ability to decolourise Astrazon Red dye. These pellets adsorbed the dye and 55% decolourisation was obtained in 24 h. But at the second cycle there was only 24% decolourisation. Our observation showed that Astrazon Red dye decolourisation by heat-killed pellets was mainly due to biosorption. The longevity of the decolourisation activity of F. trogii pellets was also investigated in repeated batch mode. Variations in the amount of pellet increased % decolourisation and stability of pellets.     

A comprehensive review on application of bioreactor for industrial wastewater treatment

In the recent past, wastewater treatment processes performed a pivotal role in accordance with maintaining the sustainable environment and health of mankind at a proper hygiene level. It has been proved indispensable by government regulations throughout the world on account of the importance of preserving freshwater bodies. Human activities, predominantly from industrial sectors, generate an immeasurable amount of industrial wastewater loaded with toxic chemicals, which not only cause dreadful environmental problems, but also leave harmful impacts on public health. Hence, industrial wastewater effluent must be treated before being released into the environment to restrain the problems related to industrial wastewater discharged to the environment. Nowadays, biological wastewater treatment methods have been considered an excellent approach for industrial wastewater treatment process because of their cost-effectiveness in the treatment, high efficiency and their potential to counteract the drawbacks of conventional wastewater treatment methods. Recently, the treatment of industrial effluent through bioreactor has been proved as one of the best methods from the presently available methods. Reactors are the principal part of any biotechnology-based method for microbial or enzymatic biodegradation, biotransformation and bioremediation. This review aims to explore and compile the assessment of the most appropriate reactors such as packed bed reactor, membrane bioreactor, rotating biological contactor, up-flow anaerobic sludge blanket reactor, photobioreactor, biological fluidized bed reactor and continuous stirred tank bioreactor that are extensively used for distinct industrial wastewater treatment.     

Molecular determinants of azo reduction activity in the strain <Emphasis Type="Italic">Pseudomonas putida</Emphasis> MET94

Azo dyes are the major group of synthetic colourants used in industry and are serious environmental pollutants. In this study, Pseudomonas putida MET94 was selected from 48 bacterial strains on the basis of its superior ability to degrade a wide range of structurally diverse azo dyes. P. putida is a versatile microorganism with a well-recognised potential for biodegradation or bioremediation applications. P. putida MET94 removes, in 24 h and under anaerobic growing conditions, more than 80% of the majority of the structurally diverse azo dyes tested. Whole cell assays performed under anaerobic conditions revealed up to 90% decolourisation in dye wastewater bath models. The involvement of a FMN dependent NADPH: dye oxidoreductase in the decolourisation process was suggested by enzymatic measurements in cell crude extracts. The gene encoding a putative azoreductase was cloned from P. putida MET94 and expressed in Escherichia coli. The purified P. putida azoreductase is a 40 kDa homodimer with broad substrate specificity for azo dye reduction. The presence of dioxygen leads to the inhibition of the decolourisation activity in agreement with the results of cell cultures. The kinetic mechanism follows a ping-pong bi–bi reaction scheme and aromatic amine products were detected in stoichiometric amounts by high-performance liquid chromatography. Overall, the results indicate that P. putida MET94 is a promising candidate for bioengineering studies aimed at generating more effective dye-reducing strains.     

Biodegradation, decolourisation and detoxification of textile wastewater enhanced by advanced oxidation processes

Recently, an increasing application of so called advanced oxidation processes (AOPs) to industrial wastewater has been observed. In particular, an integrated approach of biological and chemical treatment of wastewater is advantageous conceptually. The subject of our study was synthetic wastewater, simulating effluents from knitting industry. The wastewater contained components that are very often used in Polish textile industry: an anionic detergent Awiwaz KG conc., a softening agent Tetrapol CLB and an anthraquinone dyestuff-Acid Blue 40, CI 2125. The toxicity of the detergents and the dye was determined in terms of effective concentration EC50 using mixed cultures of activated sludge as well as pure culture of luminescent bacteria Vibrio fischerii NRRLB-11177. The dye did not undergo biodegradation without AOPs pretreatment, therefore a degree of its removal (decolourisation) by the AOPs has been determined and its bio-sorption properties on the flocks of activated sludge have been studied. The dye adsorption onto flocks of activated sludge was described by Henry's isotherm. Our investigations focussed on the influence of various oxidants like O3, H2O2 and UV light on biodegradation of single components aqueous solution as well as of the whole textile wastewater. The results of kinetic measurements of the biodegradation (by means of acclimated activated sludge) was described by Monod type of kinetic equation. The experimental evidence of the positive effect of chemical oxidation pretreatment on the biodegradation of recalcitrant compounds was quantified by estimation of the kinetic parameters of the Monod equation. Due to the AOPs pretreatment a decrease of the Monod constant and an increase of maximal specific growth rate was observed. The activity of degradative enzymes of activated sludge was assayed by the methods of 2-[4-iodophenyl]-3-[4-nitrophenyl]-5-phenyltetrazolium chloride test.     

LANCE: Laccase-nanoparticle conjugates for the elimination of micropollutants (endocrine disrupting chemicals) from wastewater in bioreactors

Elimination of recalcitrant chemicals during wastewater treatment is a difficult problem for both developing and industrialized countries. The biological elimination of very persistent xenobiotics such as endocrine disrupting chemicals from municipal and industrial sewage treatment plants is an ambitious challenge as existing physico-chemical methods, such as advanced oxidation processes, are energy-intensive and consume high amounts of chemicals. Through the entry into force of strict legislative measures, such as the Water Framework Directives (EU WFD in Directive 2000/60/EC of the European Parliament and of the Council establishing a framework for the Community action in the field of water policy, 2000) and REACH (REACH EU in European Community Regulation on chemicals and their safe use (EC 1907/2006), 2007), the market for wastewater treatment is exploding. For instance the European market potential for the membrane bioreactor technology is estimated to 57 M€ per year. Based on recent progresses in nanotechnology, new developments in catalysis and environmental applications can be foreseen for the near future. Indeed, because of high surface area-to-volume ratio in nano-systems, heterogeneous enzymatic or catalytic reactions can be greatly enhanced. In the LANCE project a nanoparticle (NP)-based technology is under development. Cheap and resistant oxidative enzymes, i.e. laccases are immobilized onto the surface of the particles in order to produce systems possessing a broad substrate spectrum for the degradation of cocktails of recalcitrant pollutants. One of the objectives is to produce NPs that are compatible with wastewater treatment and can be synthesised in a cost-effective and large-scale fashion, e.g. silica-based NPs using flame spray pyrolysis and emulsion-based techniques. The modified particles are applied in bioreactors where they are retained, i.e. membrane bioreactors or perfusion basket reactors to eliminate pollutants from the wastewater. Such reactors allow multi-cycle use of the NPs coated with active enzymes and thus contribute to decrease the treatment costs. The two-year activities of the LANCE project encompass the synthesis of various NP systems, the immobilization of selected low cost industrial laccases on the latter, and the technical and scientific proof of the “depollution” concept.     

Multi-faceted strategy based on enzyme immobilization with reactant adsorption and membrane technology for biocatalytic removal of pollutants: A critical review

In the modern era, the use of sustainable, environmentally friendly alternatives for removal of recalcitrant pollutants in streams resulting from industrial processes is of key importance. In this context, biodegradation of phenolic compounds, pharmaceuticals and dyes in wastewater by using oxidoreductases offers numerous benefits. Tremendous research efforts have been made to develop novel, hybrid strategies for simultaneous immobilization of oxidoreductase and removal of toxic compounds. The use of support materials with the options for combining enzyme immobilization with adsorption technology focused on phenolic pollutants and products of biocatalytic conversion seems to be of particular interest. Application of enzymatic reactors based on immobilized oxidoreductases for coupling enzyme-aided degradation and membrane separation also attract still growing attention. However, prior selection of the most suitable support/sorbent material and/or membrane as well as operational mode and immobilization technique is required in order to achieve high removal efficiency. Thus, in the framework of this review, we present an overview of the impact of support/sorbent material on the catalytic properties of immobilized enzymes and sorption of pollutants as well as parameters of membranes for effective bioconversion and separation. Finally, future perspectives of the use of processes combining enzyme immobilization and sorption technology as well as application of enzymatic reactors for removal of environmental pollutants are discussed.     

Different bioreactor configurations for the decolourisation of the azo dye reactive black 5 by Geotrichum sp. CCMI 1019

Decolourisation of the azo dye Reactive Black 5 by Geotrichum sp. CCMI 1019 was studied using stirred tank reactors (STR) and two types of bubble columns (porous plate (PP) bubble column and aeration tube (AT) bubble column). For the bubble columns, the k_La increased with the gas fractional hold-up (ε_G) and the aeration rate. A linear relationship between ε_G and superficial gas velocity was obtained for all reactors. At same aeration rates, the PP bubble columns showed higher k_La and hold-up values than the AT bubble column. In the STRs, large and dense aggregates were formed which adhered to surfaces whereas bubble columns gave smaller and less compact pellets.

Manganese peroxidase and laccase were detected in the extracellular media in all reactors. However, laccase was only detected after the onset of decolourisation, suggesting that additional enzymes may be involved. Mn peroxidase activity was detected (about 46 U/ml) in both the STRs and AT bubble columns but higher values (110 U/ml) were obtained with the PP bubble columns.

Out of the three reactor systems studied, the AT bubble columns gave the most favourable results for Reactive Black 5 decolourisation. Rapid and complete colour removal was obtained throughout the visible spectrum. Bubble columns are simple in design as well as operation and may be useful for the bioremediation of textile wastewater.     

Hydrodynamic performance of pilot-scale duckweed,algal-based,rock filter and attached-growth media reactors used for waste stabilisation pond research

The specific nature of fluid dynamics within waste stabilisation ponds can have a determining influence on their functional treatment performance. This paper presents the results of hydraulic tracer experiments undertaken to characterise the hydrodynamic behaviour of several pilot-scale advanced pond treatment systems (a duckweed-based pond, a conventional algal-based ‘open’ pond, a rock filter and a novel horizontal-flow attached-growth media reactor) investigated for their effectiveness at polishing a full-scale tertiary pond effluent. Duplicate tracer studies were undertaken for each of the four experimental reactors with the aid of the fluorescent dye rhodamine WT. Results from tracer studies showed flow distribution in all reactors to be highly dispersed with varying degrees of dead space volume and short-circuiting in all pilot reactors, indicating that a percentage of the total reactor volume across all treatment systems was inactive. Results from a number of calculated parameters of hydraulic performance showed that hydrodynamic efficiency was greatest in the novel horizontal-flow attached-growth media system, where a possible baffling of inflowing wastewater was thought to have promoted improved hydraulic operation. Outcomes from this research in general highlight the importance of undertaking detailed characterisations of the hydrodynamic operation of experimental pond systems and also emphasise the value of pre-validating the hydraulic design of experimental reactors used for stabilisation pond research.     

Treatment and kinetic modelling of a simulated dye house effluent by enzymatic catalysis

Biocatalytic treatment of a synthetic dye house effluent, simulating a textile wastewater containing various reactive dyestuffs (Reactive Yellow 15, Reactive Red 239 and Reactive Black 5) and auxiliary chemicals, was investigated in a batch reactor using a commercial laccase. A high decolourisation (above 86%) was achieved at the maximum wavelength of Reactive Black 5. The decolourisation at the other dyes wavelengths (above 63% for RY15 and around 41% for RR239) and the total decolourisation based on all the visible spectrum (around 55%) were not so good, being somewhat lower than in the case of a mixture of the dyes (above 89% for RB5, 77% for RY15, 68% for RR239 and above 84% for total decolourisation). Even so, these results suggest the applicability of this method to treat textile dyeing wastewaters. Kinetic models were developed to simulate the synthetic effluent decolourisation by commercial laccase. The kinetic constants of the models were estimated by minimizing the difference between the predicted and the experimental time courses. The close correlation between the experimental data and the simulated values seems to demonstrate that the models are able to describe with remarkable accuracy the simulated effluent degradation. Water quality parameters such as TOC, COD, BOD₅ and toxicity were found to be under the maximum permissible discharge limits for textile industries wastewaters.     

Potential of combined fungal and bacterial treatment for color removal in textile wastewater

Low efficiency of dye removal by mixed bacterial communities and high rates of dye decolorization by white-rot fungi suggest a combination of both processes to be an option of treatment of textile wastewaters containing dyes and high concentrations of organics. Bacteria were able to remove mono-azo dye but not other chemically different dyes whereas decolorization rates using Irpex lacteus mostly exceeded 90% within less than one week irrespective of dye structure. Decolorization rates for industrial textile wastewaters containing 2-3 different dyes by fungal trickling filters (FTF) attained 91%, 86%, 35% within 5-12 d. Sequential two-step application of FTF and bacterial reactors resulted in efficient decolorization in 1st step (various single dyes, 94-99% within 5 d; wastewater I, 90% within 7 d) and TOC reduction of 95-97% in the two steps. Large potential of combined use of white-rot fungi and traditional bacterial treatment systems for bioremediation of textile wastewaters was demonstrated.     

The use of solid waste of a nylon-6 plant as a nutrient for bacterial decolourisation of dyes

Three caprolactam-degrading bacterial isolates grew in liquid synthetic medium containing solubilised solid waste of a nylon-6 production plant as the sole source of carbon and nitrogen. Typically, the caprolactam content of solid waste was decreased by 95% in 72 h by Alcaligenes faecalis. A. faecalis was the most potent caprolactam-degrading bacterium out of the three isolates. The biomass of the bacteria obtained by growth in the solubilised solid waste medium had the ability to decolourise some synthetic azo and triphenylmethane dyes. Decolourisation of dyes was obtained in static condition, in synthetic medium which contained only the components of the solid waste as the sole sources of carbon and nitrogen and also in nutritionally rich medium. The supplementation of yeast extract to solid waste medium did not increase the efficiency of decolourisation in case of two of the bacterial cultures. Depending on the dye, medium and bacteria used, decolourisation in the range of 35–94% was achieved in 48–96 h. The decolourisation was not due to the adsorption of the dyes by the bacterial biomass except in case of Procion Blue MR and Black B. Based on these observations, the simultaneous biological treatment of the solid waste of nylon-6 plant and the decolourisation of synthetic dyes present in wastewater or solid waste is envisaged.     

Batch tests for assessing decolourisation of azo dyes by methanogenic and mixed cultures

Most of the published studies on azo dye colour removal involve anaerobic mixed cultures and there is some interest in the knowledge of how dye reduction occurs, if by facultative, strictly anaerobic or both bacterial trophic groups present in classic anaerobic digestors. This paper describes the behaviour of methanogenic and mixed bacteria cultures on the colour removal in batch systems, of a commercial azo dye, C.I. Acid Orange 7, used in paper and textile industries. The aim of this study is to demonstrate, by analysing dye decolourisation, that it occurs with mixed cultures as well as with strictly anaerobic (methanogenic) cultures. Tests were performed with a range of dye concentrations between 60 and 300 mg l⁻¹. The influence of dye concentration on the carbon source removal and decolourisation processes was studied. The effect of carbon source concentration on colour removal was also analysed for both cultures. The degradation rates in mixed and methanogenic cultures were compared. The consumption of carbon source was monitored by COD analysis and dye degradation by ultraviolet-visible spectrophotometry and thin layer chromatography.