Effect of SBR feeding strategy and feed composition on the stability of aerobic granular sludge in the treatment of a simulated textile wastewater.

Treatment of the highly polluting and variable textile industry wastewater using aerobic granular sludge (AGS) sequencing batch reactors (SBRs) has been recently suggested. Aiming to develop this technology application, two feeding strategies were compared regarding the capacity of anaerobic-aerobic SBRs to deal with disturbances in the composition of the simulated textile wastewater feed. Both a statically fed, anaerobic-aerobic SBR and an anaerobic plug-flow fed, anaerobic-aerobic SBR could cope with shocks of high azo dye concentration and organic load, the overall chemical oxygen demand and color removal yields being rapidly restored to 80%. Yet, subsequent azo dye metabolite bioconversion was not observed, along the 315-day run. Moreover, switching from a starch-based substrate to acetate in the feed composition deteriorated AGS stability. Overall, the plug-flow fed SBR recovered more rapidly from the imposed disturbances. Further research is needed towards guaranteeing long-term AGS stability during the treatment of textile wastewater.

An electronic device to record consensual reflex in human pupil.

Examination of the pupil offers an objective evaluation of visual function as well as the vegetative pathways to the eye. This work proposes the development of an effective method and a portable device to test the consensual pupillary reflex. The first results demonstrate the success of a new device construction and methodology to record the consensual reflex with different stimulus, in a situation of complete blockage of light.

Dual-mode cultivation of Chlorella protothecoides applying inter-reactors gas transfer improves microalgae biodiesel production.

Chlorella protothecoides, a lipid-producing microalga, was grown heterotrophically and autotrophically in separate reactors, the off-gases exiting the former being used to aerate the latter. Autotrophic biomass productivity with the two-reactor association, 0.0249gL(-1)h(-1), was 2.2-fold the value obtained in a control autotrophic culture, aerated with ambient air. Fatty acid productivity was 1.7-fold the control value. C. protothecoides heterotrophic biomass productivity was 0.229gL(-1)h(-1). This biomass' fatty acid content was 34.5% (w/w) with a profile suitable for biodiesel production, according to European Standards. The carbon dioxide fixed by the autotrophic biomass was 45mgCO2L(-1)h(-1) in the symbiotic arrangement, 2.1 times the control reactor value. The avoided CO2 atmospheric emission represented 30% of the CO2 produced in the heterotrophic stage, while the released O2 represented 49% of the oxygen demand in that stage. Thus, an increased efficiency in the glucose carbon source use and a higher environmental sustainability were achieved in microalgal biodiesel production using the proposed assembly.

Bioreactor monitoring with spectroscopy and chemometrics: a review.

Biotechnological processes are crucial to the development of any economy striving to ensure a relevant position in future markets. The cultivation of microorganisms in bioreactors is one of the most important unit operations of biotechnological processes, and real-time monitoring of bioreactors is essential for effective bioprocess control. In this review, published material on the potential application of different spectroscopic techniques for bioreactor monitoring is critically discussed, with particular emphasis on optical fiber technology, reported for in situ bioprocess monitoring. Application examples are presented by spectroscopy type, specifically focusing on ultraviolet-visible, near-infrared, mid-infrared, Raman, and fluorescence spectroscopy. The spectra acquisition devices available and the major advantages and disadvantages of each spectroscopy are discussed. The type of information contained in the spectra and the available chemometric methods for extracting that information are also addressed, including wavelength selection, spectra pre-processing, principal component analysis, and partial least-squares. Sample handling techniques (flow and sequential injection analysis) that include transport to spectroscopic sensors for ex-situ on-line monitoring are not covered in this review.

Development of PLS calibration models from UV-Vis spectra for TOC estimation at the outlet of a fuel park wastewater treatment plant.

In the present work ultraviolet (UV)-visible spectra of water samples collected at the outlet of a fuel park wastewater treatment plant, including biological treatment, were acquired and used for the development of partial least squares (PLS) calibration models for the fast and simple estimation of total organic carbon (TOC). Three different PLS models were developed and compared on the basis of a common spectral range. The first model was obtained using spectra of raw samples, the second using spectra of diluted samples, to assess signal saturation in the UV region, and the third using spectra of both diluted and raw samples, in order to expand the narrow interval of TOC concentration values present in the original dataset. The root mean squared error of cross-validation values for the developed PLS models were 2.3, 1.0 and 4.4 mg Cl(-1), respectively, and the validation results where highly satisfactory (root mean squared error of prediction values of 1.8, 0.8 and 4.5 mg Cl(-1), respectively).

Scanning electron microscopy investigations on bis(2-ethylhexyl)phthalate treated Mycobacterium cells.

Comparative investigation of steroid transforming activity and ultrastructural changes of bis(2-ethylhexyl)phthalate (BEHP, phthalate) treated Mycobacterium sp. NRRL B-3805 cells was carried out. Transformation of beta-sitosterol into androstenedione (AD) and androstadienedione (ADD) was performed in phthalate medium by resting cells preincubated in the organic solvent for a period from 3 to 24 h. It was observed that a preincubation greater than 12 h leads to the development of dense formations on the cells surface, reduction in the cell turgor, disruption in the cell walls, and formation of zones with reduced electron density. The preincubation for 24 h causes deeper changes in the cell ultrastructure but the treated cells retain their steroid transforming activity, allowing up to 80% of the substrate to be converted into AD and ADD. A preincubation of the resting Mycobacterium cells in BEHP for 6 h might be recommended as it leads to an achievement of stoichiometrical transformation of the substrate into AD and ADD and slightly higher initial rate of the reaction performed.

UV spectra analysis for water quality monitoring in a fuel park wastewater treatment plant.

In the context of the high application potentials for on-line measurements in wastewater quality monitoring, UV spectroscopy has received recent attention. In the present work UV spectrophotometric analyses were coupled to principal component analysis (PCA) and cluster analysis (CA) to characterize samples taken from a fuel park wastewater treatment plant and to attempt preliminary contaminant identification in the treated wastewater. The score plot resulting from PCA identified two different groups of spectra, one including the influents to the biological reactor and the other the treated wastewater samples. Among the latter, weekday and weekend samples could be further distinguished. The same groups of samples were identified in a dendrogram from CA. The score plot and the dendrogram also allowed the tentative identification of employed process chemicals (lubricant and detergents) as residual contaminants in the treated effluent.

Monoazo and diazo dye decolourisation studies in a methanogenic UASB reactor.

Mixed anaerobic bacterial consortia have been show to reduce azo dyes and batch decolourisation tests have also demonstrated that predominantly methanogenic cultures also perform azo bond cleavage. The anaerobic treatment of wool dyeing effluents, which contain acetic acid, could thus be improved with a better knowledge of methanogenic dye degradation. Therefore, the decolourisation of two azo textile dyes, a monoazo dye (Acid Orange 7, AO7) and a diazo dye (Direct Red 254, DR254), was investigated in a methanogenic laboratory-scale Upflow Anaerobic Sludge Blanket (UASB), fed with acetate as primary carbon source. As dye concentration was increased a decrease in total COD removal was observed, but the acetate load removal (90%) remained almost constant. A colour removal level higher than 88% was achieved for both dyes at a HRT of 24h. The identification by HPLC analysis of sulfanilic acid, a dye reduction metabolite, in the treated effluent, confirmed that the decolourisation process was due mainly to azo bond reduction. Although, HPLC chromatograms showed that 1-amino-2-naphthol, the other AO7 cleavage metabolite, was removed, aeration batch assays demonstrated that this could be due to auto-oxidation and not biological mineralization. At a HRT of 8h, a more extensive reductive biotransformation was observed for DR254 (82%) than for AO7 (56%). In order to explain this behaviour, the influence of the dye aggregation process and chemical structure of the dye molecules are discussed in the present work.

Model development and application for surfactant biodegradation in an acclimatising activated sludge system.

A model for the biodegradation of non-ionic surfactants in an activated sludge system during acclimatisation was developed, based on respirometric and titrimetric experimental data. The data were obtained in a sequencing batch reactor (SBR) using a non-ionic surfactant as sole carbon source and sludge previously acclimatised to a different surfactant. The model was successfully applied to successive SBR-cycles of sludge acclimatisation processes subjected to two ethoxylated surfactants. The model was validated using the corresponding total organic carbon data. The evolution of the model parameters along the acclimatisation process was examined. An acclimatisation model was developed using the evolution of two of these parameters (affinity constant and inhibition constant), supported by two independently calculated acclimatisation indicators. This acclimatisation model was then applied to determine an optimal surfactant concentration sequence to feed non-acclimatised sludge during a period of 41 days, in order to induce pre-acclimatisation to this surfactant before it replaces another one in the wastewater. The model was also useful in the economical assessment of this and alternative procedures to cope with frequent changes in activated sludge feed composition.

Behaviour of Mycobacterium sp. NRRL B-3805 whole cells in aqueous, organic-aqueous and organic media studied by fluorescence microscopy.

The present work aimed at quantifying the viability and morphological changes occurring during the time course of the side-chain cleavage of beta-sitosterol, in aqueous, two-phase organic-aqueous and organic media by free resting cells of Mycobacterium sp. NRRL B-3805. The solvent used was bis(2-ethylhexyl) phthalate (BEHP). A 66.3% reduction in cell viability was observed after 24 h when the cells were incubated in phosphate buffer only, but the percentage of viable cells was constant thereafter. In biphasic systems with BEHP, cell viability was maintained at higher values in the first 48 h, during which complete degradation of substrate was achieved. The availability of oxygen, which should be higher in the biphasic system than in the aqueous system, and of a carbon and energy source, thus seem important for the cells to retain their viability. In biphasic systems, cells tended to shrink and decrease their surface roughness, i.e. to decrease their surface area, possibly as a way to protect themselves from mechanical stress due to the presence of organic-aqueous interfacial forces, which resulted in disaggregation of cell clusters. A method used to visualise BEHP droplets with a standard optical microscope showed that the cells adhered to the surface of the solvent droplets, but no cells were observed inside these. In pure BEHP medium, cells retained their viability level for at least 150 h, independently of a pre-incubation period, which did not seem to induce any adaptation effect. Solvent biocompatibility, higher oxygen availability and reduced interfacial stress could have contributed to this maintenance of viability.

Analysis of secondary metabolite fate during anaerobic-aerobic azo dye biodegradation in a sequential batch reactor.

A great number of the reported examples of azo dye biodegradation comprise two main steps, the reductive cleavage of the azo bond under anaerobic conditions and the subsequent aerobic mineralization of the produced aromatic amines. Based on this possible metabolism a Sequencing Batch Reactor was chosen to study biologicalcolor removal from simulated cotton textile effluents containing a reactive azo dye. In previous studies high color removal levels of the azo dye Remazol Brilliant Violet 5R were achieved (up to 90% with an initial dye concentration of 100 mg l(-1)) during the anaerobic phase of Sequencing Batch Reactor operation. However, HPLC analyses revealed that the aromatic amines formed in the anaerobic phase were not mineralized during the subsequent aerobic phase. In an attempt to promote the aerobic biodegradation of these aromatic amines three different approaches were tested, the increase of the relative duration of the aerobic phase, the increase of the hydraulic retention time through the decrease of the daily fill flow and finally the increase of the dye/carbon source concentration ratio through the decrease of the fed volumetric organic load. The two aromatic amines directly resulting from azo bond reduction were detected by HPLC analysis. However, a third metabolite with significant peak area was also detected with a time profile suggesting an equilibrium with one of the aromatic amines In spite of the conversions occurring between metabolites during the cycles of the tested approaches, no effective biodegradation of these metabolites was observed during the experimental period of over 810 days.

Activated sludge acclimatisation kinetics to non-ionic surfactants.

The biodegradation of surfactants is a frequent and complex problem in domestic and industrial wastewater treatment processes. In addition to the resulting metabolites being sometimes refractory, the complete biodegradation of many of the most employed non-ionic surfactants requires long hydraulic retention times and the presence of specialised bacterial consortia. Preliminary acclimatisation tests highlighted the importance of the sludge acclimatisation state to a specific surfactant substrate for biotreatment efficiency. This paper reports on studies aimed at quantifying activated sludge acclimatisation and memory retention levels when subjected to changes in the type of surfactant included in the feed. Several transitions were tested, namely from an alkylphenol ethoxylate to a linear alkyl ethoxylate and the reverse, and between alkyl ethoxylates with different hydrophobic and hydrophilic molecular chain lengths. The kinetic results showed that sludge activation and memory loss were more dynamic for primary biodegradation It was found that the sludge was harder to adapt to alkylphenol ethoxylate than to alkyl ethoxylate. The former also apparently introduced an inhibitory effect, resulting in very slow degradation kinetics when imposed to alkyl ethoxylate acclimatised sludge. When replacing an alkyl ethoxylate with another surfactant of the same family, a longer ethoxylate chain reduced the degradation rates. This effect was further enhanced by simultaneously increasing the hydrophobic chain length of the substrate. The acclimatisation kinetic after the replacement of an alkyl ethoxylate by a longer counterpart was slower than the reverse case, and memory was also more easily lost.

Isolation of a biodegradable sterol-rich fraction from industrial wastes.

Several industrial waste materials were screened for their sterol content. The possibility of using these industrial by-products as sterol sources for the microbiological production of 4-androsten-3,17-dione (AD) and 1,4-androsta-diene-3,17-dione (ADD) was investigated. Two methods of obtaining the sterol fraction from wastes were developed. Sterol-rich (96-98%) fractions were isolated in a yield above 70%, from a tall-oil effluent of a paper pulp industry and from edible-oil deodorizates. These fractions were subsequently used as a substrate for microbial degradation by a Mycobacterium sp. strain and proved to be easily converted to AD and ADD.

Optimal operation for timely adaptation of activated sludge plants to changes in the surfactant composition of wastewater.

The composition of a textile industry wastewater is highly variable, as the industrial process has to follow fashion and season trends. Surfactants represent one of the largest COD fractions in a typical textile wastewater. Therefore, it was the aim of this paper to model the acclimatisation behaviour of an activated sludge system when subjected to composition variations in the surfactant containing feed. The model was based on data obtained in SBR experiments in which a linear alkyl ethoxylate as sole carbon source in the feed was replaced by another with a longer ethoxylate chain. A previously developed model (Fractionated Degradation Model) was applied to each of the 21 SBR cycles carried out in this study. The resulting best-fit parameters were investigated and sub-models were further developed, to create an acclimatisation model, able to predict the sludge acclimatisation level. Using the information given by this model, it was possible to propose an optimal operation scheme to pre-acclimatise the sludge before a surfactant replacement is made in the textile process. A cost analysis was carried out to compare different scenarios, with and without the application of this operation scheme. It was concluded that the proposed pre-acclimatisation process may be cost effective as compared to other scenarios if a cheap surfactant-containing product was employed.

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 x 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.

Effect of some operational parameters on textile dye biodegradation in a sequential batch reactor.

The combination of anaerobic and aerobic periods in the operation cycle of a Sequencing Batch Reactor (SBR) was chosen to study biological color removal from simulated textile effluents containing reactive, sulfonated, monoazo and diazo dyes, respectively, Remazol Brilliant Violet 5R and Remazol Black B. 90% color removal was obtained for the violet dye in a 24-h cycle with a Sludge Retention Time (SRT) of 15 days and an aerated reaction phase of 10 h. For the black dye only 75% color removal was achieved with the same operational conditions and no improvement was observed with the increase of the SRT to 20 days. For the violet dye a reduction of the color removal values from 90 to 75% was observed with the increase of the aerated reaction phase from 10 to 12 h. However, this increase did not promote the aerobic biodegradation of the produced aromatic amines. Abiotic tests were performed with sterilized SBR samples and no color removal was observed in cell-free supernatants. However color removal values of 30 and 12% were observed in the presence of sterilized cells and supernatants with violet and black dye, respectively and could be attributed to the presence of active reducing principles in the sterilized samples.

Modelling of activated sludge acclimisation to a non-ionic surfactant.

A model is proposed to describe activated sludge acclimatisation to a non-ionic surfactant. The model was calibrated automatically, using WEST, a specific software environment for wastewater treatment model building, simulation and parameter estimation. The assays have been performed in a sequencing-batch reactor (SBR), using a non-ionic surfactant as sole carbon source and non-acclimatised sludge. The best fitting model was based on the assumption of three sequentially degraded COD fractions, where the second fraction is a metabolite of the original molecule and the third fraction is a more slowly biodegradable metabolite resulting from the secondary degradation. For primary degradation, hydrolysis with no associated growth was assumed. The growth of microorganisms responsible for degradation of the second and third COD fractions was presumed to follow Haldane and first order kinetics, respectively. The model was able to fit four consecutive assays of the same acclimatisation process, using Brij 30 as carbon source, with different food/microorganism ratios. The parameters obtained showed that the (self-)inhibition of the growth on the second COD fraction decreased along acclimatisation.

Sterol side-chain cleavage with immobilized Mycobacterium cells in water-immiscible organic solvents.

The possibility of using whole Mycobacterium sp. cells for the selective degradation of the side-chain of sitosterol in an organic bioconversion medium was investigated. Sterol solubility limits were estimated and free-cell biocompatibility tests were carried out, using a range of water-immiscible solvents. Among these, phthalates exhibited good biocompatibility and sterol-solubilizing capacities. Several organic and inorganic matrices were tested for the immobilization of Mycobacterium sp. cells by surface adhesion. Celite led to the best results, being thus selected for beta-sitosterol side-chain degradation tests in phthalates and in aqueous medium. Cells entrapped in kappa-carrageenan and in polyurethane foams were also used in these tests. The highest degradation activities were obtained with cells immobilized in Celite, with bis(2-ethylhexyl)phthalate as the conversion medium, resulting in molar conversion yields up to 70%, with respect to beta-sitosterol (5 g l-1). Further activity and stability tests revealed that this bioconversion system is markedly temperature dependent.

Activity and stability of an entrapped-cell system for the Delta(1)-dehydrogenation of steroids in organic media.

Whole Arthrobacter simplex cells entrapped in kappa-carra-geenan or in two types of polyurethane foam, or adsorbed on silanized Celite, were tested for the Delta(1)-dehydrogenation of hydrocortisone and its derivatives in organic media. Catalytic activity and stability levels were evaluated for the immobilized cells in buffer with 2.5% (vol/vol) methanol, and in three buffer-saturated solvents (n-octan-1-ol, n-decan-1-ol, and chloroform). The addition of glutamate to the immobilization support stabilized the activity of the immobilized cells in the tested organic media. The system with polyurethane (HYPOL6100)-entrapped cells (with coimmobilized glutamate) in n-decan-1-ol provided the highest long-term activity levels. Several factors involved in the polyurethane-entrapment procedure were also studied.

Screening of whole-cell immobilization procedures for the delta 1-dehydrogenation of steroids in organic medium.

Different whole-cell immobilization methods and conditions were tested for the delta 1-dehydrogenation of 6-alpha-methyl-hydrocortisone-21-acetate with Arthrobacter simplex cells, in n-decane-1-ol and chloroform. Among the entrapment methods, polyurethane foams gave the best productivity of n-decane-1-ol, but could not be used in chloroform. Partition and diffusional barriers appeared to be the factors limiting productivity in entrapped-cell systems. The coentrapment of enzyme-stabilizing (glycerol, sucrose, sodium sulfate, monosodium glutamate) or hydrophobic additives did not significantly improve dehydrogenation rates in chloroform, although in some cases higher activities resulted in n-decane-1-ol. Dehydration of kappa-carrageenan-immobilized cells lowered the productivity in both solvents. The use of cell adsorption methods on silica-based carriers produced biocatalysts that in some cases were more active in chloroform than the entrapped cells. However, the surface-attached cells appeared to be more sensitive to solvent toxicity, more hydrophilic supports generally giving higher dehydrogenation rates. The estimated partition coefficients for the substrate between several of the tested supports and the two solvents are also presented. High partition ratios followed high measured activities in entrapment matrices but not in Celite (cell adsorption matrix). Activities in chloroform were always very low (less than 1 mumol product per hour and gram of cell dry weight). Polyurethane entrapment with n-decane-1-ol as a reaction medium was the most promising system, with measured dehydrogenation rates up to 40 times higher.