Microaeration improves the removal/biotransformation of organic micropollutants in anaerobic wastewater treatment systems.

This work assessed the effect of increasing microaeration flow rates (1-6 mL min-1 at 28 °C and 1 atm, equivalent to 0.025-0.152 L O2 L-1 feed) on the removal/biotransformation of seven organic micropollutants (OMPs) (three hormones, one xenoestrogen, and three pharmaceuticals), at 200 µg L-1 each, in a lab-scale upflow anaerobic sludge blanket reactor operated at a hydraulic retention time (HRT) of 7.4 h. Additionally, the operational stability of the system and the evolution of its microbial community under microaerobic conditions were evaluated. Microaeration was demonstrated to be an effective strategy to improve the limited removal/biotransformation of the evaluated OMPs in short-HRT anaerobic wastewater treatment systems. The rise in the airflow rate considerably increased the removal efficiencies of all OMPs. However, there seems to be a saturation limit for the biochemical reactions. Then, the best results were obtained with 4 mL air min-1 (0.101 L O2 L-1 feed) (~90%) because, above this flow rate, the efficiency increase was negligible. The long-term exposure to microaerobic conditions (249 days) led the microbiota to a gradual evolution. Consequently, there was some enrichment with species potentially associated with the biotransformation of OMPs, which may explain the better performance at the end of the microaerobic term even with the lowest airflow rate tested.

Evaluation of the production of alginate-like exopolysaccharides (ALE) and tryptophan in aerobic granular sludge systems.

The engineering and microbiological aspects involved in the production of alginate-like exopolysaccharides (ALE) and tryptophan (TRY) in aerobic granular sludge systems were evaluated. The inclusion of short anoxic phase (A/O/A cycle-anaerobic, oxic, and anoxic phase) and the control of sludge retention time (SRT ≈ 10 days) proved to be an important strategy to increase the content of these bioproducts in granules. The substrate concentration also has a relevant impact on the production of ALE and TRY. The results of the microbiological analysis showed that slow-growing heterotrophic microbial groups (i.e., PAOs and GAOs) might be associated with the production of ALE, and the EPS-producing fermentative bacteria might be associated with the TRY production. The preliminary economic evaluation indicated the potential of ALE recovery in AGS systems in decreasing the OPEX (operational expenditure) of the treatment, especially for larger sewage treatment plants or industrial wastewaters with a high organic load.

Redox mediator, microaeration, and nitrate addition as engineering approaches to enhance the biotransformation of antibiotics in anaerobic reactors.

The present work assessed some engineering approaches, such as the addition of the redox mediator anthraquinone-2,6-disulfonate (AQDS) (50 and 100 µM), microaeration (1 mL air min-1), and nitrate (100-400 mg L-1), for enhancing the biotransformation of the antibiotics sulfamethoxazole (SMX) and trimethoprim (TMP) (200 µg L-1 each) in anaerobic reactors operated at a short hydraulic retention time (7.4 h). Initially, very low removal efficiencies (REs) of SMX and TMP were obtained under anaerobic conditions (∼6%). After adding AQDS, the anaerobic biotransformation of these antibiotics significantly improved, with an increase of approximately 70% in the REs with 100 µM of AQDS. Microaeration also enhanced the biotransformation of SMX and TMP, especially when associated with AQDS, which provided REs above 70%, particularly for TMP (∼91% with 1 mL air min-1 and 50 µM of AQDS). Concerning nitrate, the higher the added concentration, the higher the REs of the antibiotics (∼86% with 400 mg L-1). Therefore, all the assessed approaches were demonstrated to be very effective in improving the limited biotransformation of SMX and TMP in anaerobic reactors, ensuring REs comparable to those found in higher-cost wastewater treatment technologies, such as conventional activated sludge, membrane bioreactors, and hybrid processes.

Parabens in aerobic granular sludge systems: Impacts on granulation and insights into removal mechanisms.

This work assessed the impact of methylparaben, ethylparaben, propylparaben, and butylparaben (200 µg L-1 each) on the granulation process as well as on the organic matter and nutrient removal of an aerobic granular sludge (AGS) system (6-h cycle). Additionally, some insights into the main paraben removal mechanisms were provided. In the presence of parabens, aerobic granules with good settleability, but with fragile and irregular structure, were grown. No significant effect of parabens on organic matter (>90%) and nitrogen (~70%) removal was evidenced. On the other hand, phosphorus removal was slightly impaired, although high removal efficiencies (~70%) were reached. High paraben removal efficiencies were achieved (>85%) in the AGS system, with methylparaben being the most recalcitrant compound. Concerning the removal mechanisms, biotransformation was the main mechanism in the removal of all parabens (85.5% for methylparaben and 100% for the others), whereas, apparently, adsorption played a role only in the removal of methylparaben. In addition, this compound was also suggested as a probable intermediate of the degradation of the larger alkyl-chain parabens. Lastly, regarding the microbial community, with the exception of Mycobacterium, the reactors shared the same genera, which may explain their comparable operational performances. Additionally, some genera that developed more in the presence of parabens may be related to their degradation. Therefore, although antimicrobial agents such as parabens compromised the granule structure, AGS system maintained a good operational performance and showed to be very efficient in paraben removal.

Effect of calcium addition to aerobic granular sludge systems under high (conventional SBR) and low (simultaneous fill/draw SBR) selection pressure.

This paper investigated the effect of calcium addition on the formation and properties of aerobic granules under high (conventional SBR) and low (simultaneous fill/draw SBR) selection pressure. Additionally, the simultaneous removal of carbon, nitrogen, and phosphorus, and the operational stability were assessed. The conventional SBRs showed earlier granule development (20 days) than the simultaneous fill/draw SBRs. The effect of calcium on granulation was more accentuated in conventional SBRs, forming larger granules in a shorter interval of time due to the higher EPS production. Additionally, higher amounts of calcium were found in the EPS matrix, mainly during the formation of granules. The operation regime and the addition of calcium did not affect the removal of carbon, nitrogen, and phosphorus. However, they both influenced the granulation time, settleability characteristics, size, and granule composition.

Autotrophic denitrification via nitrate as an effective approach for removal of dissolved sulfide in anaerobic reactors.

This work assessed the effect of adding different concentrations of nitrate (50-300 mg NO3 -·L-1) on the removal of dissolved and gaseous sulfide in an anaerobic reactor treating synthetic effluent containing sulfate (100 mg SO4 2-·L-1) and organic matter (1 g COD·L-1). Autotrophic denitrification, stimulated by the addition of nitrate, was demonstrated to be a very effective approach for removal of dissolved sulfide even in the presence of a high concentration of organic matter (complete removal with 50 mg NO3 - mg·L-1). However, it had a minor effect on H2S(g). Sulfide remained partially oxidized to elemental sulfur even with excess nitrate (100-300 mg NO3 - mg·L-1). Therefore, the competition for this electron acceptor between the autotrophic and heterotrophic denitrification pathways may have prevented the conversion of the generated sulfide into sulfate again. No evidence of inhibition of methanogenesis and sulfidogenesis was found during nitrate supplementation.

Effects of coal ash supplementation on aerobic granular sludge cultivated in a simultaneous fill/draw sequencing batch reactor / Efeitos da suplementação de cinzas de carvão mineral em lodo granular aeróbio cultivado em reator em batelada sequencial de alimentação/descarte simultâneos

ABSTRACT This study aimed to verify if coal ash, a residue from thermal power plants, could act as a granulation nucleus, cations source, and abrasive element to favor granules formation and stability in aerobic granular sludge (AGS) systems. Two simultaneous fill/draw sequencing batch reactors (SBRs) (R1 and R2) were operated with 6-h cycles, i.e., the filling and drawing phases occurred simultaneously, followed by the reaction and settling phases. R1 was maintained as control, while R2 was supplemented with coal ash (1 g·L-1) on the first day of operation. Granulation was achieved in both reactors, and no significant differences were observed in terms of settleability, biomass retention, morphology, resistance to shear, and composition of the EPS matrix. However, the ash addition did not change the settleability, biomass retention, granule morphology, shear resistance, and extracellular polymeric substances (EPS) content significantly. COD removal was high (≥ 90%), while nitrogen (~50%) and phosphorus (~40%) removals were low, possibly due to the presence of nitrate during the anaerobic phase. With granulation, microbial population profile was altered, mainly at the genus level. In general, the operational conditions had a more considerable influence over granulation than the ash addition. The possible reasons are because the ash supplementation was performed in a single step, the low sedimentation rate of this particular residue, and the weak interaction between the ash and the EPS formed in the granular sludge. These factors appear to have decreased or prevented the action of the ash as granulation nucleus, source of cations, and abrasive element.

RESUMO O objetivo deste estudo foi verificar se a cinza de carvão mineral, um resíduo de usinas termelétricas, poderia atuar como núcleo de granulação, fonte de cátions e elemento abrasivo em sistemas de lodo granular aeróbio (LGA) para favorecer a formação e estabilidade dos grânulos. Dois reatores em batelada sequencial (RBS) (R1 e R2) foram operados em regime de alimentação/descarte simultâneos com ciclos de 6 h, ou seja, as fases de alimentação e descarte do efluente ocorreram simultaneamente, seguidas das fases de reação e de decantação. O R1 foi mantido como controle, enquanto o R2 foi suplementado com as cinzas (1 g·L-1) no primeiro dia de operação. A granulação foi alcançada em ambos os reatores, não havendo diferenças marcantes em termos de sedimentabilidade, retenção de biomassa, morfologia do grânulo, resistência ao cisalhamento e conteúdo de substâncias poliméricas extracelulares (SPE). A remoção de DQO foi alta (≥ 90%), enquanto as remoções de nitrogênio (~50%) e fósforo (~40%) foram baixas, possivelmente pela presença de nitrato na fase anaeróbia. Após a granulação, o perfil da comunidade microbiana mudou, especialmente em termos de gênero. Globalmente, as condições operacionais tiveram maior influência sobre a granulação do que a adição das cinzas, possivelmente porque elas só foram adicionadas uma vez e possuem baixa velocidade de sedimentação, bem como devido a uma fraca interação das cinzas com a matriz de SPE formada no lodo aeróbio. Esses fatores podem ter diminuído, ou mesmo impedido, a ação das cinzas como núcleo de grânulo, fonte de cátions ou elemento abrasivo.

Biogas valorization via continuous polyhydroxybutyrate production by Methylocystis hirsuta in a bubble column bioreactor.

Creating additional value out of biogas during waste treatment has become a priority in past years. Biogas bioconversion into valuable bioproducts such as biopolymers has emerged as a promising strategy. This work assessed the operational feasibility of a bubble column bioreactor (BCB) implemented with gas recirculation and inoculated with a polyhydroxybutyrate (PHB)-producing strain using biogas as substrate. The BCB was initially operated at empty bed residence times (EBRTs) ranging from 30 to 120 min and gas recirculation ratios (R) from 0 to 30 to assess the gas-to-liquid mass transfer and bioconversion of CH4. Subsequently, the BCB was continuously operated at a R of 30 and an EBRT of 60 min under excess of nitrogen and nitrogen feast-famine cycles of 24 h:24 h to trigger PHB synthesis. Gas recirculation played a major role in CH4 gas-liquid transfer, providing almost fourfold higher CH4 elimination capacities (~41 g CH4 m-3 h-1) at the highest R and EBRT of 60 min. The long-term operation under N excess conditions entailed nitrite accumulation (induced by O2 limiting conditions) and concurrent methanotrophic activity inhibition above ~60 mg N-NO2- L-1. Adjusting the N-NO3- supply to match microbial N demand successfully prevented nitrite accumulation. Finally, the N feast-famine 24 h:24 h strategy supported a stable CH4 conversion with a removal efficiency of 70% along with a continuous PHB production, which yielded PHB accumulations of 14.5 ± 2.9% (mg PHB mg-1 total suspended solids × 100). These outcomes represent the first step towards the integration of biogas biorefineries into conventional anaerobic digestion plants.

Tecnologia de lodo granular aeróbio no tratamento de esgoto doméstico: oportunidades e desafios / Aerobic granular sludge technology in domestic wastewater treatment: opportunities and challenges

RESUMO Considerado por muitos profissionais como um dos maiores avanços do século 21 na área de tratamento de esgotos, os reatores de lodo granular aeróbio (LGA) vêm recebendo bastante atenção em termos de pesquisa e instalação em escala plena em diferentes continentes e condições climáticas. São frequentes os relatos na literatura de eficiências de remoção acima de 90% em termos de demanda química de oxigênio, nitrogênio total e fósforo total, além da manutenção no reator de elevadas concentrações de sólidos (> 8 g SSV/L) sem a necessidade de decantador secundário e recirculação de lodo. Contudo, há também diversos relatos de problemas de instabilidade da biomassa, longo período de formação dos grânulos (principalmente quando se utiliza esgoto real), formação de grânulos pequenos, acúmulo de nitrito e outras questões. Esta revisão explora os mecanismos necessários para granulação em estações de tratamento de esgoto em escala plena no tratamento de esgoto sanitário, incluindo os principais grupos microbianos presentes no LGA, parâmetros-chave para a formação dos grânulos, configurações de reator etc. Além disso, discutem-se algumas questões sobre a operação e a manutenção desses sistemas em escala plena.

ABSTRACT Considered by many professionals as one of the greatest advances in wastewater treatment in the 21st century, the aerobic granular sludge (AGS) reactors have received great attention in terms of research and full-scale installation in different continents and weather conditions. There are frequent reports in the literature on removal efficiencies above 90% in terms of COD, total nitrogen and total phosphorus, as well as the maintenance of high solids concentrations (> 8 g VSS/L) in the reactor without the need for secondary clarifier and sludge recirculation. However, there are also several reports on problems of biomass instability, long periods of granule formation (mainly when using real sewage), formation of small granules, nitrite accumulation (incomplete denitrification), and other issues. This review explores the mechanisms required for granulation in full-scale WWTP treating sanitary wastewater, including the main microbial groups present in the AGS, key granule formation parameters, reactor configurations, etc. In addition, some issues on the operation and maintenance of these full-scale systems are discussed.

Impact of cycle type on aerobic granular sludge formation, stability, removal mechanisms and system performance.

This paper aimed to assess the impact of the cycle type on aerobic granular sludge (AGS) formation, stability and system performance. Six AGS reactors were operated either on A/O cycles (anaerobic followed by oxic phase) or A/O/A cycles (anaerobic, followed by oxic and anoxic phases), changing only the phase time distribution. Reactors with high percentage of aerobic phase (65% of the total cycle time) generated granules with better settleability and resistance, however denitrification was impaired. On the other hand, reactors with long anaerobic or anoxic phases presented excellent nutrients removals, but the granules were fluffy and unstable. The best results in terms of performance and stability were achieved in an A/O/A reactor with short anoxic phase (10% of the total cycle) and medium aerobic phase (55% of the total cycle). Therefore, in AGS reactors, it is indispensable to optimize the cycle, aiming at fast biomass formation, long-term granule stability and high-rate pollutants removal.

Elucidating the influence of environmental factors on biogas-based polyhydroxybutyrate production by Methylocystis hirsuta CSC1.

The valorization of biogas as a feedstock for the generation of added-value bioproducts will play a key role on the sustainability of anaerobic digestion. The present work assessed the influence of key environmental parameters (O2:CH4 ratio, temperature and nitrogen source) on the growth and polyhydroxybutyrate (PHB) synthesis under nitrogen limiting conditions of the type II methanotroph Methylocystis hirsuta CSC1 using biogas as a feedstock. The O2:CH4 ratios tested (1:1, 1.5:1 and 2:1) did not affect significantly M. hirsuta CSC1 growth yields (~5 g TSS mol-1 CH4), although lower CH4 removal rates were reached under O2-limiting conditions (ratio 1:1). The highest PHB content (45 wt%) was achieved at a ratio 2:1 and was threefold higher than those obtained at lower ratios (~15 wt%). The increase in temperature from 15 to 25 °C resulted in increases in the growth yield (from 5 to 6 g TSS mol-1 CH4) and PHB content (from 32 to 40 wt%). Conversely, the lowest PHB content (30 wt%) was reached at 37 °C, together with a negligible growth under nutrient sufficient conditions. The nitrogen source also played a key role on both M. hirsuta CSC1 growth and PHB synthesis. Thus, ammonium resulted in the highest growth yield (7 g TSS mol-1 CH4), although the maximum PHB content was achieved when biomass was previously grown in nitrate as the nitrogen source (41 wt%). Nitrite exerted an inhibitory effect on M. hirsuta CSC1 growth.

Effect of calcium addition on the formation and maintenance of aerobic granular sludge (AGS) in simultaneous fill/draw mode sequencing batch reactors (SBRs).

This work investigated the effect of Ca2+ (100 mg L-1) addition on the formation and maintenance of aerobic granular sludge in a simultaneous fill/draw mode sequencing batch reactor (SBR), operated with a low liquid upflow velocity (0.92 m h-1), in order to verify if Ca2+ presence compensates the low selection pressure imposed. Additionally, carbon and nutrients removals, granules characteristics and microbial community were evaluated. For this, two SBRs (R1, control, and R2, Ca2+-supplemented) were operated (6-h cycle). In general, Ca2+ supplementation affected positively the sludge settleability, although a larger fraction of inert solids was found in the granules. The total extracellular polymeric substances were the same for both reactors, and no remarkable differences were observed between their polysaccharides and proteins contents. Overall, Ca2+ addition in a simultaneous fill/draw mode SBR neither accelerated the granule formation nor improved the operational performance. The microbial community structure, especially in terms of bioactivity, was not affected as well. Therefore, the effect of divalent cations might be more pronounced in conventional SBRs, in which the selection pressure is higher.

Comparison of the dynamics, biokinetics and microbial diversity between activated sludge flocs and aerobic granular sludge.

This work aimed to compare the dynamics, biokinetics, and microbial diversity between activated sludge flocs (ASF) and aerobic granular sludge (AGS) whose systems were operated under similar experimental conditions in terms of inoculum, feeding, substrate source, etc. Therefore, the kinetic parameters involved in the organic matter removal, nitrification, denitrification, and dephosphatation were determined, as well as the microbial changes were assessed by metagenomics analysis. Regarding the kinetic parameter yield coefficient (Y), values of 0.55 and 0.36 g VSS/g COD were found for ASF and AGS, respectively, showing a higher sludge production in ASF and the importance of feast/famine periods for lowering sludge production in AGS systems. AGS presented a lower sludge production and a higher endogenous consumption rate than ASF. The activity of phosphorus-accumulating bacteria was remarkably higher in AGS. Although both biomasses were aerobic, their kinetic parameters had significant differences.

Enhanced removal of emerging micropollutants by applying microaeration to an anaerobic reactor / Remoção acelerada de micropoluentes emergentes pela aplicação de microaeração em reator anaeróbio

ABSTRACT The present paper aimed to evaluate the impact of microaeration on both the removal performance of some emerging micropollutants (pharmaceuticals, hormones, and bisphenol A) and the microbial community structure of an anaerobic reactor treating synthetic wastewater. Under anaerobic conditions, the removal efficiencies of the micropollutants were very low (< 10%). However, the microaeration (1.0 mL air·min-1 at 27 °C and 1 atm, equivalent to a QAIR/QINF ratio of 0.1) expressively improved the removal efficiencies of all compounds (> 50%). Therefore, supplementing anaerobic reactors with low amounts of oxygen seems to be an interesting strategy to enhance the removal of the micropollutants tested. However, further studies should be carried out with other compounds in order to evaluate the wide applicability of microaeration to different classes of micropollutants in lab- and full-scale treatment systems. Concerning the microbiota structure, both bacterial and archaeal communities were not compromised by the different operational conditions and preserved their functional organization with high richness during the whole experiment.

RESUMO O presente trabalho teve como objetivo avaliar o impacto da microaeração tanto no desempenho de remoção de alguns micropoluentes emergentes (fármacos, hormônios e bisfenol A) quanto na estrutura da comunidade microbiana de um reator anaeróbio tratando uma água residuária sintética. Sob condições anaeróbias, as eficiências de remoção dos micropoluentes foram muito baixas (< 10%). Entretanto, a microaeração (1,0 mL de ar·min-1 a 27 °C e 1 atm, equivalente a uma relação QAR/QAF de 0,1) melhorou expressivamente as eficiências de remoção de todos os compostos (> 50%). Portanto, a suplementação de reatores anaeróbios com baixas quantidades de oxigênio parece ser uma estratégia interessante para melhorar a remoção dos micropoluentes testados. Entretanto, mais estudos devem ser realizados com outros compostos para avaliar a ampla aplicabilidade da microaeração a diferentes classes de micropoluentes em sistemas de tratamento em escala laboratorial e real. Com relação à estrutura da microbiota, tanto as comunidades de bactérias quanto as de arqueias não foram comprometidas pelas diferentes condições operacionais e preservaram sua organização funcional com elevada riqueza durante todo o experimento.

Effects of carbon source on the formation, stability, bioactivity and biodiversity of the aerobic granule sludge.

Three aerobic granular sludge systems were operated as sequencing batch reactors (SBR) with acetate, ethanol and glucose as carbon source. The SBR cycle was 6 h, with an anaerobic phase followed by an aerobic phase. The acetate granules (>1.5 mm) had the greatest microbial diversity and better results in terms of removal efficiency for carbon and nutrients (TN ≈ 72% and TP ≈ 42%) and also in the resistance tests. However, partial disintegration was observed. On the other hand, when ethanol was the substrate, the granules were stable, good nitrogen removal was achieved (TN ≈ 53%), but phosphorus removal was not favored (TP ≈ 31%). Glucose presented the lowest efficiency values for nitrogen (TN ≈ 44%) and phosphorous removal (TP ≈ 21%), and the granules formed (<1 mm) had the lowest microbial diversity. Therefore, the carbon source had a high impact on the characteristics of the granules.

Reductive decolourisation of sulphonated mono and diazo dyes in one- and two-stage anaerobic systems.

This work assessed the application of one- and two-stage mesophilic anaerobic systems to colour removal of sulphonated mono and diazo dyes with ethanol as electron donor. The dyes Congo Red (CR), Reactive Black 5 (RB5) and Reactive Red 2 (RR2) were selected as model compounds and tested separately in seven different periods. The one-stage system (R(1)) consisted of a single up-flow anaerobic sludge blanket (UASB) reactor, whereas the two-stage system (R(2)) consisted of an acidogenic UASB reactor (R(A)), a settler and a methanogenic UASB reactor (R(M)). For CR and RB5, no remarkable difference was observed between the colour removal performance of both anaerobic systems R(1) and R(2). The experiments with RR2 revealed that R(2) was more efficient on colour removal than R(1), showing efficiencies almost 2-fold (period VI) and 2.5-fold (period VII) higher than those found by R(1). Additionally, R(2) showed a higher stability, giving a good prospect for application to textile wastewaters. Finally, the acidogenic reactor (R(A)) had an important role in the overall decolourisation achieved by R(2) during the experiments with CR and RB5 (>78 %), whereas for RR2, a more recalcitrant dye, R(A) was responsible for up to 38 % of the total colour removal.

Impact of the redox mediator sodium anthraquinone-2,6-disulphonate (AQDS) on the reductive decolourisation of the azo dye Reactive Red 2 (RR2) in one- and two-stage anaerobic systems.

This work assessed the impact of the redox mediator sodium anthraquinone-2,6-disulphonate (AQDS) on the reductive decolourisation of the azo dye Reactive Red 2 (RR2) in one- and two-stage anaerobic systems (R(1) and R(2), respectively). The two-stage system achieved better colour removal efficiencies (52-62%) than the single-stage system (23-33%) in the absence of AQDS. Addition of AQDS accelerated the electrons transfer from the substrate (ethanol) to the dye, which increased the colour removal efficiency of both anaerobic systems (≈ 85%). Finally, the impact of acidogenic and methanogenic phases separation was masked by AQDS supplementation.

Sequential anaerobic/aerobic treatment of dye-containing wastewaters: colour and COD removals, and ecotoxicity tests.

Colour and COD removals of the azo dyes Congo Red (CR) and Reactive Black 5 (RB5) were individually evaluated in a sequential anaerobic/aerobic treatment system. Additionally, dye toxicity was assessed by using acute ecotoxicity tests with Daphnia magna as the indicator-organism. The anaerobic reactor was operated at approximately 27 °C and with hydraulic retention times of 12 and 24 h. The aerobic reactor was operated in batch mode with a total cycle of 24 h. During anaerobic step, high colour removals were obtained, 96.3% for CR (400 mg/L) and 75% for RB5 (200 mg/L). During the aerobic phase, COD effluent was considerably reduced, with an average removal efficiency of 52% for CR and 85% for RB5, which resulted in an overall COD removal of 88% for both dyes. Ecotoxicity tests with CR revealed that the anaerobic effluent presented a higher toxicity compared with the influent, and an aerobic post-treatment was not efficient in reducing toxicity. However, the results with RB5 showed that both anaerobic and aerobic steps could decrease dye toxicity, especially the aerobic phase, which removed completely the toxicity in D. magna. Therefore, the anaerobic/aerobic treatment is not always effective in detoxifying dye-containing wastewaters, sometimes even increasing dye toxicity.