Azo dye reduction by thermophilic anaerobic granular sludge,and the impact of the redox mediator anthraquinone-2,6-disulfonate (AQDS) on the reductive biochemical transformation

Azo dye reduction at 55°C by thermophilic anaerobic granular sludge was investigated distinguishing between the biotic and abiotic mechanisms. The impact of the redox mediator anthraquinone-2,6-disulfonate (AQDS) on colour removal and co-substrate oxidation was also investigated. Metabolic activities of the thermophilic inoculum induced a fast azo dye reduction and indicated a biotic predominance in the process. The addition of co-substrate enhanced the decolourisation rates 1.7-fold compared with the bottles free of co-substrate. Addition of AQDS together with co-substrate enhanced the k value 1.5-fold, compared with the incubation containing co-substrate in the absence of AQDS. During a comparative study between sludge samples incubated under mesophilic (30°C) and thermophilic (55°C) conditions, the decolourisation rate at 55°C reached values up to sixfold higher than at 30°C. Biological treatment at 55°C showed a fast initial generation of reducing compounds via co-substrate oxidation, with AQDS increasing the azo dye reduction rate in all the incubations tested. Nevertheless, high concentrations of AQDS showed severe inhibition of thermophilic acetate and propionate oxidation and methane production rates. These promising results indicate that there may be good prospects for thermophilic anaerobic treatment of other reductive transformations such as reduction of nitroaromatics and dehalogenation.     

The transformation and toxicity of anthraquinone dyes during thermophilic (55 degrees C) and mesophilic (30 degrees C) anaerobic treatments

We studied in batch assays the transformation and toxicity of anthraquinone dyes during incubations with anaerobic granular sludge under mesophilic (30 degrees C) and thermophilic (55 degrees C) conditions. Additionally, the electron shuttling capacity of the redox mediator anthraquinone-2-sulfonic acid (AQS) and subsequent increase on decolourisation rates was investigated on anthraquinone dyes. Compared with incubations at 30 degrees C, serum bottles at 55 degrees C presented distinctly higher decolourisation rates not only with an industrial wastewater containing anthraquinone dyes, but also with model compounds. Compared with batch assays at 30 degrees C, the first-order rate constant "k" of the Reactive Blue 5 (RB5) was enhanced 11-fold and 6-fold for bottles at 55 degrees C supplemented and free of AQS, respectively. However, the anthraquinone dye Reactive Blue 19 (RB19) demonstrated a very strong toxic effect on volatile fatty acids (VFA) degradation and methanogenesis at both 30 degrees C and 55 degrees C. The apparent inhibitory concentrations of RB19 exerting 50% reduction in methanogenic activity (IC50-value) were 55 mg l(-1) at 30 degrees C and 45 mg l(-1) at 55 degrees C. Further experiments at both temperatures revealed that RB19 was mainly toxic to methanogens, because the glucose oxidizers including acetogens, propionate-forming, butyrate-forming and ethanol-forming microorganisms were not affected by the dye toxicity.     

Azo dye reduction by mesophilic and thermophilic anaerobic consortia

The reduction of the azo dye model compounds Reactive Red 2 (RR2) and Reactive Orange 14 (RO14) by mesophilic (30 degrees C) and thermophilic (55 degrees C) anaerobic consortia was studied in batch assays. The contribution of fermentative and methanogenic microorganisms in both temperatures was evaluated in the presence of the fermentative substrate glucose and the methanogenic substrates acetate, H2/CO2, methanol, and formate. Additionally, the effect of the redox mediator riboflavin on electron shuttling was assessed. We concluded that the application of thermophilic anaerobic treatment is an interesting option for the reductive decolorization of azo dyes compared to mesophilic conditions. The use of high temperature may decrease or even take the place of the need for continuous redox mediator dosage in bioreactors, contrarily to the evident effect of those compounds on dye reduction under mesophilic conditions. Both fermenters and methanogens may play an important role during reductive decolorization of dyes, in which mediators are important not only for allowing the different microbes to participate more effectively in this complex reductive biochemistry but also for assisting in the competition for electrons between dyes and other organic and inorganic electron acceptors.     

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.     

Application of redox mediators to accelerate the transformation of reactive azo dyes in anaerobic bioreactors.

Azo dyes are nonspecifically reduced under anaerobic conditions but the slow rates at which reactive azo dyes are converted presents a serious problem for the application of anaerobic technology as a first stage in the complete biodegradation of these compounds. As quinones have been found to catalyze reductive transfers by acting as redox mediators, the application of anthraquinone-2,6-disulfonic acid (AQDS) during continuous anaerobic treatment of the reactive azo dye, Reactive Red 2 (RR2), was evaluated. A mixture of volatile fatty acids was used as the electron-donating primary substrate. Batch experiments demonstrated that AQDS could increase the first-order rate constant of RR2 reductive cleavage by one order of magnitude. In the continuous experiment, treatment of RR2 containing synthetic wastewater in a lab-scale upflow anaerobic sludge blanket (UASB) reactor yielded low dye removal efficiencies (<30%). Consequently, severe toxicity problems occurred, eventually resulting in almost complete inhibition of the methanogenic activity. Addition of catalytic concentrations of AQDS (19 microM) to the reactor influent caused an immediate increase in the dye removal efficiency and recovery of biological activity. Ultimately, RR2 removal efficiency stabilized at 88%, and higher AQDS loads resulted in higher RR2 removal efficiencies (up to 98% at 155 microM AQDS). Examination of the RR2 decolorizing properties of dye-adapted reactor sludge and of nonadapted reactor seed sludge revealed that RR2 decolorization was principally a biologically driven transfer of reducing equivalents from endogenous and added substrates to the dye. Hydrogen, added in bulk, was clearly the preferred electron donor. Bacteria that couple dye decolorization to hydrogen oxidation were naturally present in seed sludge. However, enrichment was required for the utilization of electrons from volatile fatty acids for dye reduction. The stimulatory effect of AQDS on RR2 decolorization by AQDS-unadapted sludge was mainly due to assisting the electron transfer from endogenous substrates in the sludge to the dye. The stimulatory effect of AQDS on RR2 decolorization by sludge from the AQDS-exposed reactor was, in addition, strongly associated with the transfer of electrons from hydrogen and acetate to the dye, probably due to enrichment of specialized AQDS-reducing bacteria.     

Humic substances act as electron acceptor and redox mediator for microbial dissimilatory azoreduction by Shewanella decolorationis S12

The potential for humic substances to serve as terminal electron acceptors in microbial respiration and the effects of humic substances on microbial azoreduction were investigated. The dissimilatory azoreducing microorganism Shewanella decolorationis S12 was able to conserve energy to support growth from electron transport to humics coupled to the oxidation of various organic substances or H2. Batch experiments suggested that when the concentration of anthraquinone-2-sulfonate (AQS), a humics analog, was lower than 3 mmol/l, azoreduction of strain S12 was accelerated under anaerobic condition. However, there was obvious inhibition to azoreduction when the concentration of the AQS was higher than 5 mmol/l. Another humics analog, anthraquinone-2-sulfonate (AQDS), could still prominently accelerate azoreduction, even when the concentration was up to 12 mmol/l, but the rate of acceleration gradually decreased with the increasing concentration of the AQDS. Toxic experiments revealed that AQS can inhibit growth of strain S12 if the concentration past a critical one, but AQDS had no effect on the metabolism and growth of strain S12 although the concentration was up to 20 mmol/l. These results demonstrated that a low concentration of humic substances not only could serve as the terminal electron acceptors for conserving energy for growth, but also act as redox mediator shuttling electrons for the anaerobic azoreduction by S. decolorationis S12. However, a high concentration of humic substances could inhibit the bacterial azoreduction, resulting on the one hand from the toxic effect on cell metabolism and growth, and on the other hand from competion with azo dyes for electrons as electron acceptor.     

Thermophilic anaerobic digestion of Lurgi coal gasification wastewater in a UASB reactor

Lurgi coal gasification wastewater (LCGW) is a refractory wastewater, whose anaerobic treatment has been a severe problem due to its toxicity and poor biodegradability. Using a mesophilic (35 ± 2 °C) reactor as a control, thermophilic anaerobic digestion (55 ± 2 °C) of LCGW was investigated in a UASB reactor. After 120 days of operation, the removal of COD and total phenols by the thermophilic reactor could reach 50-55% and 50-60% respectively, at an organic loading rate of 2.5 kg COD/(m³ d) and HRT of 24 h; the corresponding efficiencies were both only 20-30% in the mesophilic reactor. After thermophilic digestion, the wastewater concentrations of the aerobic effluent COD could reach below 200 mg/L compared with around 294 mg/L if mesophilic digestion was done and around 375 mg/L if sole aerobic pretreatment was done. The results suggested that thermophilic anaerobic digestion improved significantly both anaerobic and aerobic biodegradation of LCGW.     

Methanogenic population structure in a variety of anaerobic bioreactors

The methanogenic community structures of six anaerobic sludges were examined using culture-independent techniques. The sludges were obtained from full-scale and laboratory-scale bioreactors, treating a variety of low- and high-strength, simple and complex wastewaters at psychrophilic (10-14 degrees C), mesophilic (37 degrees C) and thermophilic (55 degrees C) temperatures. Amplified rDNA restriction analysis identified 18 methanogenic operational taxonomic units in the six samples. 16S rRNA gene sequencing and phylogenetic reconstruction demonstrated that five separate groups of methanogens were represented with Methanosaeta-like species dominant in all sludges, but particularly in samples from a psychrophilic bioreactor treating low-strength synthetic sewage (75% of all clones detected).     

Comparative performance and microbial diversity of hyperthermophilic and thermophilic co-digestion of kitchen garbage and excess sludge

The objective of this study was to evaluate the performance characteristics of a hyperthermophilic digester system that consists of an acidogenic reactor operated at hyperthermophilic (70 degrees C) conditions in series with a methane reactor operated at mesophilic (35 degrees C), thermophilic (55 degrees C), and hyperthermophilic (65 degrees C) conditions. Lab-scale reactors were operated continuously, and were fed with co-substrates composed of artificial kitchen garbage (TS 9.8%) and excess sludge (TS 0.5%) at the volumetric ratio of 20:80. In the acidification step, COD solubilization was in the range of 22-46% at 70 degrees C, while it was 21-29% at 55 degrees C. The average protein solubilization was 44% at 70 degrees C. The double bond fatty acid removal ratio at 70 degrees C was much higher than at 55 degrees C. These results suggested that the optimal operation conditions for the acidogenic fermenter were about 3.1 days of HRT and 4 days of SRT at 70 degrees C. Methane conversion efficiency and the VS removal percentage in the methanogenic step following acidification was around 65% and 64% on average at 55 degrees C, respectively. The optimal operational conditions for this system are acidogenesis performed at 70 degrees C and methanogenesis at 55 degrees C. The key microbes determined in the hyperthermophilic acidification step were Anaerobic thermophile IC-BH at 6.4 days of HRT and Thermoanaerobacter thermohydrosulfuricus DSM 567 at 2.4 days of HRT. These results indicated that the hyperthermophilic system provides considerable advantages in treating co-substrates containing high concentrations of proteins, lipids, and nonbiodegradable solid matter.     

Influence of temperature on the performance of anaerobic fixed-film reactors utilizing screened dairy manure

Thermophilic (55°C) and mesophilic (35°C and 22°C) anaerobic digestions in laboratory scale (4 litre) fixed-film reactors fed with screened dairy manure were successfully operated over a range of hydraulic retention times, from 1 to 20 days. Maximum methane production rates of 1·82, 1·68 and 1·28 litres CH₄ litre⁻¹ day⁻¹ occurred at 1, 1·5 and 1 days HRT for the respective 55°C, 35°C and 22°C reactors. Both thermophilic and mesophilic digestions achieved maximum biodegradation efficiency at 10 days HRT. The thermophilic fixed-film reactor performed better than completely-mixed reactors in terms of methane production at HRTs shorter than 2 days. From the results, mesophilic fixed-film reactor operated at 35°C provided optimum methane production and net energy output between 1 and 5 days HRT.     

Effect of process temperature on bacterial and archaeal communities in two methanogenic bioreactors treating organic household waste

The bacterial and archaeal community structure was examined in two methanogenic anaerobic digestion processes degrading organic household waste at mesophilic (37 degrees C) and thermophilic (55 degrees C) temperatures. Analysis of bacterial clone libraries revealed a predominance of Bacteroidetes (34% of total clones) and Chloroflexi (27%) at the mesophilic temperature. In contrast, in the thermophilic clone library, the major group of clones were affiliated with Thermotogae (61%). Within the domain Archaea, the phyla Euryarchaeota and Crenarchaeota were both represented, the latter only at the mesophilic temperature. The dominating archaeons grouped with Methanospirillum and Methanosarcina species at the mesophilic and thermophilic temperature, respectively. Generally, there was a higher frequency of different sequences at the lower temperature, suggesting a higher diversity compared to the community present at the thermophilic temperature. Furthermore, it was not only the species richness that was affected by temperature, but also the phylogenetic distribution of the microbial populations.     

厌氧条件下希瓦氏菌腐殖质还原对偶氮还原的影响

以希瓦氏菌属的3个代表种为研究对象,研究了在厌氧条件下腐殖质的存在对偶氮还原的影响。实验结果表明:3个代表菌株在厌氧条件下都有高效的偶氮还原和腐殖质还原功能,1mmol/L偶氮染料在24h内完全脱色,并且偶氮还原与电子供体氧化存在着紧密的偶联关系。腐殖质物质模式物2-磺酸蒽醌AQS在小于1~2mmol/L条件下能显著加速偶氮还原,12h就完全脱色,3mmol/L时18h完全脱色。但当浓度大于3mmol/L时则对偶氮还原产生明显抑制作用。另一腐殖质模式物2,6-双磺酸蒽醌AQDS其浓度在1~3mmol/L以内亦使脱色在12h内完成,4~6mmol/L时15h左右完成脱色。7~12mmol/L仍有一定的脱色促进作用,但随着浓度的提高,其促进作用也逐渐减弱。这说明腐殖质的确可以作为氧化还原中间体穿梭于电子供体与染料的偶氮双键之间促进偶氮还原。但当其浓度达到某一阈值时它就显出与偶氮键竞争电子的本质,从而使偶氮还原速率下降。原因在于他们的氧化还原电势的差异,导致细菌呼吸链的电子递体对腐殖质物质和偶氮键的亲和力不同,从而使不同腐殖质浓度对偶氮键还原产生了不同的影响。     

Kinetics during the redox biotransformation of pollutants mediated by immobilized and soluble humic acids

The aim of this study was to elucidate the kinetic constraints during the redox biotransformation of the azo dye, Reactive Red 2 (RR2), and carbon tetrachloride (CT) mediated by soluble humic acids (HA_s) and immobilized humic acids (HA_i), as well as by the quinoid model compounds, anthraquinone-2,6-disulfonate (AQDS) and 1,2-naphthoquinone-4-sulfonate (NQS). The microbial reduction of both HA_s and HA_i by anaerobic granular sludge (AGS) was the rate-limiting step during decolorization of RR2 since the reduction of RR2 by reduced HA_i proceeded at more than three orders of magnitute faster than the electron-transferring rate observed during the microbial reduction of HA_i by AGS. Similarly, the reduction of RR2 by reduced AQDS proceeded 1.6- and 1.9-fold faster than the microbial reduction of AQDS by AGS when this redox mediator (RM) was supplied in soluble and immobilized form, respectively. In contrast, the reduction of NQS by AGS occurred 1.6- and 19.2-fold faster than the chemical reduction of RR2 by reduced NQS when this RM was supplied in soluble and immobilized form, respectively. The microbial reduction of HA_s and HA_i by a humus-reducing consortium proceeded 1,400- and 790-fold faster than the transfer of electrons from reduced HA_s and HA_i, respectively, to achieve the reductive dechlorination of CT to chloroform. Overall, the present study provides elucidation on the rate-limiting steps involved in the redox biotransformation of priority pollutants mediated by both HA_s and HA_i and offers technical suggestions to overcome the kinetic restrictions identified in the redox reactions evaluated.     

A comparison of the performance of mesophilic and thermophilic anaerobic filters treating papermill wastewater

Two anaerobic filters, one mesophilic (35 degrees C) and one thermophilic (55 degrees C), were operated with a papermill wastewater at a series of organic loadings. The hydraulic retention time (HRT) ranged from 6 to 24 h with organic loading rates (OLR) 1.07-12.25 g/l per day. At loading rates up to 8.4 g COD/l d, there was no difference in terms of the removal of soluble COD (SCOD) and gas production. At the higher organic loading rate, the SCOD removal performance of thermophilic digester was slightly better compare to mesophilic digester. Similar trend was also observed in terms of the daily methane production. The stability of thermophilic digester was also better than mesophilic digester particularly for the higher organic loadings. Volatile fatty acid accumulation was observed in the effluent of the mesophilic filter at the higher organic loading rates. The Stover-Kincannon model was applied to both digesters and it was found that model was applicable to both digesters for papermill wastewater. K(B) and U(max) constants from the Stover-Kincannon model were also derived.     

Humic analog AQDS and AQS as an electron mediator can enhance chromate reduction by Bacillus sp. strain 3C3

Humus as an electron mediator is recognized as an effective strategy to improve the biological transformation and degradation of toxic substances, yet the action of humus in microbial detoxification of chromate is still unknown. In this study, a humus-reducing strain 3C₃ was isolated from mangrove sediment. Based on the analyses of morphology, physiobiochemical characteristics, and 16S rRNA gene sequence, this strain was identified Bacillus sp. Strain 3C₃ can effectively reduce humic analog anthraquinone-2,6-disulfonate (AQDS) and anthraquinone-2-sulfonate (AQS) with lactate, formate, or glucose as electron donors. When the cells were killed by incubation at 95°C for 30 min or an electron donor was absent, the humic reduction did not occur, showing that the humic reduction was a biochemical process. However, strain 3C₃ had low capability of chromate reduction under anaerobic conditions, despite of having strong tolerance of the toxic metal. But in the presence of humic substances AQDS or AQS, we found that chromate reduction by strain 3C₃ was enhanced greatly. Because strain 3C₃ is an effective humus-reducing bacterium, it is proposed that humic substances could serve as electron mediator to interact with chromate and accelerate chromate reduction. Our results suggest that chromate contaminations can be detoxified by adding humic analog (low to 0.1 mM) as an electron mediator in the microbial incubation.     

Effect of iron(III), humic acids and anthraquinone-2,6-disulfonate on biodegradation of cyclic nitramines by Clostridium sp. EDB2

AIMS: To determine the biodegradation of cyclic nitramines by an anaerobic marine bacterium, Clostridium sp. EDB2, in the presence of Fe(III), humic acids (HA) and anthraquinone-2,6-disulfonate (AQDS). METHODS AND RESULTS: An obligate anaerobic bacterium, Clostridium sp. EDB2, degraded RDX and HMX, and produced similar product distribution including nitrite, methylenedinitramine, nitrous oxide, ammonium, formaldehyde, formic acid and carbon dioxide. Carbon (C) and nitrogen (N) mass balance for RDX products were 87% and 82%, respectively, and for HMX were 88% and 74%, respectively. Bacterial growth and biodegradation of RDX and HMX were stimulated in the presence of Fe(III), HA and AQDS suggesting that strain EDB2 utilized Fe(III), HA and AQDS as redox mediators to transfer electrons to cyclic nitramines. CONCLUSIONS: Strain EDB2 demonstrated a multidimensional approach to degrade RDX and HMX: first, direct degradation of the chemicals; second, indirect degradation by reducing Fe(III) to produce reactive-Fe(II); third, indirect degradation by reducing HA and AQDS which act as electron shuttles to transfer electrons to the cyclic nitramines. SIGNIFICANCE AND IMPACT OF THE STUDY: The present study could be helpful in determining the fate of cyclic nitramine energetic chemicals in the environments rich in Fe(III) and HA.     

Autocatalysis in Reactive Black 5 biodecolorization by <Emphasis Type="Italic">Rhodopseudomonas palustris</Emphasis> W1

Autocatalysis in biological decolorization of Reactive Black 5 (RB5) by Rhodopseudomonas palustris W1 was investigated in batch assays. An improvement of 1.5-fold in decolorization rate of RB5 was obtained by supplementing decolorization metabolites from 200 mg l(-1) RB5. Liquid chromatography-mass spectrometry and cyclic voltammetric analysis revealed that the constituent of dye precursors, from azo bonds breakage, with quinone-like structure and reversible oxidation-reduction activity can be used as redox mediators and was responsible for the catalytic reduction of RB5. The required amount of metabolites for catalytic decolorization was quite small, indicating its possible application in real textile wastewater treatment. Furthermore, decolorization metabolites of RB5 were shown as effective in catalyzing anaerobic decolorization of Direct Yellow 11, an azo dye without autocatalyic activity.     

Improvement of the decolorization of azo dye by anaerobic sludge bioaugmented with<Emphasis Type="Italic">Desulfovibrio desulfuricans</Emphasis>

A culture of anaerobic sludge was bioaugmented withDesulfovibrio desulfuricans for the color removal of authentic textile wastewater containing a substantial amount of sulfate, in order to improve the decolorization process. The sulfide produced by sulfate respiration ofD. desulfuricans can chemically reduce azo bonds to produce a colorless metabolite in the form of aromatic amines. In the case where the culture of anaerobic sludge was bioaugmented withD. desulfuricans, the decolorization of C.I. Reactive Black 5 showed an increase of more than 14% after 48 h in comparison with that in the culture of anaerobic sludge alone. In the decolorization of authentic textile wastewater, the color removal (about 69.0%) was improved by the mixed culture of anaerobic sludge andD. desulfuricans, compared with results obtained with only anaerobic sludge as reported in our previous work, suggesting that bioaugmentation byD. desulfuricans can be useful for the decolorization of wastewater that contains complex dye compounds and sulfate.     

Decolorization of the textile dyes by newly isolated bacterial strains

Six bacterial strains with the capability of degrading textile dyes were isolated from sludge samples and mud lakes. Aeromonas hydrophila was selected and identified because it exhibited the greatest color removal from various dyes. Although A. hydrophila displayed good growth in aerobic or agitation culture (AGI culture), color removal was the best in anoxic or anaerobic culture (ANA culture). For color removal, the most suitable pH and temperature were pH 5.5-10.0 and 20-35 degrees C under anoxic culture (ANO culture). More than 90% of RED RBN was reduced in color within 8 days at a dye concentration of 3,000 mg l(-1). This strain could also decolorize the media containing a mixture of dyes within 2 days of incubation. Nitrogen sources such as yeast extract or peptone could enhance strongly the decolorization efficiency. In contrast to a nitrogen source, glucose inhibited decolorization activity because the consumed glucose was converted to organic acids that might decrease the pH of the culture medium, thus inhibiting the cell growth and decolorization activity. Decolorization appeared to proceed primarily by biological degradation.