Isolation and characterization of a Klebsiella oxytoca strain for simultaneous azo-dye anaerobic reduction and bio-hydrogen production

A facultative anaerobic bacteria strain GS-4-08, isolated from an anaerobic sequence batch reactor for synthetic dye wastewater treatment, was investigated for azo-dye decolorization. This bacterium was identified as a member of Klebsiella oxytoca based on Gram staining, morphology characterization and 16S rRNA gene analysis. It exhibited a good capacity of simultaneous decolorization and hydrogen production in the presence of electron donor. The hydrogen production was less affected even at a high Methyl Orange (MO) concentration of 0.5 mM, indicating a superior tolerability of this strain to MO. This efficient bio-hydrogen production from electron donor can not only avoid bacterial inhibition due to accumulation of volatile fatty acids during MO decolorization, but also can recover considerable energy from dye wastewater.     

Decolorization of Orange I under alkaline and anaerobic conditions by a newly isolated humus-reducing bacterium,Planococcus sp. MC01

Printing and dyeing wastewater (PDW) normally has a high pH of 9.0–13.0, but alkaliphilic bacteria capable of treating PDW have rarely been isolated. Here we report an alkaliphilic and halotolerant, humus-reducing facultative anaerobe, Planococcus sp. MC01 (CGMCC 4771 = KCTC 33120), which can effectively reduce AQDS (anthraquinone-2, 6-disulphonate, humus analog) and decolorize Orange I (>94.0%) under alkaline and anaerobic conditions. The decolorization process of Orange I fits a pseudo-first-order kinetics well, and the rate constants (k) were 0.12, 0.17, 0.14, and 0.12 h⁻¹ when acetate, glucose, sucrose, and lactate, respectively, served as electron donor. When 0.5 mmol l⁻¹ AQDS and 2.0 mmol l⁻¹ γ-FeOOH were added as electron shuttles, the decolorization process was stimulated by 44.4% and 32.8%, respectively. Additionally, strain MC01 showed high decolorizing activity with low initial concentrations of Orange I (0.01–0.2 mmol l⁻¹), and the optimal glucose concentration for decolorization was 10.0 mmol l⁻¹. Results of UV/vis spectra suggested the cleavage of the double azo bond during decolorization. To the best of our knowledge, this is the first report of an alkaliphilic facultative anaerobe capable of decolorizing Orange I under alkaline conditions.     

Fe(III)-enhanced Azo Reduction by <Emphasis Type="Italic">Shewanella decolorationis</Emphasis> S12

Shewanella decolorationis S12 is capable of high rates of azo dye decolorization and dissimilatory Fe(III) reduction. Under anaerobic conditions, when Fe(III) and azo dye were copresent in S12 cultures, dissimilatory Fe(III) reduction and azo dye biodecolorization occurred simultaneously. Furthermore, the dye decolorization was enhanced by the presence of Fe(III). When 1 mM Fe(III) was added, the methyl red decolorizing efficiency was 72.1% after cultivation for 3 h, whereas the decolorizing efficiency was only 60.5% in Fe(III)-free medium. The decolorizing efficiencies increased as the concentration of Fe(III) was increased from 0 to 6 mM. Enzyme activities, which mediate the dye decolorization and Fe(III) reduction, were not affected by preadaption of cells to Fe(III) and azo dye nor by the addition of chloramphenicol. Both the Fe(III) reductase and the azo reductase were membrane associated. The respiratory electron transport chain inhibitors metyrapone, dicumarol, and stigmatellin showed significantly different effects on Fe(III) reduction than on azo dye decolorization.     

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.     

Microbial decolorization and degradation of synthetic dyes: a review

The synthesis of dyes and pigments used in textiles and other industries generate the hazardous wastes. A dye is used to impart color to materials of which it becomes an integral part. The waste generated during the process and operation of the dyes commonly found to contain the inorganic and organic contaminant leading to the hazard to ecosystem and biodiversity causing impact on the environment. The amount of azo dyes concentration present in wastewater varied from lower to higher concentration that lead to color dye effluent causing toxicity to biological ecosystem. The physico-chemical treatment does not remove the color and dye compound concentration. The decolorization of the dye takes place either by adsorption on the microbial biomass or biodegradation by the cells. Bioremediation takes place by anaerobic and/or aerobic process. The anaerobic process converts dye in toxic amino compounds which on further treatment with aerobic reaction convert the intermediate into CO₂ biomass and inorganics. In the present review the decolorization and degradation of azo dyes by fungi, algae, yeast and bacteria have been cited along with the anaerobic to aerobic treatment processes. The factors affecting decolorization and biodegradation of azo dye compounds such as pH, temperature, dye concentration, effects of CO₂ and Nitrogen, agitation, effect of dye structure, electron donor and enzymes involved in microbial decolorization of azo dyes have been discussed. This paper will have the application for the decolorization and degradation of azo dye compound into environmental friendly compounds.     

Degradation of azo dyes containing aminonaphthol by Sphingomonas sp strain 1CX

Sphingomonas sp strain 1CX was isolated from a wastewater treatment plant and is capable of aerobically degrading a suite of azo dyes, using them as a sole source of carbon and nitrogen. All azo dyes known to be decolorized by strain 1CX (Orange II, Acid Orange 8, Acid Orange 10, Acid Red 4, and Acid Red 88) have in their structure either 1-amino-2-naphthol or 2-amino-1-naphthol. In addition, an analysis of the structures of the dyes degraded suggests that there are certain positions and types of substituents on the azo dye which determine if degradation will occur. Growth and dye decolorization occurs only aerobically and does not occur under fermentative or denitrification conditions. The mechanism by which 1CX decolorizes azo dyes appears to be through reductive cleavage of the azo bond. In the case of Orange II, the initial degradation products were sulfanilic acid and 1-amino-2-naphthol. Sulfanilic acid, however, was not used by 1CX as a growth substrate. The addition of glucose or inorganic nitrogen inhibited growth and decoloration of azo dyes by 1CX. Attempts to grow the organism on chemically defined media containing several different amino acids and sugars as sources of nitrogen and carbon were not successful. Phylogenetic analysis of Sphingomonas sp strain 1CX shows it to be related to, but distinct from, other azo dye-decolorizing Sphingomonas spp strains isolated previously from the same wastewater treatment facility. Received 19 May 1999/ Accepted in revised form 11 August 1999     

耐盐偶氮染料脱色菌株GYW的筛选及特性

从某印染厂排水沟的底泥中分离筛选到1株对偶氮染料具有脱色能力的耐盐菌株GYW, 经16S rDNA序列分析, 鉴定为盐单胞菌属(Halomonas)中度耐盐菌。实验结果表明, 菌株GYW可以耐受10%以上的高盐度, 对酸性大红GR和其它偶氮染料具有广谱的脱色能力, 处于对数生长期的细胞脱色能力最强。对酸性大红GR的最佳脱色条件为：温度30°C, pH 7.5, LB培养基。氯离子对酸性大红GR脱色的抑制作用较强, 硫酸盐对脱色影响不大, 添加甜菜碱可提高染料的脱色速率, 最佳添加量为200 mg/L。     

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.     

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.     

Effects of electron donors and acceptors on anaerobic reduction of azo dyes by <Emphasis Type="Italic">Shewanella decolorationis</Emphasis> S12

Shewanella decolorationis S12 was able to reduce various azo dyes in a defined medium with formate, lactate, and pyruvate or H₂ as electron donors under anaerobic conditions. Purified membranous, periplasmic, and cytoplasmic fractions from strain S12 analyzed, respectively, only membranous fraction was capable of reducing azo dye in the presence of electron donor, indicating that the enzyme system for anaerobic azoreduction was located on cellular membrane. Respiratory inhibitor Cu²⁺, dicumarol, stigmatellin, and metyrapone inhibited anaerobic azoreduction by purified membrane fraction, suggesting that the bacterial anaerobic azoreduction by strain S12 was a biochemical process that oxidizes the electron donors and transfers the electrons to the acceptors through a multicompound system related to electron transport chain. Dehydrogenases, cytochromes, and menaquinones were essential electron transport components for the azoreduction. The electron transport process for azoreduction was almost fully inhibited by O₂, 6 mM of , and 0.9 mM of , but not by 10 mM of Fe³⁺. The inhibition may be a result from the competition for electrons from electron donors. These findings impact on the understanding of the mechanism of bacterial anaerobic azoreduction and have implication for improving treatment methods of wastewater contaminated by azo dyes.     

Alkaline extracellular reduction: isolation and characterization of an alkaliphilic and halotolerant bacterium, Bacillus pseudofirmus MC02

Aims: To isolate an alkaliphilic bacterium and to investigate its ability of extracellular reduction. Methods and Results: An alkaliphilic and halotolerant humus‐reducing anaerobe, Bacillus pseudofirmus MC02, was successfully isolated from a pH 10·0 microbial fuel cell. To examine its ability of extracellular reduction, AQDS (anthraquinone‐2, 6‐disulfonae), humic acids (HA) and Fe(III) oxides were chosen as representative electron acceptors. All the experiments were conducted in a pH 9·5 carbonate buffer. The results are as follows: (i) Sucrose, lactate, glucose and glycerol were the favourable electron donors for AQDS reduction by the strain MC02; (ii) The strain had the ability of reducing HA in the presence of sucrose; (iii) It could effectively reduce Fe(III) oxides coupled with sucrose fermentation when AQDS was added as electron shuttle and its Fe(III) reducing capacity ranked as: lepidocrocite (γ‐FeOOH) > goethite (α‐FeOOH) > haematite(α‐Fe₂O₃); (iv) The strain could decolourize azo dye Orange I. Conclusions: Bacillus pseudofirmus MC02 was capable of extracellular reduction in AQDS, HA and Fe(III) oxides, and it can be used for decolourizing azo dye (Orange I) in alkaline conditions. Significance and Impact of the Study: This is the first report of an alkaliphlic strain of B. pseudofirmus capable of extracellular reduction in AQDS, HA, Fe(III) oxides and decolourization of Orange I. This study could provide valuable information on alkaline biotransformation in the printing and dyeing wastewater and saline‐alkali soil.     

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.     

Rapid biodegradation and decolorization of Direct Orange 39 (Orange TGLL) by an isolated bacterium <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> strain BCH

A newly isolated novel bacterium from sediments contaminated with dyestuff was identified as Pseudomonas aeruginosa strain BCH by 16S rRNA gene sequence analysis. The bacterium was extraordinarily active and operative over a wide rage of temperature (10–60°C) and salinity (5–6%), for decolorization of Direct Orange 39 (Orange TGLL) at optimum pH 7. This strain was capable of decolorizing Direct Orange 39; 50 mg l⁻¹ within 45 ± 5 min, with 93.06% decolorization, while maximally it could decolorize 1.5 g l⁻¹ of dye within 48 h with 60% decolorization. Analytical studies as, UV–Vis spectroscopy, FTIR, HPLC were employed to confirm the biodegradation of dye and formation of new metabolites. Induction in the activities of lignin peroxidases, DCIP reductase as well as tyrosinase was observed, indicating the significant role of these enzymes in biodegradation of Direct Orange 39. Toxicity studies with Phaseolus mungo and Triticum aestivum revealed the non-toxic nature of degraded metabolites.     

Decolorization of azo dyes by <Emphasis Type="Italic">Shewanella oneidensis</Emphasis> MR-1 in the presence of humic acids

The effects of humic acid (HA) on azo dye decolorization by Shewanella oneidensis MR-1 were studied. It was found that HA species isolated from different sources could all accelerate the decolorization of Acid Red 27 (AR27). Anoxic and anaerobic conditions were required for the enhancement of azo dye decolorization by HA. In the presence of 50 mg DOC L⁻¹ Aldrich HA, 15–29% increases in decolorization efficiencies of azo dyes with different structures were achieved in 11 h. The enhancing effects increased with the increase of HA concentrations ranging from 25 to 150 mg DOC L⁻¹, and the decolorization rates were directly proportional to the HA concentrations when they were below 100 mg DOC L⁻¹. Lactate and formate were good electron donors for AR27 decolorization in the presence of HA. Both nitrate (0.1–3.0 mM) and nitrite (0.3–1.2 mM) inhibited AR27 decolorization in the presence of HA, and negligible decolorization was observed before their removal. Soluble FeCl₃ could accelerate the decolorization process in the presence of HA, whereas insoluble hematite could not. These findings may affect the understanding of bioremediation of azo dye-polluted environments and help improve the treatment of azo dye wastewaters.     

Reactive azo dye reduction by Shewanella strain J18 143

A bacterial isolate designated strain J18 143, originally isolated from soil contaminated with textile wastewater, was shown to reduce intensely coloured solutions of the reactive azo dye, Remazol Black B to colourless solutions. Phylogenetic placement based on 16S rRNA gene sequence homology identified the bacterium as a Shewanella species. Based on results from analyses of the end products of dye decoloration of Remazol Black B and the simpler molecule, Acid Orange 7, using capillary electrophoresis, UV-visible spectrophotometry and liquid chromatography-mass spectrometry, we suggest that colour removal by this organism was a result of microbially mediated reduction of the chromophore in the dye molecules. Anaerobic dye reduction by Shewanella strain J18 143 was 30 times more efficient than the reduction carried out by aerated cultures. Whole cells used a range of electron donors for dye reduction, including acetate, formate, lactate, and nicotinamide adenine dinucleotide (NADH), with formate being the optimal electron donor. The impact of a range of process variables was assessed (including nitrate, pH, temperature, substrate concentration, presence of an extracellular mediator) and results suggest that whole cells of Shewanella J18 143 offer several advantages over other biocatalysts with the potential to treat azo dyes.     

耐碱的偶氮染料脱色菌筛选及其特性的研究

从浙江某污水处理厂的活性污泥中筛选出若干株在高pH条件下对偶氮染料酸性大红GR有脱色能力的菌株,经脱色验证得到一株具有高效脱色活性的菌株Z1,经鉴定为巴斯德葡萄球菌(Staphylococcus pasteuri),并对此菌株的脱色特性进行了初步研究。结果表明,在厌氧条件下,Z1在pH7~12,40h对50mg/L的酸性大红GR脱色率均可达90%以上。该菌株对染料有较强的耐受力,在酸性大红GR浓度为300mg/L时,48h的脱色率仍可达93%。此外,该菌株能够对多种偶氮染料脱色,具有较好的脱色广谱性,有望应用于处理工业废水中的偶氮染料。     

脱水污泥中脱色偶氮染料功能菌群的驯化分离

【目的】获得降解混合偶氮染料的高效降解菌,应用于印染行业偶氮染料废水的生物处理和资源化。【方法】以某污水处理厂的脱水污泥作为分离源,经偶氮染料废水驯化后,分离筛选出9株偶氮染料脱色株(命名为T-1-T-9),通过形态观察、生理特征及基于16S rRNA基因序列的分子生物学鉴定,初步认定分离株分属于芽孢杆菌属(Bacillus)、微小杆菌属(Exiguobacterium)、寡单胞菌属(Stenotrophomonas)和副球菌属(Paracoccus)。【结果】所得分离株纯培养均可不同程度地脱色单一偶氮染料和混合偶氮染料,其中T-8对甲基橙和金橙I的脱色速率最大,40 h的脱色率分别为85.9%和86.2%,T-8菌株干粉也可在无外源碳源的条件下完全脱色金橙I。分离株混合培养脱色混合偶氮染料的效率明显高于纯培养,可达90.1%。【结论】脱水污泥作为脱色偶氮染料功能菌群的新来源具有良好的应用价值。     

Biodecolorization of textile azo dyes by isolated yeast from activated sludge: Issatchenkia orientalis JKS6

An ascomycetous yeast strain isolated from activated sludge could decolorize Reactive Black 5 azo dye at 200 mg l^?1 up to 90 % within 12–18 h under agitated condition. Yeast decolorization ability was investigated at different RB5 concentrations and, at higher dye concentration, 500 mg l^?1, the decolorization was found to be 98 % after 36 h incubation time. Extensive decolorization (95–99 %) was obtained in presence of five other azo dyes, Reactive Orange 16, Reactive Red 198, Direct Blue 71, Direct Yellow 12, and Direct Black 22, by isolated yeast. HPLC analysis, UV–vis spectra and colorless biomass obtained after complete decolorization showed that the decolorization occured through a biodegradation mechanism. Decolorization was occurred during the exponential growth phase which is associated to primary metabolism. Laccase production by the yeast cells was not detected. The isolated yeast was characterized according to phenotypical and molecular procedures and was closely related (99 % identity) to Issatchenkia orientalis.     

偶氮染料的微生物脱色研究进展

微生物法是染料废水治理的重要方法。本文综述了特异性酶作用下好氧细菌和真菌对偶氮染料的脱色以及厌氧条件下氧化还原介质作为电子穿梭体时偶氮染料的非特异性还原过程。指出厌氧偶氮还原是偶氮染料还原的主要形式, 电子供体不同脱色效率不同。对目前生物法去除偶氮染料存在的问题进行了分析, 提出了相应的对策措施。     

Decolorization of reactive dyes by <Emphasis Type="Italic">Clostridium bifermentans</Emphasis> SL186 isolated from contaminated soil

Decolorization of textile reactive azo dyes by a strain of bacteria (SL186) isolated from a contaminated site was investigated. SL186 was identified as Clostridium bifermentans by phenotypic characterization and 16S rDNA sequence comparison. Under anaerobic conditions, SL186 had decolorized the dyes Reactive Red 3B-A, Reactive Black 5, and Reactive Yellow 3G-P by over 90% after 36 h post-inoculation. The bacterium retained decolorizing activity over a wide range of pH values (6–12), with peak activity at pH 10. Additionally, SL186 decolorized a relatively high concentration of Reactive Red 3B-A dye (1,000 ppm) by over 80% and raw industrial effluent effectively. The addition of glucose increased the decolorization rate a little. Spectrophotometric analyses of the reactive dyes showed no distinct peak indicating aromatic amines. However, a new peak was detected between 300 and 450 nm from the decolorized raw industrial effluent. These results suggest that C. bifermentans SL186 is a suitable bacterium for the biological processing of dye-containing wastewater.