Enhanced decolorization of sulfonated azo dye methyl orange by single and mixed bacterial strains AK1, AK2 and VKY1

Abstract

Methyl orange, a sulfonated azo dye having various industrial applications was decolorized by three bacteria Bacillus sp. strain AK1, Lysinibacillus sp. strain AK2 and Kerstersia sp. strain VKY1. The effect of various factors such as dye concentration, pH, temperature and NaCl concentration on decolorization was investigated. At 200?mg/L methyl orange concentration, the strains AK1, AK2 and VKY1 exhibited maximum decolorizing potential of 93, 95 and 96%, respectively, at temperature 35?°C and pH 7.0 within 18?h of incubation. These strains decolorized the dye over a wide range of pH (5–10), temperature (15–55?°C), and NaCl concentration (5–20?g/L). Further, these strains decolorize up to 800?mg/L concentrations of methyl orange within 24?h. The dye decolorization efficiency was further increased by using different consortia of these three strains which could decolorize the dye completely within 12?h of incubation. The cell-free extracts of the strains AK1, AK2 and VKY1 grown on methyl orange exhibited the azoreductase activity of 0.4794, 1.56 and 1.01?µM/min/mg protein, respectively. HPLC and FTIR analysis of the dye decolorized sample indicated the formation of 4-aminobenzenesulfonic acid and N,N-dimethyl-p-phenylenediamine as breakdown products of azo bond. The high decolorization potential of these bacterial strains individually and in consortia has potential application in remediation of dye effluent.     

Hydrogen production and anaerobic decolorization of wastewater containing Reactive Blue 4 by a bacterial consortium of Salmonella subterranea and Paenibacillus polymyxa

Anaerobic biodegradability of wastewater (3,000 mg CODcr/l) containing 300 mg/l Reactive Blue 4, with different co-substrates, glucose, butyrate and propionate by a bacterial consortium of Salmonella subterranea and Paenibacillus polymyxa, concomitantly with hydrogen production was investigated at 35°C. The accumulative hydrogen production at 3,067 mg CODcr/l was obtained after 7 days of incubation with glucose, sludge, the bacterial consortium. The volatile fatty acids, residual glucose and the total organic carbon were correlated to hydrogen obtained. Interestingly, the bacterial consortium possess decolorization ability showing approximately 24% dye removal after 24 h incubation using glucose as a co-substrate, which was about two and eight times those of butyrate (10%), propionate (12%) and control (3%), respectively. RB4 decolorization occurred through acidogenesis, as high volatile fatty acids but low methane was detected. The bacterial consortium will be the bacterial strains of interest for further decolorization and hydrogen production of industrial waste water.     

Bacterial Decolorization of Azo Dyes by Rhodopseudomonas palustris

Summary The ability of Rhodopseudomonas palustris AS1.2352 possessing azoreductase activity to decolorize azo dyes was investigated. It was demonstrated that anaerobic conditions were necessary for bacterial decolorization, and the optimal pH and temperature were pH 8 and 30–35 °C, respectively. Decolorization of dyes with different molecular structures was performed to compare their degradability. The strain could decolorize azo dye up to 1250 mg l⁻¹, and the correlation between the specific decolorization rate and dye concentration could be described by Michaelis–Menten kinetics. Long-term repeated operations showed that the strain was stable and efficient during five runs. Cell extracts from the strain demonstrated oxygen-insensitive azoreductase activity in vitro.     

Bacillus sp. mutant for improved biodegradation of Congo red: Random mutagenesis approach

This study presents the improved biodegradation of Congo red, a toxic azo dye, using mutant Bacillus sp. obtained by random mutagenesis of wild Bacillus sp. using UV and ethidium bromide. The mutants obtained were screened based on their decolorization performance and best mutants were selected for further studies. Better decolorization was observed in the initial Congo red concentration range 100–1000 mg/l for wild species whereas mutant strain was found to offer better decolorization up to 3000 mg/l. Mutant strain offered 12–30% reduction in time required for the complete decolorization by wild strain. The optimum pH and temperature were found to be 7.0 and 37 °C, respectively. Two efficient strains such as Bacillus sp. ACT 1 and Bacillus sp. ACT 2 were isolated from the various mutants obtained. Bacillus sp. ACT 2 showed improved enzymatic production and Bacillus sp. ACT 1 showed improved growth compared to wild strain. The enzyme responsible for the degradation was found to be azoreductase by SDS–PAGE and about 53% increased production of enzyme was achieved with mutant species. The experimental data were modeled using growth and substrate inhibition models.     

Degradation of a tannery and textile dye,Navitan Fast Blue S5R by Pseudomonas aeruginosa

The degradation of Navitan Fast Blue S5R, a very important commercial diazo dye in the tannery and textile industries was investigated. Pseudomonas aeruginosa decolourized this dye at concentrations upto 1200 mg l^–1 and the organism was also able to decolourize various other tannery dyes at different levels. The organism required ammonium salts and glucose to co-metabolize the dye. Organic nitrogen sources did not support appreciable decolourization whereas, combined with inorganic nitrogen (NH₄NO₃) there was an increased effect on both growth and decolourization. Decolourization of this dye started when the organism reached late exponential growth phase and after 24 h of incubation nearly 90% of 100 mg l^–1 of the dye was decolourized. An oxygen insensitive azoreductase was involved in the decolourization mechanism. HPLC analysis confirmed the formation of metanilic acid from the dye, which on further incubation was completely metabolized under shaken culture condition.     

Biodegradation of methyl violet by Pseudomonas mendocina MCM B-402

Pseudomonas mendocina MCM B-402 was found to utilize a triphenylmethane dye, methyl violet as the sole source of carbon when incorporated in synthetic medium. Almost complete decolorization of methyl violet by P. mendocina was observed within 48 h of incubation at ambient temperature (28 ± 2 °C) under aerated culture conditions, when the bacteria were inoculated into Davis Mingioli's synthetic medium at a concentration of 100 mg/l medium. Methyl violet was mineralized to CO₂ through three unknown intermediate metabolites and phenol. The decolorization of the dye involved demethylation. Received: 27 November 1998 / Received revision: 2 March 1999 / Accepted: 5 March 1999     

Coordinate action of exiguobacterial oxidoreductive enzymes in biodegradation of reactive yellow 84A dye

A novel bacterial species identified as Exiguobacterium sp. RD3 degraded the diazo dye reactive yellow 84A (50 mg l⁻¹) within 48 h at static condition, at 30°C and pH 7. Lower salinity conditions were found to be favorable for growth and decolorization. Enzymatic activities of an H₂O₂ independent oxidase along with laccase and an azoreductase suggest their prominent role during the decolorization of reactive yellow 84A. Presence of an H₂O₂ independent oxidase in Exiguobacterium sp. RD3 was confirmed and hydrogen peroxide produced was detected by a coupled iodometric assay. Azoreductase activity was prominent in presence of cofactors NADH and NADP in mineral salt medium. Considerable depletion of COD of the dye solution during degradation of dye was indicative of conversion of complex dye into simple oxidizable products. Products of degradation were analyzed by HPLC, FTIR and GCMS. A possible product of the degradation was identified by GCMS. Degradation of dye resulted with significant reduction of phytotoxicity, confirming the environmentally safe nature of the degradation metabolites.     

Decolorization of Fast red by metabolizing cells of <Emphasis Type="Italic">Oenococcus oeni</Emphasis> ML34

Three different azo dyes such as Fast red, metanil yellow and Fast orange were examined for their decolorization by O. oeni ML34. Fast red (FR) was decolorized by 68%, whereas the other dyes were removed by only about 30%. The effects of glucose addition, substrate (dye) concentration and environmental factors (temperature, pH) on decolorization were investigated by two-level factorial design. The statistical analyses revealed that glucose specifically increases the extent of FR decolorization. A glucose level of 5 g/l was the optimum concentration for removal of, FR reaching a decolorization percentage of up to 93%.     

红平菇产漆酶条件优化及对孔雀绿染料脱色的研究

采用LNAS(低氮天冬酰胺-琥珀酸)培养基添加方式,对红平菇Pleurotus djamor HP1进行培养,检测不同时间培养液对不同底物的氧化作用,进而得到光密度值的变化情况,作为漆酶的产生及活性测定的主要依据。结果表明:在含Cu2+的培养液中漆酶最大酶活为235.4 U/L。含Cu2+的培养液添加底物木屑后漆酶最大酶活为458.8 U/L。提取经优化筛选后的培养基培养出的漆酶粗酶液,对4种具有不同化学结构的染料进行了脱色试验。结果表明:三苯基甲烷类的孔雀绿在6 h时脱色率为87.5%,蒽醌类的SN4R在24 h时脱色率为49.4%,偶氮类的甲基橙在24 h时脱色率为45%,杂环类的中性红在24 h时脱色率为23.6%。因此,显示出红平菇漆酶对孔雀绿染料脱色具有较大的应用潜力,进而对废水处理具有更好的应用前景。     

Decolorization kinetics of a recombinant Escherichia coli strain harboring azo-dye-decolorizing determinants from Rhodococcus sp.

A 6.3 kb DNA fragment containing genes responsible for azo-dye decolorization was cloned and expressed in Escherichia coli. The resulting recombinant strain E. coli CY1 decolorized 200 mg azo dye (C.I. Reactive Red 22) l^–1 at 28 °C at 8.2 mg g cell^–1 h^–1, while the host (E. coli DH5) had no color-removal activity. Addition of 0.5 mM isopropyl--d-thiogalacto-pyranoside (IPTG) increased the decolorization rate 3.4-fold. The dependence of the decolorization rate on initial dye concentration essentially followed Monod-type kinetics and the maximal rate occurred with the dye at 600 mg l^–1. The decolorization rate of E. coli CY1 was optimal at 40 °C and pH 11. Aeration (increased dissolved O₂ level) strongly inhibited the decolorization, but decolorization occurred effectively under static incubation conditions (no agitation was employed). The CY1 strain also exhibited excellent stability during repeated-batch operations.     

Decolorization of azo dyes by Rhodobacter sphaeroides

Rhodobacter sphaeroides AS1.1737 decolorized more than 90% of several azo dyes (200 mg dyes l^–1) in 24 h. The optimal culture conditions were: anaerobic illumination (1990 lx), peptone as carbon source, temperature 35–40 °C and pH 7–8. Intracellular crude enzyme from this strain had azoreductase activity, optimized temperature as 45–50 °C, and decolorization kinetics which were consistent with a ping-pong mechanism.     

Bioaugmentation of the decolorization rate of acid red GR by genetically engineered microorganism <Emphasis Type="Italic">Escherichia coli</Emphasis> JM109 (pGEX-AZR)

The study showed that the genetically engineered microorganism (GEM) bioaugment successfully the dye wastewater biotreatment systems to enhance acid red GR (ARGR) removal. Escherichia coli JM109 (pGEX-AZR) was the GEM with higher azoreductase activity. The kinetics of the ARGR decolorization by the E. coli JM109 (pGEX-AZR) agreed with Andrews model. The kinetic parameters, r _dye,max, K _s and K _i , were found to be 42.45 mg g⁻¹ h⁻¹, 584.93 mg L⁻¹ and 556.89 mg L⁻¹, respectively. The E. coli JM109 (pGEX-AZR) was tested in anaerobic sequencing batch reactors (AnSBR) in order to enhance the ARGR decolorization. The decolorization rate of ARGR was affected by the amount of E. coli JM109 (pGEX-AZR) inoculation and the best amount of inoculation was 10%. The continuous operations of the four bioreactors with different E. coli JM109 (pGEX-AZR) immobilization supports showed that the E. coli JM109 (pGEX-AZR) could bioaugment decolorization in AnSBRs with suspended and immobilized on macroporous foam carriers. For 42 days continuous operation in the AnSBRs, both the tolerance to ARGR concentration shock and the decolorization rate in these two bioaugmented AnSBRs are higher than those of the other two systems, control system and bioaugmented AnSBRs system with the sodium-alginate immobilized cells, the decolorization rate reached 90%. Changes in microbial community were detected by ribosomal intergenic spacer analysis (RISA) and amplified ribosomal DNA restriction analysis (ARDRA), which revealed that the introduced E. coli JM109 (pGEX-AZR) was persistent in the augmented systems and maintained higher metabolic activity.     

Efficient decolorization and detoxification of sulphonated azo dye Ponceau 4R by using single and mixed bacterial consortia

Abstract

The decolorization of toxic azo dye Ponceau 4R by three strains of bacteria Bacillus sp. strain AK1, Lysinibacillus sp. strain AK2 and Kerstersia sp. strain VKY1 individually and in consortia was studied. At optimal conditions, up to 95%, 93% and 87% of the dye was decolorized by the strains AK1, AK2 and VKY1, respectively, in 24?h at 200?mg/L of the dye. Decolorization of the dye was optimized for different parameters such as the concentration of dye, pH, temperature and NaCl concentration. These strains were able to decolorize Ponceau 4R up to an initial concentration of 800?mg/L in the pH range of 5–10, temperature 25–55?°C and NaCl concentration up to 30?g/L. The dye decolorization efficiency of these strains was further enhanced by using different consortia of AK1, AK2 and VKY1 in various combinations. The complete decolorization of the dye by a consortium was achieved within 18?h at 200?mg/L. The cell-free extract of these strains grown on this dye exhibited a remarkable activity of azoreductase which is involved in the breakage of the azo bond. The steady-state kinetics of azoreductase, validated the ping pong Bi-Bi mechanism of enzyme action. UV–Vis spectra, HPLC, FTIR and LC-MS analysis of the dye decolorized samples showed the formation of 4-aminonaphthalene-1-sulphonic acid and 5-amino-6-hydroxynaphthalene-2, 4-disulphonic acid as the products of azo bond breakage. The phytotoxicity test of decolorized sample revealed a considerable reduction in the toxicity in comparison with the parent dye.     

Decolorization of Acid Black 210 by <Emphasis Type="Italic">Vibrio harveyi</Emphasis> TEMS1, a newly isolated bioluminescent bacterium from Izmir Bay,Turkey

The present study deals with the decolorization of Acid Black 210 by a bioluminescent bacterium, Vibrio harveyi TEMS1, isolated from coastal seawater of Izmir Bay, Turkey. Maximum rate of decolorization of Acid Black 210 was observed when Luria Bertani medium was used. Decolorization of Acid Black 210 was 38.9% and 93.9% at 24 h under shaking and static conditions, respectively. The optimum dye-decolorizing activity of the culture was obtained at 100 ppm initial dye concentration and incubation temperature of 20°C. Vibrio harveyi TEMS1 was also tested for its ability to decolorize four azo dyes (Acid Black 24, Acid Blue 7, Acid Green 20, Acid Yellow 36) in addition to Acid Black 210.     

Enhanced transformation of malachite green by laccase of <Emphasis Type="Italic">Ganoderma lucidum</Emphasis> in the presence of natural phenolic compounds

In this study, we investigated the efficacy of phenolic extract of wheat bran and lignin-related phenolic compounds as natural redox mediators on laccase-mediated transformation of malachite green (MG) using purified laccase from the white-rot fungus Ganoderma lucidum. G. lucidum laccase was able to decolorize 40.7% MG dye (at 25 mg l⁻¹) after 24 h of incubation. Whereas, the addition of phenolic extract of wheat bran enhanced the decolorization significantly (p < 0.001) by two- to threefold than that of purified laccase alone. Among various natural phenolic compounds, acetovanillone, p-coumaric acid, ferulic acid, syringaldehyde, and vanillin were the most efficient mediators, as effective as the synthetic mediator 1-hydroxybenzotriazole. Characterization of MG transformation products by HPLC, UV–Vis, and liquid chromatography-mass spectrometry-electrospray ionization analysis revealed that N-demethylation was the key mechanism of decolorization of MG by laccase. Growth inhibition test based on mycelial growth inhibition of white rot fungus Phanerochaete chrysosporium revealed that treatment with laccase plus natural mediators effectively reduced the growth inhibitory levels of MG than that of untreated one. Among all the tested compounds, syringaldehyde showed the highest enhanced decolorization, as a consequence reduced growth inhibition was observed in syringaldehyde-treated samples. The results of the present study revealed that the natural phenolic compounds could alternatively be used as potential redox mediators for effective laccase-mediated decolorization of MG.     

Decolorization of textile dyestuffs by a mixed bacterial consortium

A mixed anaerobic bacterial culture decolorized Drimaren Orange K-GL, Everzol Red RBN and Everdirect Supra Yellow PG dyestuffs at 200 mg dyestuff l^–1 over 24 h. Improved performance with complete decolorization within 24 h was achieved by incubation with 5 g yeast extract l^–1 compared to glucose, lactose and sucrose though 50 mg yeast extract l^–1 supplemented with 5 g lactose l^–1 or 5 g sucrose l^–1 also resulted in complete decolorization within 24 h.     

Biodegradation of Basic Violet 3 by <Emphasis Type="Italic">Candida krusei</Emphasis> isolated from textile wastewater

Basic Violet 3 (BV) belongs to the most important group of synthetic colorants and is used extensively in textile industries. It is considered as xenobiotic compound which is recalcitrant to biodegradation. As Candida krusei could not use BV as sole carbon source, experiments were conducted to study the effect of cosubstrates on decolorization of BV in semi synthetic medium using glucose, sucrose, lactose, maltose, yeast extract, peptone, urea and ammonium sulphate. Maximum decolorization (74%) was observed in media supplemented with sucrose. Use of sugarcane bagasse extract as sole nutrient source showed 100% decolorization of BV within 24 h under optimized condition. UV–visible, FTIR spectral analysis and HPLC analysis confirmed the biodegradation of BV. Six degradation products were isolated and identified. We propose the biodegradation pathway for BV which occurs via stepwise reduction and demethylation process to yield mono-, di-, tri-, tetra-, penta- and hexa-demethylated BV species which was degraded completely. The study of the enzymes responsible for decolorization showed the activities of lignin peroxidase, lacasse, tyrosinase, NADH-DCIP reductase, MG reductase and azoreductase in cells before and after decolorization. A significant increase in activities of NADH-DCIP reductase and laccase was observed in the cells after decolorization. The yeast C. krusei could show the ability to decolorize the textile dye BV using inexpensive source like sugarcane bagasse extract for decolorization.     

Biodegradation of diazo reactive dye Navy blue HE2R (Reactive blue 172) by an isolated <Emphasis Type="Italic">Exiguobacterium</Emphasis> sp. RD3

The diazo reactive dye Navy blue HE2R (50 mg/L) was decolorized up to 91.2% within 48 h at static condition by the Exiguobacterium sp. isolated from the dyestuff contaminated soil, collected from the textile industrial area Solapur, India. It showed ability to decolorize seven different reactive textile dyes. Maximum decolorization was observed at 30°C and pH 7. The presence and significant increase in the activity of enzymes lignin peroxidase, laccase, and azoreductase indicated prominent role of these enzymes in the decolorization of Navy blue HE2R. The degradation metabolites were analyzed by UV-Vis spectroscopy, TLC, HPLC, and FTIR spectroscopy. A possible pathway for biodegradation of this diazo reactive dye was proposed with the help of GC-MS analysis. The phytotoxicity studies confirmed the environmentally safe nature of degradation products.     

Clozapine Administration Modifies Neurotensin Effect on Synaptosomal Membrane Na<Superscript>+</Superscript>, K<Superscript>+</Superscript> -ATPase Activity

Na⁺, K⁺-ATPase is inhibited by neurotensin, an effect which involves the peptide high affinity receptor (NTS1). Neurotensin effect on cerebral cortex synaptosomal membrane Na⁺, K⁺-ATPase activity of rats injected i.p. with antipsychotic clozapine was studied. Whereas 3.5 × 10⁻⁶ M neurotensin decreased 44% Na⁺, K⁺-ATPase activity in the controls, the peptide failed to modify enzyme activity 30 min after a single 3.0, 10.0 and 30.0 mg/kg clozapine dose. Neurotensin decreased Na⁺, K⁺-ATPase activity 40 or 20% 18 h after 3.0 or 5.6 mg/kg clozapine administration, respectively, and lacked inhibitory effect 18 h after 17.8 and 30.0 mg/kg clozapine doses. Results indicated that the clozapine treatment differentially modifies the further effect of neurotensin on synaptosomal membrane Na⁺, K⁺-ATPase activity according to time and dose conditions employed. Taken into account that clozapine blocks the dopaminergic D2 receptor, findings obtained favor the view of an interplay among neurotensinergic receptor, dopaminergic D2 receptor and Na⁺, K⁺-ATPase at synaptic membranes.     

Evidence on manganese peroxidase and tyrosinase expression during decolorization of textile industry dyes by Trichosporon akiyoshidainum

Textile dyes are engineered to be resistant to environmental conditions. During recent years the treatment of textile dye effluents has been the focus of significant research because of the potentially low cost of the process. Mechanisms of biological textile dye decolorization depend greatly on the chemical structure of the dye and the microorganisms used. While basidiomycetous filamentous fungi are well recognized for dye decolorization through ligninolytic enzymes, reports on textile dye decolorization mechanisms of basidiomycetous yeasts have been scarce. Decolorization of several textile dyes by Trichosporon akiyoshidainum occurs during the first 12 h of cultivation. This fast decolorization process could not be solely related to siderophore production or dye sorption to biomass; it was shown to be a co-metabolic process. T. akiyoshidainum could use glucose, sucrose, and maltose as alternative carbon sources, and urea as an alternative nitrogen source with similar decolorization rates. The activity of two enzymes, manganese peroxidase and tyrosinase, were induced by the presence of dyes in the culture media, pointing to their potential role during the decolorization process. Manganese peroxidase titers reached 666 U l⁻¹ to 10538 U l⁻¹, while tyrosinase titers ranged between 84 U l⁻¹ and 786 U l⁻¹, depending on the dye tested. The present work provides a useful background to propose new eco-friendly alternatives for wastewater treatment in textile dying industries.