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.     

Biodegradation of reactive textile dye Red BLI by an isolated bacterium Pseudomonas sp. SUK1

A novel bacterial strain capable of decolorizing reactive textile dye Red BLI is isolated from the soil sample collected from contaminated sites of textile industry from Solapur, India. The bacterial isolate was identified as Pseudomonas sp. SUK1 on the basis of 16S rDNA analysis. The Pseudomonas sp. SUK1 decolorized Red BLI (50 mg l(-1)) 99.28% within 1h under static anoxic condition at pH range from 6.5 to 7.0 and 30 degrees C. This strain has ability to decolorize various reactive textile dyes. UV-Vis spectroscopy, FTIR and TLC analysis of samples before and after dye decolorization in culture medium confirmed decolorization of Red BLI. A significant increase in the activities of aminopyrine N-demethylase and NADH-DCIP reductase in cells obtained after decolorization indicates involvement of these enzymes in the decolorization process. Phytotoxicity testing with the seeds of Sorghum vulgare and Phaseolus mungo, showed more sensitivity towards the dye, while the products obtained after dye decolorization does not have any inhibitory effects.     

Purification, characterization and decolorization of bilirubin oxidase from Myrothecium verrucaria 3.2190

Myrothecium verrucaria 3.2190 is a nonligninolytic fungus that produces bilirubin oxidase. Both M. verrucaria and the extracellular bilirubin oxidase were tested for their ability to decolorize indigo carmine. The biosorption and biodegradation of the dye were detected during the process of decolorization; more than 98% decolorization efficiency was achieved after 7 days at 26°C. Additionally, the crude bilirubin oxidase can efficiently decolorize indigo carmine at 30°C~50°C, pH 5.5~9.5 with dye concentrations of 50 mg l(-1)~200 mg l(-1). Bilirubin oxidase was purified and visualized as a single band on native polyacrylamide gel electrophoresis (PAGE). Several enzymatic properties of the purified enzyme were investigated. Moreover, the identity of the purified bilirubin oxidase (BOD) was confirmed by matrix assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF-MS). These results demonstrate that the purified bilirubin oxidase in M. verrucaria strain has potential application in dye effluent decolorization.     

Fed-batch bioreactor strategies for microbial decolorization of azo dye using a Pseudomonas luteola strain

A Pseudomonas luteola strain possessing azoreductase activity was utilized to decolorize a reactive azo dye (C. I. Reactive Red 22) with fed-batch processes consisting of an aerobic cell growth stage and an anaerobic fed-batch decolorization stage. The fed-batch decolorization was conducted with different agitation and aeration rates, initial culture volumes, dye loading strategies, and yeast extract to dye (Y/D) ratios, and the effect of those operation parameters on azo dye decolorization was evaluated. Dissolved oxygen strongly inhibited the azo reduction activity; thus aeration should be avoided during decolorization but slight agitation (around 50 rpm) was needed. With the periodical feeding strategy, the specific decolorization rate (v(dye)) and overall decolorization efficiency (eta(dye)) tended to increase with increasing feeding concentrations of dye, whereas substrate inhibition seems to arise when the feeding concentration exceeded 600 mg dye/L. In the continuous feeding mode, higher initial culture volume resulted in better eta(dye) due to higher biomass loading, but lower v(dye) due to lower dye concentration in the bioreactor. With a volumetric flow rate (F) of 25 mL/h, both v(dye) and eta(dye) increased almost linearly with the increase in the loading rate of dye (F(dye)) over the range of 50-200 mg/h, while further increase in F(dye) (400 mg/h) gave rise to a decline in v(dye) and eta(dye). As the F was doubled (50 mL/h), the v(dye) and eta(dye) increased with F(dye) only for F(dye) < 80 mg/h. The best v(dye) (113.7 mg dye g cell(-)(1) h(-)(1)) and eta(dye) (86.3 mg dye L(-)(1) h(-)(1)) were achieved at F(dye) = 200 mg/h and F = 25 mL/h. The yield coefficient representing the relation between dye decolorized and yeast extract consumed was estimated as 0.8 g/g. With F(dye) = 75 mg/h, the Y/D ratio should be higher than 0.5 to ensure sufficient supply of yeast extract for stable fed-batch operations. However, performance of the fed-batch decolorization process was not appreciably improved by raising the Y/D ratio from 0.5 to 1.875 but was more sensitive to the changes in the dye loading rate.     

Decolorization of azo dyes and simulated dye bath wastewater using acclimatized microbial consortium--biostimulation and halo tolerance

Anaerobic acclimatization of activated sludge from a textile effluent treatment plant to high concentration of RB5 could effectively decolorize RB5 dye solution. The strains viz. Pseudomonas aeruginosa and Bacillus circulans and other unidentified laboratory isolates (NAD1 and NAD6) were predominantly present in the microbial consortium. The conditions for efficient decolorization, biostimulation to increase effectiveness of microbial consortium, its tolerance to high salt concentration and non-specific ability towards decolorization of eight azo dyes, are reported. The optimum inoculums concentration for maximum decolorization were found to be 1-5 ml of 1800+/-50 mg l(-1) MLSS and 37 degrees C, respectively. The decolorization efficiency was 70-90% during 48 h. The biomass showed efficient decolorization even in the presence of 10% NaCl, as tested with RB5. In the presence of flavin adenine dinucleotide (FAD) more than 99% decolorization occurred in 8h. The decolorization of RB5 was traced to extracellular enzymes. The effectiveness of acclimatized biomass under optimized conditions towards decolorization of two types of synthetic dye bath wastewaters that were prepared using chosen azo dyes is reported.     

Enhancing the electron transfer capacity and subsequent color removal in bioreactors by applying thermophilic anaerobic treatment and redox mediators

The effect of temperature, hydraulic retention time (HRT) and the redox mediator anthraquinone-2,6-disulfonate (AQDS), on electron transfer and subsequent color removal from textile wastewater was assessed in mesophilic and thermophilic anaerobic bioreactors. The results clearly show that compared with mesophilic anaerobic treatment, thermophilic treatment at 55 degrees C is an effective approach for increasing the electron transfer capacity in bioreactors, and thus improving the decolorization rates. Furthermore, similar color removals were found at 55 degrees C between the AQDS-free and AQDS-supplemented reactors, whereas a significant difference (up to 3.6-fold) on decolorization rates occurred at 30 degrees C. For instance, at an HRT of 2.5 h and in the absence of AQDS, the color removal was 5.3-fold higher at 55 degrees C compared with 30 degrees C. The impact of a mix of mediators with different redox potentials on the decolorization rate was investigated with both industrial textile wastewater and the azo dye Reactive Red 2 (RR2). Color removal of RR2 in the presence of anthraquinone-2-sulfonate (AQS) (standard redox potential E(0)' of -225 mV) was 3.8-fold and 2.3-fold higher at 30 degrees C and 55 degrees C, respectively, than the values found in the absence of AQS. Furthermore, when the mediators 1,4-benzoquinone (BQ) (E(0)' of +280 mV), and AQS were incubated together, there was no improvement on the decolorization rates compared with the bottles solely supplemented with AQS. Results imply that the use of mixed redox mediators with positive and negative E(0)' under anaerobic conditions is not an efficient approach to improve color removal in textile wastewaters.     

Azo dye decolorization with a mutant Escherichia coli strain

A recombinant Escherichia coli strain (E. coli NO3) containing genomic DNA fragments from azo-reducing wild-type Pseudomonas luteola strain decolorized a reactive azo dye (C.I. Reactive Red 22) at approx. 17 mg dye h^–1 g cell. The ability to decolorize the azo dye probably did not originate from the plasmid DNA. Acclimation in azo-dye-containing media gave a nearly 10% increase in the decolorization rate of E. coli NO3. Growth with 1.25 g glucose l^–1 completely stopped the decolorization activity. When the decolorization metabolites from E. coli NO3 were analyzed by HPLC and MS, the results suggested that decolorization of the azo dye may be due to cleavage of the azo bond.     

Decolorization of azo dyes by Shewanella sp. under saline conditions

Wastewaters from textile processing and dye-stuff manufacture industries contain substantial amounts of salts in addition to azo dye residues. To examine salinity effects on dye-degrading bacteria, a study was carried out with four azo dyes in the presence of varying concentrations of NaCl (0-100 g l(-1)) with a previously isolated bacterium, Shewanella putrefaciens strain AS96. Under static, low oxygen conditions, the bacterium decolorized 100 mg dye l(-1) at salt concentrations up to 60 g NaCl l(-1). There was an inverse relationship between the velocity of the decolorization reaction and salt concentration over the range between 5 and 60 g NaCl l(-1) and at dye concentrations between 100 and 500 mg l(-1). The addition of either glucose (C source) or NH(4)NO(3) (N source) to the medium strongly inhibited the decolorization process, while yeast extract (4 g l(-1)) and Ca(H(2)PO(4))(2).H(2)O (1 g l(-1)) both enhanced decolorization rates. High-performance liquid chromatography analysis demonstrated the presence of 1-amino-2-naphthol, sulfanilic acid and nitroaniline as the major metabolic products of the azo dyes, which could be further degraded by a shift to aerobic conditions. These findings show that Shewanella could be effective for the treatment of dye-containing industrial effluents containing high concentrations of salt.     

Effects of alkalinity and co-substrate on the performance of an upflow anaerobic sludge blanket (UASB) reactor through decolorization of Congo Red azo dye

The effect of substrate (glucose) concentrations and alkalinitiy (NaHCO3) on the decolorization of a synthetic wastewater containing Congo Red (CR) azo dye was performed in an upflow anaerobic sludge blanket (UASB). Color removal efficiencies approaching 100% were obtained at glucose-COD concentrations varying between 0 and 3000 mg/l. The methane production rate and total aromatic amine (TAA) removal efficiencies were found to be 120 ml per day and 43%, respectively, while the color was completely removed during glucose-COD free operation of the UASB reactor. The complete decolorization of CR dye under co-substrate free operation could be attributed to TAA metabolism which may provide the electrons required for the cleavage of azo bond in CR dye exist in the UASB reactor. No significant differences in pH levels (6.6-7.4), methane production rates (2000-2700 ml/day) and COD removal efficiencies (82-90%) were obtained for NAHCO3 concentrations ranging between 550 and 3000 mg/l. However, decolorization efficiency remained at 100% with decreasing NaHCO3 concentrations as low as 250 mg/l in the feed. An alkalinity/COD ratio of 0.163 in the feed was suggested for simultaneous optimum COD and color removal.     

Expression of a temperature-sensitive esterase in a novel chaperone-based Escherichia coli strain

A new principle for expression of heat-sensitive recombinant proteins in Escherichia coli at temperatures close to 4 degrees C was experimentally evaluated. This principle was based on simultaneous expression of the target protein with chaperones (Cpn60 and Cpn10) from a psychrophilic bacterium, Oleispira antarctica RB8(T), that allow E. coli to grow at high rates at 4 degrees C (maximum growth rate, 0.28 h(-1)). The expression of a temperature-sensitive esterase in this host at 4 to 10 degrees C yielded enzyme specific activity that was 180-fold higher than the activity purified from the non-chaperonin-producing E. coli strain grown at 37 degrees C (32,380 versus 190 micromol min(-1) g(-1)). We present evidence that the increased specific activity was not due to the low growth temperature per se but was due to the fact that low temperature was beneficial to folding, with or without chaperones. This is the first report of successful use of a chaperone-based E. coli strain to express heat-labile recombinant proteins at temperatures below the theoretical minimum growth temperature of a common E. coli strain (7.5 degrees C).     

Improved biodegradation of Congored by using Bacillus sp

The biodegradation of Congored, a toxic azo dye, was studied by using a hybrid technique involving sonolysis as pretreatment followed by biological treatment. The experiments were carried out with and without pretreatment using dye solution as a sole source of nutrition with an isolated and acclimatized strain of Bacillus sp. obtained from tannery industry effluent. The pretreatment time was varied as 30, 60, 90, 120, 150 and 180 min and then the pretreated dye solution was subjected to biological treatment. The effectiveness of pretreatment was compared with the results of biological degradation of non pretreated Congored and the results showed that the pretreatment improved the efficiency of the biodegradation of Congored. During the biological degradation, the increase in initial dye concentration decreased the decolorization rate and at high concentrations (1500 and 2000 mg/l), the inhibition was observed. The optimum pH and temperature were determined to be 7.0 and 37 degrees C, respectively. The data obtained through biodegradation experiments were fitted with five different kinetic models and the results were analyzed.     

Comparative study on methyl orange removal by growing cells and washed cell suspensions of <Emphasis Type="Italic">Lactobacillus casei</Emphasis> TISTR 1500

Lactobacillus casei TISTR 1500 possesses cytoplasmic azoreductase, and converts methyl orange to N,N-dimethyl-p-phenylenediamine and 4-aminobenzenesulfonic acid. In culture growth, the strain completely degraded methyl orange at 200 mg/l, even though the pH value was lower than 4. The decolorization was inhibited in the growing culture with 800 mg of the dye/l after incubation for 12 h. The percentage of decolorization and specific decolorization rate with 400 and 800 mg/l were 66 and 15%, and 14.2, and 8.7 mg/gCell/h, respectively. Additionally, a growing culture is more tolerant to a high initial dye concentration than when using washed cell suspensions supplied with only sucrose. Moreover, incubation of a low cell density in 600 μM of Na⁺ and 20 mM of sucrose increased the specific decolorization rate from 2.34 mg/gCell/h (without Na⁺) to 4.32 mg/gCell/h. However, Na⁺ had no effect on the enhancement of azoreductase activity in the reaction mixture.     

Decolorization of azo dye using PVA-immobilized microorganisms

A microbial consortium having a high capacity for rapid decolorization of azo dye (RED RBN) was immobilized by a phosphorylated polyvinyl alcohol (PVA) gel. The immobilized-cell beads exhibited a color removal capability of 75%, even at a high concentration of RED RBN (500 mg l(-1)) within 12 h using flask culture. The continuous operation was conducted at a hydraulic retention time (HRT) of 5-20 h in which the dye loading rate ranged from 240 to 60 mg dye h(-1). A removal efficiency exceeding 90% was obtained at the HRT higher than 10 h. No recognizable destruction of bead appearance was observed in the 6-month operation. Examination of the mechanism of the decolorization process by cell beads indicated that it proceeded primarily by biological decolorization associated with partial adsorption of the dye onto the entrapped cells and gel matrix. Microscopic observation revealed that the microbial consortium contained in the gel beads was at least made up of three kinds of bacterial species. From the economical viewpoint, alternative cheaper nitrogen sources such as fish meal, soybean meal, pharmamedia and vita yeast powder were examined.     

短双歧杆菌α-D-半乳糖苷酶基因aga1在大肠杆菌中的高效表达

短双歧杆菌（Bifidobacterium breve 203）α_D_半乳糖苷酶基因(aga1)被克隆到大肠杆菌温度诱导表达质粒pBV220中,构建重组质粒pBVaga1,转入大肠杆菌进行温度诱导表达,得到的重组酶Aga1在大肠杆菌DH5α、DH10B和BL21中的比活分别为28.08、19.44和13.85U/mg, 均高于短双歧杆菌α_D_半乳糖苷酶的比活1.76U/mg。重组质粒pBVaga1在E. coli BL21中稳定性较好。重组酶Aga1蛋白亚基分子量约67kD,最适反应温度为45℃,酶在40℃以下稳定,60℃仅剩余约5%的酶活性,70℃时酶全部失活;最适反应pH为4.0～4.4,酶在pH 3.6～6.0范围内稳定;酶对p_硝基苯酚_α_半乳糖苷的Km＝1.43mmol/L,Vmax＝35.71μmol/(L·min),对蜜二糖的Km＝261mmol/L,Vmax＝63.69μmol/(L·min);酶在蜜二糖、棉子糖水解体系中不显示转糖基活性。结果说明Aga1与已经报道的一种短双歧杆菌的α_D_半乳糖苷酶不同,是新发现的一种短双歧杆菌的α_D_半乳糖苷酶。     

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.     

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。     

开放条件下烟管菌XX-2对孔雀石绿染料的高效降解

[目的]评价白腐真菌Bjerkandera adusta XX-2处理孔雀石绿染料废水的能力,为其在染料废水中的应用提供参考依据.[方法]采用批次实验在开放条件下研究通气、pH、温度、染料初始浓度、培养时间、碳源、氮源、金属离子、盐度等因子对该菌降解孔雀石绿的影响.同时利用植物萌发、微生物抑菌和水生动物致死实验对降解产物进行毒性测试.[结果]B.adusta XX-2菌株在开放的非灭菌条件下也能高效降解孔雀石绿.例如,在初始浓度为120 mg/L且以孔雀石绿为唯一营养源的条件下降解率也能达到60％.静置培养和摇动培养呈现出几乎相同的降解率,这可以为技术应用节约动力成本.最适降解pH与温度分别为7.0和25℃.在上述参数体系的优化基础上,分别进行了碳源、氮源与金属离子的添加优化实验,结果显示低浓度的碳源(如柠檬酸钠)、氮源(如氯化铵)和金属离子(如Zn2+)均可大大提高B.adusta XX-2对孔雀石绿的脱色效率.同时B.adusta XX-2的降解也能在很高的盐浓度下进行.毒性测试表明降解后的染料对植物、微生物、水生生物的毒性大大减少.[结论]B.adusta XX-2菌株在处理染料废水方面具有很大的应用潜力.     

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.     

RAPID detection and quantification of E. coli O157/O26/O111 in minced beef by real-time PCR

AIM: To develop a real-time PCR detection procedure for Escherichia coli O111, O26 and O157 from minced meat. METHODS AND RESULTS: Strains (n = 8) of each of E. coli O26, E. coli O111 and E. coli O157 were inoculated at ca 10-20 CFU g(-1) into minced retail meat and enriched for 6 h at 41.5 degrees C as follows: E. coli O26 in tryptone soya broth (TSB) supplemented with cefixime (50 microg l(-1)), vancomycin (40 mg l(-1)) and potassium tellurite (2.5 mg l(-1)); E. coli O111 in TSB supplemented with cefixime (50 microg l(-1)) and vancomycin (40 mg l(-1)); E. coli O157 in E. coli broth supplemented with novobiocin (20 mg l(-1)). DNA was extracted from the enriched cultures, and detected and quantified by real-time PCR using verotoxin (vt1 and vt2) and serogroup (O157 per gene; O26 fliC-fliA genes and O111 wzy gene) specific primers. CONCLUSIONS: The methods outlined were found to be sensitive and specific for the routine detection of E. coli O111, O26 and O157 in minced beef. SIGNIFICANCE AND IMPACT OF THE STUDY: The enrichment, isolation and detection procedures used in this study provide a rapid routine-based molecular method for the detection and differentiation of E. coli O26, O111 and O157 from minced meat.     

Decolorization of the anthraquinone dye Cibacron Blue 3G-A with immobilized Coprinus cinereus in fluidized bed bioreactor

Coprinus cinereus, which was able to decolorize the anthraquinone dye Cibacron Blue 3G-A (CB) enzymatically, was used as a biocatalyst for the decolorization of synthetic solutions containing this reactive dye. Coprinus cinereus was immobilized in both calcium alginate and polyacrylamide gels, and was used for the decolorization of CB from synthetic water by using a fluidized bed bioreactor. The highest specific decolorization rate was obtained when Coprinus cinereus was entrapped in calcium alginate beads, and was of about 3.84 mg g(-1) h(-1) with a 50% conversion time (t1/2) of about 2.60 h. Moreover, immobilized fungal biomass in calcium alginate continuously decolorized CB even after 7 repeated experiments without significant loss of activity, while polyacrylamide-immobilized fungal biomass retained only 67% of its original activity. The effects of some physicochemical parameters such as temperature, pH and dye concentration on decolorization performance of isolated fungal strain were also investigated.