Methylene Blue Active Substances Assay Research Articles

Bioremediation of Hexanoic Acid and Phenanthrene in Oil Sands Tailings by the Microbial Consortium BioTiger™.

Polycyclic aromatic hydrocarbons (PAHs) and naphthenic acids (NAs) are toxic contaminants of environmental concern found in process water and mature fine tailings, or tailings, from the oil sands industry. BioTiger™, a patented microbial consortium of twelve natural environmental isolates, was found to cometabolically biodegrade the NA hexanoic acid and the PAH phenanthrene in the presence of tailings. Hexanoamide was found to be produced and consumed during cometabolism of hexanoic acid. Mechanistic analysis demonstrated three of the BioTiger™ strains generated biosurfactants with the bacterial adhesion to hydrocarbons assay, seven with the methylene blue active substances assay, and nine with a hemolysis assay. Serial transfers of the BioTiger™ consortium demonstrated the stability of hexanoic acid degradation over several generations. The results demonstrate that BioTiger™ cometabolically biodegrades combinations of phenanthrene and hexanoic acid in tailings. This work reveals the potential for in situ bioremediation of tailings with this natural microbial consortium.

Optimization of Ultrasonication Process for the Degradation of Linear Alkyl Benzene Sulfonic Acid by Response Surface Methodology

The optimization of ultrasonication process applied to linear alkyl benzene sulfonic acid (LABSA) degradation is investigated with response surface methodology in this study. Methylene blue active substance assay is used as a mean to monitor the degradation of LABSA. The effects of independent process variables (initial LABSA concentration, pH, and reaction time) on main material degradation are investigated. Among the independent variables, the greatest effect on LABSA degradation is observed for reaction time. Besides this, the effects of both reaction time and initial LABSA concentration are quite similar. The initial pH value of the solution has a lower effect on degradation performance when compared to other parameters. The predicted maximum detergent degradation was 75% at the optimal conditions of pH 7.8, initial LABSA concentration of 20 mg L−1, and reaction time of 70 min. Specific cost of LABSA degradation by ultrasonic treatment varies from 0.579 to 4.657 Euro g−1 removal LABSA. According to the results of sonication treatment applied to real wastewater, approximately 40% of the LABSA degradation is achieved at three pH values (5.27, 7.50, and 9.59). Because the original pH of the real wastewater is 9.59, it is more feasible to run experiments at this pH value.

Higher alkyl sulfatase activity required by microbial inhabitants to remove anionic surfactants in the contaminated surface waters.

Biodegradation of anionic surfactants, like sodium dodecyl sulfate (SDS) are challenged by some bacteria through the function of the enzyme alkyl sulfatases. Therefore, identifying and characterizing bacteria capable of degrading SDS with high alkyl sulfatase enzyme activity are pivotal. In this study, bacteria isolated from surfactant contaminated river water were screened for their potential to degrade SDS. Primary screening carried out by the conventional enrichment culture technique and assessment of SDS-degrading ability through methylene blue active substance assay revealed 12, out of 290, SDS-degrading surface water bacteria with maximum SDS degrading abilities of 46-94% in 24-54 h. The isolates exhibited optimum growth at SDS concentration of 1 g/L, but tolerated up to 15-75 g/L. Eleven isolates were identified as the species of Pseudomonas and one isolate was identified as Aeromonas through 16S rRNA sequencing. Proteolytic activity of alkyl sulfatases in the identified isolates was shown by using native-PAGE analysis. The determined enzyme activities changed in between 1.32 and 2.90 U/mg in the crude extracts. Preliminary experiments showed that the isolates with the alkyl sulfatase enzyme activities ≥2.50 U/mg were strong gratuitous degraders. However, their relative importance in soil, sewage, and wastewater treatment plants remains to be assessed.

Molybdate-reducing and SDS-degrading Enterobacter sp. Strain Neni-13

AbstractToxicants removal through microorganism’s action is intensely being sought due to economic reasons. The aim of this paper is to isolate a bacterium that is able to reduce molybdenum blue and at the same time can grow on the detergent Sodium Dodecyl Sulfate (SDS). Biochemical analysis resulted in a tentative identification of the bacterium asEnterobactersp. strain Neni-13. Growth on SDS showed a 100 % removal at 800 mg/L SDS within 12 days. The removal of SDS from media was confirmed through Methylene Blue Active Substances Assay. Molybdenum reduction using sodium molybdate as a substrate was characterized using a microplate assay. The optimum pH and temperature for molybdenum reduction was between 6.0 and 6.5, and at 37 °C, respectively. Glucose was the best electron donor for molybdate reduction. Phosphate and molybdate concentrations of between 2.5 and 5.0 mM and at 15 mM, were optimal for molybdate reduction, respectively. Molybdate reduction was inhibited by the heavy metals mercury, silver, copper and chromium at 2 ppm. The ability of this bacterium to detoxify molybdate and degrade the SDS makes this bacterium an important tool for bioremediation of toxicants in soil.

Bioattenuation of Detergent Plant Effluents Enhanced via Single Microbial Augmentations

AbstractDue to natural attenuation, anionic detergents in surface waters are not inferred as big environmental issues. However, the effluents from large industrial areas with high detergent concentrations can have significant local impacts. These circumstances can be diminished by using efficient detergent‐degrading bacterial isolates through bioaugmentation. In this study, detergent plant effluents were analysed by using a methylene blue active substance assay to determine detergent content during natural attenuation processes, and after single augmentations of 12 anionic detergent‐degrading bacterial isolates with high detergent tolerating abilities in batch microcosms. Maximum bioattenuation of detergents was determined as 56 % after 66 h incubation under the conditions that mimicked the natural environment. Bioattenuation was enhanced as much as 83 and 91 % in 78 h incubation time through single microbial augmentations of filter‐sterilized and non‐sterilized effluents, respectively. Eight Pseudomonas and one Aeromonas species were found to be highly competitive by showing high biodegradation abilities in pure culture experiments as well as enhancing degradation of detergents in both filter‐sterilized and non‐sterilized effluents through their single augmentations. Although remaining three isolates, namely Pseudomonas fluorescens SDS6, P. resinovorans SDS10‐2, and P. corrugata SDS10‐3 displayed lower degrading abilities in pure culture experiments than the natural attenuation, they later turned out to be actively enhancing the degradation of detergents during their single augmentations.

Investigating the removal of linear alkyl benzene sulfonate from aqueous solution by ultraviolet irradiation and hydrogen peroxide process

This study investigated the efficiency of ultraviolet radiation and hydrogen peroxide for the removal of linear alkylbenzene sulfonate (LAS) from aqueous solutions. Methylene blue active substances assay and spectrophotometry were used to determine the amount of anionic and residual surfactants. The effect of H2O2 concentration, initial concentration of surfactant, pH, and length of UV radiation were examined. The potential for use of UV/H2O2 to degrade LAS was analyzed statistically using multiple linear regression analysis. The results showed that after 20 min of contact time, ultraviolet radiation alone removed 38.44% of the LAS. The use of hydrogen peroxide alone for 10, 20, and 30 min showed no effect on the removal of LAS. The removal rates for the combination UV/H2O2 at 10, 20, and 30 min were 86.2, 90, and 96.5%, respectively. The results of this study showed that the use of ultraviolet radiation and hydrogen peroxide individually for degradation of anionic surfactant was not effective, but the combination of UV/H2O2 was effective for the removal of anionic detergents.

Pseudomonas plecoglossicida S5, a novel nonpathogenic isolate for sodium dodecyl sulfate degradation

Microbial remediation is commonly used to treat environmental samples polluted by anionic surfactants. However, this method is often limited due to the pathogenicity of the microbes used to clean environmental samples. Therefore, we aimed to isolate nonpathogenic bacterial strains capable of degrading sodium dodecyl sulfate. We used conventional enrichment culture. Strains were screened by methylene blue active substance assay and HPLC. The primary sodium dodecyl sulfate splitting enzyme alkylsulfatase was isolated by column chromatography. The enzymatic conversion of sodium dodecyl sulfate to dodecanol was followed by zymogram analysis. The biosurfactant producing ability of the isolate was measured using Siegmund Wagner blue agar, and the emulsification index was measured. The isolate was identified as Pseudomonas plecoglossicida S5 (JN700182), based on biochemical analysis and 16S rRNA sequencing. This strain S5 was able to degrade 89.28 ± 0.56 % of sodium dodecyl sulfate at 10 g/L. This strain produced a single type of alkylsulfatase enzyme and rhamnolipid biosurfactant in response to sodium dodecyl sulfate. Moreover, a 20 day fish study proved the nonpathogenicity of S5. This is the first report of sodium dodecyl sulfate degradation by P. plecoglossicida.

In-syringe magnetic stirring-assisted dispersive liquid–liquid microextraction for automation and downscaling of methylene blue active substances assay

A simple and rapid method for the determination of the methylene blue active substances assay based on in-syringe automation of magnetic stirring-assisted dispersive liquid–liquid microextraction was developed. The proposed method proved to be valid for the determination of anionic surfactant in waste, pond, well, tap, and drinking water samples.Sample mixing with reagents, extraction and phase separation were performed within the syringe of an automated syringe pump containing a magnetic stirring bar for homogenization and solvent dispersion. The syringe module was used upside-down to enable the use of chloroform as an extraction solvent of higher density than water.The calibration was found to be linear up to 0.3mg/L using only 200µL of solvent and 4mL of sample. The limits of detection (3σ) and quantification (10σ) were 7.0µg/L and 22µg/L, respectively. The relative standard deviation for 10 replicate determinations of 0.1mg/L SBDS was below 3%. Concentrations of anionic surfactants in natural water samples were in the range of 0.032–0.213mg/L and no significant differences towards the standard method were found. Standard additions gave analyte recoveries between 95% and 106% proving the general applicability and adequateness of the system to MBSA index determination. Compared to the tedious standard method requiring up to 50mL of chloroform, the entire procedure took only 345s using 250-times less solvent.

Production and characterization of rhamnolipid biosurfactant from waste frying coconut oil using a novel Pseudomonas aeruginosa D

To improve biosurfactant production economics by the utilization of potential low-cost materials. In an attempt to utilize cost-effective carbon sources in the fermentative production of biosurfactants, various pure and waste frying oils were screened by a standard biosurfactant producing strain. Considering the regional significance, easy availability and the economical advantages, waste frying coconut oil was selected as the substrate for further studies. On isolation of more competent strains that could use waste frying coconut oil efficiently as a carbon source, six bacterial strains were isolated on cetyltrimethyl ammonium bromide-methylene blue agar plate, from a soil sample collected from the premises of a coconut oil mill. Among these, Pseudomonas aeruginosa D was selected as the potential producer of rhamnolipid. Spectrophotometric method, TLC, methylene blue active substance assay, drop collapse technique, surface tension measurement by Du Nouy ring method and emulsifying test confirmed the rhamnolipid producing ability of the selected strain and various process parameters were optimized for the production of maximum amount of biosurfactant. Rhamnolipid components purified and separated by ethyl acetate extraction, preparative silica gel column chromatography, HPLC and TLC were characterized by fast atom bombardment mass spectrometry as a mixture of dirhamnolipids and monorhamnolipids. The rhamnolipid homologues detected were Rha-Rha-C(10) -C(10) , Rha-C(12) -C(10) and Rha-C(10) -C(8) /Rha-C(8) -C(10) . These results indicated the possibility of waste frying coconut oil to be used as a very effective alternate substrate for the economic production of rhamnolipid by a newly isolated Ps.aeruginosa D. Results of this study throws light on the alternate use of already used cooking oil as high-energy source for producing a high value product like rhamnolipid. This would provide options for the food industry other than the recycling and reuse of waste frying oils in cooking and also furthering the value of oil nuts.

Plasmid-Mediated Biodegradation of the Anionic Surfactant Sodium Dodecyl Sulphate, by Pseudomonas aeruginosa S7

Sodium dodecyl sulphate (SDS), an anionic surfactant, has been used extensively due to its low cost and excellent foaming properties. Fifteen different bacterial isolates capable of degrading SDS were isolated from detergent contaminated soil by enrichment culture technique and the degradation efficiency was assessed by Methylene Blue Active Substances (MBAS) assay. The most efficient SDS degrading isolate was selected and identified as Pseudomonas aeruginosa S7. The selected isolate was found to harbor a single 6-kb plasmid. Acridine orange, ethidium bromide, SDS and elevated temperatures of incubation failed to cure the plasmid. The cured derivatives of SDS degrading Pseudomonas aeruginosa were obtained only when ethidium bromide and elevated temperature (40 °C) were used together. Transformation of E. coli DH5α with plasmid isolated from S7 resulted in subsequent growth of the transformants on minimal salt media with SDS (0.1%) as the sole source of carbon. The SDS degradation ability of S7 and the transformant was found to be similar as assessed by Methylene Blue Active Substance Assay. The antibiotic resistance profiles of S7, competent DH5α and transformant were analyzed and it was noted that the transfer of antibiotic resistance correlated with the transfer of plasmid as well as SDS degrading property.