Microbial Degradation, Spectral analysis and Toxicological Assessment of Malachite Green Dye by Streptomyces exfoliatus

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Abstract

Malachite green (MG) dye is a common environmental pollutant that threatens human health and the integrity of the Earth’s ecosystem. The aim of this study was to investigate the potential biodegradation of MG dye by actinomycetes species isolated from planted soil near an industrial water effluent in Cairo, Egypt. The Streptomyces isolate St 45 was selected according to its high efficiency for laccase production. It was identified as S. exfoliatus based on phenotype and 16S rRNA molecular analysis and was deposited in the NCBI GenBank with the gene accession number OL720220. Its growth kinetics were studied during an incubation time of 144 h, during which the growth rate was 0.4232 (µ/h), the duplication time (td) was 1.64 d, and multiplication rate (MR) was 0.61 h, with an MG decolorization value of 96% after 120 h of incubation at 25 °C. Eleven physical and nutritional factors (mannitol, frying oil waste, MgSO4, NH4NO3, NH4Cl, dye concentration, pH, agitation, temperature, inoculum size, and incubation time) were screened for significance in the biodegradation of MG by S. exfoliatus using PBD. Out of the eleven factors screened in PBD, five (dye concentration, frying oil waste, MgSO4, inoculum size, and pH) were shown to be significant in the decolorization process. Central composite design (CCD) was applied to optimize the biodegradation of MG. Maximum decolorization was attained using the following optimal conditions: food oil waste, 7.5 mL/L; MgSO4, 0.35 g/L; dye concentration, 0.04 g/L; pH, 4.0; and inoculum size, 12.5%. The products from the degradation of MG by S. exfoliatus were characterized using high-performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GC-MS). The results revealed the presence of several compounds, including leuco-malachite green, di(tert-butyl)(2-phenylethoxy) silane, 1,3-benzenedicarboxylic acid, bis(2-ethylhexyl) ester, 1,4-benzenedicarboxylic acid, bis(2-ethylhexyl) ester, 1,2-benzenedicarboxylic acid, di-n-octyl phthalate, and 1,2-benzenedicarboxylic acid, dioctyl ester. Moreover, the phytotoxicity, microbial toxicity, and cytotoxicity tests confirmed that the byproducts of MG degradation were not toxic to plants, microbes, or human cells. The results of this work implicate S. exfoliatus as a novel strain for MG biodegradation in different environments.

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MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods.

K Tamura, D. Peterson, N. Peterson … (2011)

Comparative analysis of molecular sequence data is essential for reconstructing the evolutionary histories of species and inferring the nature and extent of selective forces shaping the evolution of genes and species. Here, we announce the release of Molecular Evolutionary Genetics Analysis version 5 (MEGA5), which is a user-friendly software for mining online databases, building sequence alignments and phylogenetic trees, and using methods of evolutionary bioinformatics in basic biology, biomedicine, and evolution. The newest addition in MEGA5 is a collection of maximum likelihood (ML) analyses for inferring evolutionary trees, selecting best-fit substitution models (nucleotide or amino acid), inferring ancestral states and sequences (along with probabilities), and estimating evolutionary rates site-by-site. In computer simulation analyses, ML tree inference algorithms in MEGA5 compared favorably with other software packages in terms of computational efficiency and the accuracy of the estimates of phylogenetic trees, substitution parameters, and rate variation among sites. The MEGA user interface has now been enhanced to be activity driven to make it easier for the use of both beginners and experienced scientists. This version of MEGA is intended for the Windows platform, and it has been configured for effective use on Mac OS X and Linux desktops. It is available free of charge from http://www.megasoftware.net.

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CONFIDENCE LIMITS ON PHYLOGENIES: AN APPROACH USING THE BOOTSTRAP.

Joseph Felsenstein (1985)

The recently-developed statistical method known as the "bootstrap" can be used to place confidence intervals on phylogenies. It involves resampling points from one's own data, with replacement, to create a series of bootstrap samples of the same size as the original data. Each of these is analyzed, and the variation among the resulting estimates taken to indicate the size of the error involved in making estimates from the original data. In the case of phylogenies, it is argued that the proper method of resampling is to keep all of the original species while sampling characters with replacement, under the assumption that the characters have been independently drawn by the systematist and have evolved independently. Majority-rule consensus trees can be used to construct a phylogeny showing all of the inferred monophyletic groups that occurred in a majority of the bootstrap samples. If a group shows up 95% of the time or more, the evidence for it is taken to be statistically significant. Existing computer programs can be used to analyze different bootstrap samples by using weights on the characters, the weight of a character being how many times it was drawn in bootstrap sampling. When all characters are perfectly compatible, as envisioned by Hennig, bootstrap sampling becomes unnecessary; the bootstrap method would show significant evidence for a group if it is defined by three or more characters.

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New colorimetric cytotoxicity assay for anticancer-drug screening.

P Skehan, R. Storeng, D Scudiero … (1990)

We have developed a rapid, sensitive, and inexpensive method for measuring the cellular protein content of adherent and suspension cultures in 96-well microtiter plates. The method is suitable for ordinary laboratory purposes and for very large-scale applications, such as the National Cancer Institute's disease-oriented in vitro anticancer-drug discovery screen, which requires the use of several million culture wells per year. Cultures fixed with trichloroacetic acid were stained for 30 minutes with 0.4% (wt/vol) sulforhodamine B (SRB) dissolved in 1% acetic acid. Unbound dye was removed by four washes with 1% acetic acid, and protein-bound dye was extracted with 10 mM unbuffered Tris base [tris (hydroxymethyl)aminomethane] for determination of optical density in a computer-interfaced, 96-well microtiter plate reader. The SRB assay results were linear with the number of cells and with values for cellular protein measured by both the Lowry and Bradford assays at densities ranging from sparse subconfluence to multilayered supraconfluence. The signal-to-noise ratio at 564 nm was approximately 1.5 with 1,000 cells per well. The sensitivity of the SRB assay compared favorably with sensitivities of several fluorescence assays and was superior to those of both the Lowry and Bradford assays and to those of 20 other visible dyes. The SRB assay provides a colorimetric end point that is nondestructive, indefinitely stable, and visible to the naked eye. It provides a sensitive measure of drug-induced cytotoxicity, is useful in quantitating clonogenicity, and is well suited to high-volume, automated drug screening. SRB fluoresces strongly with laser excitation at 488 nm and can be measured quantitatively at the single-cell level by static fluorescence cytometry.