The dyes are complex chemical compounds that imparts colour to substances. On the urge of urbanisation, the use of synthetic dyes is increasing largely and its untreated effluent release causes serious environmental pollution affecting water bodies by disturbing the aquatic ecosystem, soil, plants, animals, humans. The recalcitrant nature of these dyes limits its treatment using conventional methods wherein biological methods using microorganisms are reported to completely mineralize the dyes lowering the release of degradation products.
The present work focuses on the biodegradation of synthetic dyes by isolating bacteria from a dyeing unit. Soil samples from the dye contaminated soils was collected, its degradation potentiality was observed using three major dye of studies congo red, brilliant green and methylene blue within 24 hr incubation. Maximum decoloarized dye (congo red and brilliant green) were chosen, serially diluted to 10-5 and plated to obtain two distinct colonies from decolorized congo red (CR1, CR2) and three distinct colonies from decolorized brilliant green (BG1, BG2, BG3). These isolates were biochemically characterized.
Molecular characterization was performed by isolating DNA from five isolates and amplified it using PCR, with the 16s rRNA gene primer. The PCR amplification product having approximately 1500bp were sequenced, edited and searched using BLAST against the known sequences within NCBI databases. The isolates were identified to be CR1 as Pseudomonas (88% identity), CR2 as Aeromonas (89% identical) and BG1/BG2 were confirmed to belong to same genus as Bacillus (99% identical). The phylogenetic tree showed a clear divergence between isolated species. Furthermore, the dye tolerance of isolates were observed by optimization analysis to be as CR1 and CR2 tolerated up to 4% of congo red dye and among BG1, BG2 and BG3; BG1 tolerated up to 0.50% of brilliant green dye. Thus, CR1 and CR2 were observed to be potent azo dye decolourizers. Recombinant DNA Technology can be applied in this field that can make the above future application more reliable.
Table of contents
Isolation and molecular characterization of dye tolerant bacteria from the dye contaminated soil in Kerala
Abstract
1. Introduction
1.1 Objectives
1.2 Scope of the study
2. Review of literature
3. Hypothesis
4. Materials and Methods
4.1 Study area
4.2 Soil sample collection
4.3 Quantitative analysis of microorganisms present in the collected soil
4.4 Preliminary screening of dye tolerant microorganisms from soil
4.5 Isolation of potential dye decolourizers
4.7 Morphological and biochemical tests
4.8 Optimization of the dye concentration
4.9 Molecular identification and isolation of DNA
4.10 Quantification of DNA
4.11 Agarose gel electrophoresis
4.12 PCR amplification using 16s rRNA
4.13 Data sequencing
4.14 Data analysis
4.15 Cross evaluation of bacterial isolates and MTCC
4.16 Statistical analysis
5. Results and discussion
5.1 Isolation of microorganism from soil
5.2 Preliminary screening of dye tolerant bacteria
5.3 Isolation of potential dye decolourizers
5.4 Pure culture preparation, morphological and biochemical identification
5.5 Optimization of dye concentration
5.5 Bacterial DNA isolation
5.6 PCR amplification using 16s rRNA and sequencing
5.7 Sequence analysis
5.8 Molecular phylogenetic analysis using maximum likelihood method
5.9 Cross evaluation of bacterial isolates and MTCC
6. Conclusions
Research Objectives and Topics
This study aims to isolate and identify dye-tolerant bacteria from dye-contaminated soil in Kerala and to evaluate their potential for the biodegradation of synthetic dyes. The research specifically focuses on the characterization of these isolates through biochemical and molecular methods, including 16S rRNA sequencing, to explore their utility in industrial bioremediation.
- Isolation of dye-tolerant bacteria from contaminated soil samples.
- Biochemical characterization of isolated bacterial strains.
- Molecular identification of isolates using PCR and 16S rRNA gene analysis.
- Evaluation of dye tolerance and biodegradation potential of the identified species.
Excerpt from the book
1. Introduction
Dyes are compounds used to colour substances. It is used widely in textile, leather, plastic, cosmetics and food industries. The use of dyes dates back to late Bronze Age, the first of which to be used was indigo. Indigo was a plant extract named Indigofera tinctoria by formation and possess a characteristic blue colour of itself. Natural dyes were obtained from extracts of fruits, leaves, roots etc from plants used for dyeing clothes long back. Since the dyeing procedure took more time using natural dye, an alternative dyeing method was implemented, the use of synthetic dyes began in 1856 synthesised with the first dye being mauve dye (aniline), a brilliant fuchsia colour. From then to the present date synthetic dyes finds its applications by many industries making itself an indispensible part of human life.
A chromophore is an unsaturated group of atoms attached to the coloured compounds. These compounds are the colour induced group present in a dye without which a substance could remain colourless (Figure 1). The intensity in the colour absorption is brought about by another group of compounds associated with chromophore in a dye structure called Auxochromes. The unshared pair of electrons in the auxochrome group interacts with the π electrons within the aromatic ring.
Summary of Chapters
1. Introduction: Outlines the historical context and industrial application of dyes, alongside the environmental challenges posed by synthetic dye effluents.
2. Review of literature: Provides an overview of existing research on dye pollution, toxicity, and various biological, physical, and chemical remediation methods.
3. Hypothesis: Presents the foundational assumptions regarding the presence and evolutionary adaptation of dye-degrading bacteria in contaminated soils.
4. Materials and Methods: Details the site location, sample collection, laboratory isolation techniques, biochemical testing, and molecular sequencing procedures used.
5. Results and discussion: Describes the experimental findings, including bacterial identification, phylogenetic analysis, and the optimized dye tolerance levels of the isolated strains.
6. Conclusions: Summarizes the key findings and the potential for applying these identified bacterial species in future bioremediation and recombinant technology applications.
Keywords
Azo dyes, Aeromonas, Bacillus, BLAST, DNA isolation, PCR, Pseudomonas, 16s rRNA, Biodegradation, Dye tolerance, Bioremediation, Contaminated soil, Molecular characterization, Microbial degradation, Textile effluent
Frequently Asked Questions
What is the core focus of this research?
The research focuses on the isolation and molecular characterization of bacteria capable of tolerating and degrading synthetic dyes found in contaminated soil.
What are the central thematic fields?
The study centers on microbiology, environmental biotechnology, and molecular biology, specifically targeting the bioremediation of textile industry pollutants.
What is the primary research goal?
The primary goal is to identify potent dye-degrading bacterial strains from contaminated soil in Kerala that can be utilized for the efficient bioremediation of industrial effluents.
Which scientific methods were employed?
The study utilized standard microbiological isolation techniques, biochemical characterization, PCR amplification of the 16S rRNA gene, and phylogenetic analysis via software like MEGA7.
What is addressed in the main body?
The main body covers the detailed methodology for isolating these organisms, the systematic biochemical testing of their properties, and the molecular analysis used to determine their identity.
What key terms characterize the work?
Essential terms include azo dye biodegradation, 16S rRNA, bacterial identification, and dye tolerance, which describe the systematic approach to solving industrial pollution problems.
How were the bacterial species identified?
Bacterial species (CR1, CR2, BG1, and BG2) were identified by comparing their 16S rRNA gene sequences with known sequences in the NCBI database using the BLAST tool.
What was the result of the dye tolerance testing?
The study found that isolates CR1 and CR2 could tolerate congo red dye concentrations up to 4%, while isolate BG1 showed tolerance to brilliant green dye up to 0.50%.
What is the significance of the phylogenetic tree?
The phylogenetic tree illustrates the evolutionary relationship between the isolated strains, confirming their classification into distinct genera such as Pseudomonas, Aeromonas, and Bacillus.
- Quote paper
- Dr. Prem Jose Vazhacharickal (Author), Sajeshkumar N.K (Author), Jiby John Mathew (Author), Nimisha Vinod (Author), 2017, Isolation and molecular characterization of dye tolerant bacteria from dye contaminated soil in Kerala, Munich, GRIN Verlag, https://www.grin.com/document/359357
