A thesis submitted to the faculty of the Korea Maritime and Ocean University in partial fulfillment of the requirements for the Master's degree in the Department of Environmental Science and Engineering. Colonial and cellular morphologies of cultures isolated and identified in this study ···14 Figure 4.1 Time course for azo dye decolorization rates for different pure cultures and consortia of bacteria and yeast. a) bacteria, (b) yeast, and (c) consortia ···23 Figure 4.3 Comparative analysis of consortium culture growth and associated decolorization of the azo dye Acid Blue 113 (50 mg/L) over time. The aim of this study was to select the most appropriate microbial consortium for the efficient bioremediation of wastewater with azo dyes.
It was concluded that the data for decolorization, COD removal, cell growth, and FT-IR spectra collectively provide evidence for azo dye decolorization and potential mineralization of the dye by the bacterial and yeast consortium. Moreover, transcriptomic analysis using RNA sequencing further elucidated the potential mechanisms of azo dye biodegradation by Sphingomonas melonis. The selected microbial consortia could be used for the bioremediation of azo dye wastewater on the industrial scale of varying environmental conditions.
Introduction
In contrast, the use of microbial treatment technologies has advantages in the treatment of azo dye wastewater because they are environmentally friendly, cost competitive, and can produce less sludge and yield end products that are non-toxic or can mineralize target chemicals and require less. water consumption compared to physico-chemical methods [13]. The aim of this study was to evaluate the decolorization efficiency of candidate microbial cultures for the effective bioremediation of azo-colored wastewater. Here, two bacterial and two yeast cultures were isolated from a commercial microbial consortium cultured with the azo dye AB113.
The results showed that a consortium composed of bacterial and yeast cultures appeared to more efficiently and sustainably degrade AB113, indicating the possibility of application for the bioremediation of azo dye-polluted waters on an industrial scale.
Literature Review
Principles of color chemistry
Prominent types are acid dyes for polyamide and protein substrates such as nylon, wool and silk; disperse dyes for hydrophobic substrates such as polyester and acetate, and direct and reactive dyes for cellulosic substrates such as cotton, rayon, linen and paper [26].
Azo dye decolorization and degradation
- Azo dye treatment
- Biological treatment of azo dyes
- Enzymatic decolorization and degradation of azo dyes
Several taxonomic groups of microorganisms such as bacteria, fungi, yeasts and algae have been reported for their ability to degrade azo dyes under anaerobic and aerobic conditions [32]. In most cases, the presence of oxygen prevents the cleavage of azo dyes in conventional sewage treatment plants, while anaerobic conditions are favored by various microorganisms to reduce the azo dyes to their corresponding aromatic amines, which are further degraded in the presence of oxygen [33, 34]. Azo dyes easily accumulate in the environment to a very high extent due to their high resistance to azo bond cleavage (-N=N-).
Under aerobic conditions, bacterial treatment of azo dyes usually shows low efficiency since oxygen is a stronger electron acceptor than azo dyes. Anaerobic and static condition have been reported to increase the decolorization efficiency of azo dyes. The primary step in the bacterial decolorization of azo dyes, both under anaerobic and aerobic conditions, is the reduction of the azo bond (-N=N-).
According to previous studies, two enzyme families, azoreductase and Laccases, have shown remarkable potential in enzymatic decolorization and degradation of azo dye. Recently, promising enzymes such as manganese peroxidase (MnP), lignin peroxidase (LiP) and polyphenol oxidase (PPO) have also been reported to be involved in the decolorization and degradation of azo dyes. However, the actual involvement or efficiency of intracellular azoreductase in bacterial decolorization has been questioned in recent years, since most azo dyes have complex structures and high polarities, which make them difficult to diffuse through cell membranes to reduce the process.
The typical mechanism of bacterial cells for the redox mediator-dependent reduction of azo dyes under anaerobic conditions is shown in Fig. In the cell supernatant, however; the result of the reduction of the azo dyes is a mostly chemical redox reaction, and mediators depend on cytoplasmic enzymes to supply electrons [59].
RNA sequencing for transcriptomic analysis
Materials and methods
- Isolation of efficient microorganisms for azo dye biodegradation
- Experimental culture setup for biodegradation of the azo dye
- UV-vis spectrophotometric analysis for cell growth and dye
- Monitoring of azo dye biodegradation based on FT-IR analysis
- Monitoring of the fate of organic compounds (azo dye and
- RNA isolation and sequencing procedure for RNA-seq analysis
B-2 was identified as Sphingomonas melonis DAPP-PG 224T (KB900605) according to the EzbioCloud database (Chunlab, Seoul, South Korea). Two yeast cultures (Y-1 and Y-2) were identified by the 26S rRNA sequencing method. For PCR amplification, primers Y-F (5'-GCATATCAATAAGCGGAGGAAAAG-3') and Y-R (5'-GGTCCGTGTTTC AAGACG-3') were used. Prior to the experiment, frozen cultures were obtained on TSA, then inoculated into mineral salt medium (MSM) and incubated in a shaking incubator at 27℃ and 150 rpm for 24 hours. Biodegradability test cultures were prepared according to the plan shown in Table 3.1; each culture contained 100 ml of MSM containing 0.03% (w/v) glucose and 0.0006% (w/v) yeast extract.
After autoclaving the MSM, stock solution of glucose and yeast extract, which was filter sterilized using Acrodisc syringe filters (0.2 μm; . Pall Laboratory, Westborough, MA, USA), was added to the medium with AB113 dye at concentrations of different types of paint. (20, 50 and 100 mg/L). The medium of each culture was inoculated with a small amount of each of the 24 h-grown cultures (5%, v/v) and incubated at 27 °C while shaking at 150 rpm. For monitoring dye degradation, samples were withdrawn at regular intervals and centrifuged at 8000 rpm for 15 min.
The lyophilized samples were examined in a Fourier transform infrared (FT-IR) spectrophotometer (Spectrum GX, PerkinElmer LST, South Korea) over the wavelength (4000-650 cm-1) using the attenuated total reflection (ATR) method. It was necessary to monitor changes in concentrations of the only organic compounds (glucose as reducing agent and azo dye AB113). This was because an absolute disappearance could be associated with the mineralization of these substrates through CO2 generation. COD was measured using a commercial water analysis kit (HS-COD(Mn)-L, HUMAS,.
Total RNA from the control and treated samples was isolated and contaminating DNA was eliminated. The sequencing library was assembled using TruSeq Stranded Total RNA Sample Prep Kit (Illumina, San Diego, CA, United States) according to the manufacturer's protocol.
Results and discussion
- Dye decolorization by various cultures
- Monitoring of organics through COD analysis
- Comparison of cultural growth and concomitant decolorization
- FT-IR and GC-MS analyses of the biodegradation process by microbial
- Implications of the synergistic effects of inter genus co-culture
- Transcriptomic analysis of degradation of Acid Blue 113
Also, the removal rate of COD in T-C was slightly lower than in Y-C, probably due to bacterial interference in the metabolism of organic substrates by yeast cells. At the beginning (days 1 and 2), the growth of B-C was much slower than that of yeast. There was a good positive correlation between cell growth and the rate of azo dye degradation.
FT-IR profiles for the three different consortia during dye biodegradation are shown in Fig. The analysis of the FT-IR spectrum enables the determination and understanding of the different functional groups involved in the biotransformation process [69]. It was also observed that the number of OH or NH groups increased in all consortia as the intensity of the 3210 cm-1 region increased with dye degradation.
In addition, the magnitude of the peak at 1100 cm-1 representing SO2 decreased over time in B-C (Fig. 4.4a) due to bacterial biodegradation, as shown in a previous report [70]. There should be a further experiment to elucidate more metabolites in order to identify the degradation pathway of the dye. Bacteria in particular are considered to be one of the most powerful means of bioremediation in terms of dye degradation.
In this sense, the synergistic action of a yeast-bacterial consortium can lead to increased degradation and detoxification of azo dyes and thus provide a promising method for the effective removal of azo dye contaminants [19, 74]. Multistrains of microcosms are able to attack the recalcitrant dye molecule in different stereospecific positions or together can degrade the subsequent metabolites further and can lead to eventual mineralization of the dyes with its ecological relevance [6,75]. In the actual analysis for the study, the expressed values were normalized as shown in fig. 4.6.
The eggNOG (evolutionary genealogy of genes: uncontrolled orthologous groups) functional annotation of the genes produced the 16 different categories, including 6 up-regulated categories and 10 down-regulated categories.
Conclusion
In particular, some of the highly upregulated genes, such as NAD(P)-dependent oxidoreductase from the treated group exposed to Acid Blue 113, appeared to be related to azoreductase, a key enzyme responsible for the cleavage of the azo bond in the azo bond. pigment. Govindwar, Bacterial Decolorization and Degradation of Azo Dyes: A Review, Journal of the Taiwan Institute of Chemical Engineers. Bakhrouf, In vitro mutagenicity, NMR metabolite characterization of azo- and triphenylmethane dyes by adherent bacteria and the role of the “cna” adhesion gene in activated sludge, Microbial pathogenesis.
Shi, Aerobic decolorization and degradation of azo dyes by culturing cells of a newly isolated yeast Candida tropicalis TL-F1, Bioresource Technology. Mahadevan, Metabolic pathway and role of individual species in the bacterial consortium for azo dye biodegradation: A biocalorimetric study, Chemosphere. Stolz, Basic and applied aspects in the microbial degradation of azo dyes, Appl Microbiol Biotechnol.
Khan, Biodegradation of azo dye by moderately halotolerant Bacillus megaterium and study of enzyme azoreductase involved in degradation, 2011. Mattes, Reduction of azo dyes by redox mediators originating in the naphthalenesulfonic acid degradation pathway of Sphingomonas sp. Singh, Differential expression of peroxidase and ABC transporters as the key regulatory components for azo dye degradation by Penicillium oxalicum SAR-3, Functional.
Gonçalves, Serial tests for evaluation of decolorization of azo dyes by methanogenic and mixed cultures, Journal of Biotechnology. Lu, Enhanced biodecolorization of azo dyes by a co-immobilized quinone-anthraquinone reduction consortium, Bioresource Technology. Chaeyoung Rhee · Aalfin Emmanuel · Sung-Cheol Koh, Biodegradation of the azo dye Acid Blue 113 by Sphingomonas melonis and its transcriptomic analysis of the degradation pathway, 17th International Society for Microbiological Ecology, Leipzig, Germany, 12-17 August 2018.
Chaeyoung Rhee · Aalfin Emmanuel · Hong-gi Kim · Sung-Cheol Koh, Efficient Biodegradation of Azo Dye Acid Blue 113 by Bacterial and Yeast Consortium and Molecular Analysis of Bacterial Biodegradative Pathway, 9th International Conference on Environmental Science and Technology, Houston, Texas , United States, 25–29 June 2018.
![Table 2.1 Wavelength of absorption versus color in organic dyes [25]](https://thumb-ap.123doks.com/thumbv2/123dokinfo/10547530.0/18.892.123.760.812.1090/table-wavelength-absorption-versus-color-organic-dyes-25.webp)
![Figure 2.2 Proposed mechanism for degradation of azo dyes by azoreductase [61]](https://thumb-ap.123doks.com/thumbv2/123dokinfo/10547530.0/24.892.132.769.128.1000/figure-proposed-mechanism-degradation-azo-dyes-azoreductase-61.webp)
