Influence of redox mediators and media on methyl red decolorization and its biodegradation by Providencia rettgeri
Developing efficient, effective and low-cost procedure for color removal in wastewater is a serious environmental concern. In this study, we investigated the possibility of enhancing the activities of dyes decolorizing bacterium using selected redox mediators and media (nutrient broth, yeast, glucose, starch and peptone). The isolated organism identified using 16S rRNA gene as Providencia rettgeri showed the highest decolorization of a model dye, methyl red by 97% within 6 h using nutrient broth. Preferred redox mediator was found to be quinol (95% within 5 h) than other (nicotinamide adenine dinucleotide (NAD+), reduced NAD+, nicotinamide adenine dinucleotide phosphate (NADP+) and reduced NADP+). In addition, the biodegradation of methyl red was investigated by subjecting its metabolites to UV–visible, high-performance liquid chromatography and Fourier transform infrared analyses that suggested the biodegradation. This was confirmed by the removal of signature peaks of aromatic C–H bending (645, 759 and 831 cm−1) and the N=N peak at 1509 cm−1 of the FTIR spectrum of the metabolites. In conclusion, introduction of minute amount of hydroquinone (1 mM) into wastewaters could enhance their biotreatment by this strain of P. rettgeri.
KeywordsBiodegradation Decolorization Methyl red Providencia rettgeri Redox mediators
Industrialization is vital for a nation’s economic development, but it is equally associated with the production of life-threatening effluents. Such effluents contain enormous amounts of dyestuffs, which rendered them aesthetic displeasing and environmentally harmful . About 15% of dyestuffs used in industries entered into the effluents . Approximately 10,000 different dyes and pigments are used industrially, and over 0.7 million tons of synthetic dyes are produced annually worldwide. These colored wastewaters, which are often discharged into nearby streams and rivers, have adverse effects on the ecosystem. They alter the pH and increase the biochemical oxygen demand (BOD) and chemical oxygen demand (COD) of the receiving water bodies . This alteration in the physicochemical parameters of the aquatic bodies results in hardships of aquatic life and bioaccumulation of toxic compounds. Worse still, synthetic dyes in wastewaters are usually obstinate to most treatment processes. Prominent among these synthetic dyes are the azo dyes.
Azo dyes are the major textile dyestuffs produced, and they are the most commonly used synthetic dyes in the textile, food, printing, leather, cosmetics and paper-making industries . More than 800,000 tons of these dyes are produced annually worldwide . In addition to the coloration of water bodies, many synthetic azo dyes are known to be toxic, carcinogenic and genotoxic [6, 7]. Furthermore, they reduce the dissolved oxygen in waters, thus making aquatic life difficult . Azo dyes are a group of compounds characterized by the presence of one or more azo bonds (–N=N–) in association with one or more aromatic systems . This chemical nature makes them relatively resistant to most biological and chemical treatments.
Conventional wastewater treatment processes are not usually efficient to remove recalcitrant dyestuffs, like azo dyes, from effluents; and physicochemical methods such as adsorption, chemical precipitation, photolysis, chemical oxidation and reduction, electrochemical treatment have been found worrisome due to high cost, low efficiency, production of secondary pollutants and non-suitability for a variety of dyes . These disadvantages make biological treatments of the wastewater containing dyes, such as microbial or enzymatic decolorization and degradation, a viable alternative. Several microbial enzymes are being used for azo dye degradation which require redox mediators. Presently, nanoparticle-microbial enzyme conjugate-mediated degradation of textile azo dyes is on top in global scenario for specific substrates . Although biotreatment of wastewater has been adjudged to be more eco-friendly, cheaper and safer alternative to chemical decomposition process , there is a need to optimize the processes.
There are dwindling but promising results of accelerating dye decolorization by adding mediating compounds or changing process conditions . Dos-Santos et al.  have shown acceleration of azo dye reduction by the addition of a redox mediator—anthraquinone sulfonate. Studies have also demonstrated that ubiquitous sources of electrons, such as reduced forms of nicotinamide adenine dinucleotide (phosphate) (NAD(P)H), were able to reduce azo dyes, even in the absence of microbes or enzymes . Another extracellular reducing agent—sulfide—produced via respiration by sulfate-reducing bacteria has been shown to decolorize azo dyes chemically [16, 17]. Van der Zee and Villaverde  have also reported that the initial step in the biodegradation of azo dyes is the cleavage of the azo bond, a reaction catalyzed by the enzyme azoreductase. Azoreductases generally require NAD(P)H for their catalytic activities.
Another way of increasing the rate of decolorization is the choice of media. Studies with dyes as the sole source of carbon usually take prolonged time, some taken days or weeks. In previous decolorization studies, involving the use of minimal media, the percentage decolorization was low, the highest being 47% after 14 days of incubation . This study emphasizes the importance of media choice in bacterial decolorization of color effluents.
The objective of our study was thus to investigate the possibilities of selected chemical mediators and different media to increase the removal of azo dyes in wastewater. The biodegradation of the dye as against mere decolorization was also studied.
2 Materials and methods
2.1 Dyes and chemicals
Methyl red (MR) and hydroquinone were products of BDH chemicals (Poole, England), and nicotinamide adenine dinucleotide (NAD+), reduced NAD+, nicotinamide adenine dinucleotide phosphate (NADP+) and reduced NADP+ were from Sigma Chemical Co. (St. Louis, Mo, USA). Nutrient broth and agar were those of Lab M Ltd. (Lancashire, UK). Other reagents were of analytical grade.
2.2 Screening, isolation and identification of the bacterium
Isolation of bacteria from technical grade dyes was carried out by the enrichment technique using nutrient broth and a mixture of three dyes: Reactive Blue 13, Reactive Yellow 42 and Reactive Red 58 at 50 mg/L concentration. The decolorized broth was cultured on nutrient agar, and isolates from pure colonies were tested for decolorization ability. One of the prominent decolorizers was chosen and identified using the morphological, biochemical and genetic method. The genetic identification, using 16S rRNA gene sequence of the strain, was done at geneOmbio, Pune, India. The sequence was then analyzed at NCBI server (http://www.ncbi.nlm.nih.gov) and deposited at GenBank. The phylogenetic analysis of the sequence was done using MEGA 4 . The organism was maintained on a nutrient agar slant at 4 °C and proliferated in 250 mL Erlenmeyer flask containing 13 g/L of nutrient broth at room temperature until its turbidity (OD600) is about 1.0 prior every experiment. The nutrient agar contains (g/L): peptone, 5; beef extract, 3; NaCl and agar No.2, 12, while the composition of the nutrient broth (g/L) was beef extract, 1; yeast extract, 2; peptone, 5 and NaCl, 5.
2.3 Decolorization at various dye concentrations
2.4 Effect of various media on the decolorization of MR
The effect of media composition on decolorization was investigated by adding five nutrient sources into different MR solutions: nutrient broth, NB; with yeast extract, YS; with glucose, GC; with peptone, PT; with starch, SR (13 g/L each). The media were sterilized at 121 °C for 15 min. Percentage decolorization was determined as described above. Medium with the highest decolorization activities was used for the mediator experiments.
2.5 Effect of selected redox mediators on decolorization of MR
The ability of redox mediators: quinol, NAD, NADH, NADP and NADPH to influence the decolorization of the azo dye MR was investigated using 1 mM  of the mediators in different vessel containing 50 mL of the dye-broth solution. The choice of these mediators was born out of their importance in biochemical processes and as cofactors for enzymes. An equal amount of the organism (~ 0.05 g DCM) was added to each reaction setup.
2.6 Biodegradation studies
The biodegradation studies were monitored using the Fourier transform infrared (FTIR) and the high-performance liquid chromatography (HPLC) of the dye and its 24-h metabolites. The 24-h culture of the dye was centrifuged at 4000 rpm for 20 min, and the supernatant was extracted using ethyl acetate for the analyses. The HPLC analyses were done with a dual λ UV–visible detector HPLC (Waters, Austria) on a C18 column (symmetry, 4.6 × 250 mm). The extract was dissolved in HPLC grade methanol, and sample (10 μL of dye or its metabolite) was injected and allowed to separate for 10 min at the flow rate of 1 mL/min. A methanol isocratic mobile phase was used. Both MR and its ethyl acetate-extracted metabolites were used in this study. The FTIR spectra of the MR and its metabolites were done using the solid extract on FTIR model 800 (Shimadzu, Japan). The scans were done in the mid-IR region of 400-4000 cm−1 with 16 scan repeat. The samples, mixed with IR grade KBr in the ratio 10:90, and fixed in a sample holder, were used for the scans.
2.7 Statistical analysis
All values were expressed as mean ± standard deviation (SD) of triplicate observations. Decolorization data were analyzed using the paired samples T test. Statistical analyses were performed using IBM SPSS Statistics version 20. Values were considered statistically significant at P < 0.05.
3.1 Initial screening and identity of the bacterium
3.2 Decolorization of different concentrations of MR
3.3 Effect of different media on decolorization of MR
3.4 Effect of redox mediators on the decolorization of MR
3.5 Evidence of biodegradation as against mere decolorization
Generally, the use of chemical mediators in biotreatment serves a dual purpose: firstly to understand the metabolic pathway or mechanism of action of the organism; and secondly to check the possibility of improving reaction rate. In this research, the ability of the mediators to increase the rate of decolorization of MR was investigated.
The result of the 16S rRNA gene sequence of the organism showed that the bacterium is a strain of Providencia rettgeri (Fig. 1), and the sequence of the organism was deposited in the NCBI GenBank with accession number GU395555. The ability of this strain of P. rettgeri to completely decolorized textile dyes and MR (100 mg/L) under static anoxic condition suggested that it could be useful in wastewater decolorization. Mahalakshmi et al.  have observed a similar trend for Congo red, though with algae. Shen et al.  also estimated that textile effluents always have dye concentration of about 60 mg/L. The prolonged lag phase experienced as the concentrations of dye increased might be connected with the inhibition of metabolic enzymes’ production by the dye molecules, since metabolic enzymes are products of the lag phase . This inhibition of enzyme productions subsequently affected the growth of the organism, thereby preventing the production of the enzyme required for the catabolism of the pollutant, MR.
The fact that the organism preferred NB to other media was an indication that peptone, amino acid and vitamins (which are components of the NB) are required for the decolorization activities. Osuntoki et al.  have shown that nutrient sources could enhance biodegradation of pollutants. The preference of hydroquinone among other redox mediators might not be unconnected with the redox potential of the mediator. Forootanfar et al.  have reported the increase in decolorization of synthetic and textile dyes in the presence of hydroxybenzotriazole. The reduction in the decolorization time from 6 h to 5 h seems significant and economical since a small concentration of the hydroquinone (1 mM) was used. However, the hydroquinone concentration needs to be optimized. The decolorization pattern is similar to Michaelis–Menten kinetics, and such trend has been interpreted as probable involvement of enzymes .
The abrupt reduction in the absorbance value at the λmax after 6-h incubation is an indication of the decolorization and degradation of the dye by the organism [3, 23]. The disappearance of the peaks at 645.21–831.35 cm−1 and the distortion of that at 759.01 cm−1 (aromatic C–H bending) in the FTIR of MR metabolites suggested removal of the aromatic rings of the dyes. Similarly, Jadhav et al.  have reported changes in the peaks around 1500 cm−1 as evidence of azo bonds reduction. In this study, there was complete removal of the 1509.35 cm−1 peak in the FTIR of MR metabolites (Fig. 5).
The presence of several peaks in the HPLC profile of MR metabolites and the low AU values suggested biodegradation rather than mere decolorization. This result differs from studies by Wong and Yuen  and Moutaouakkil et al.  in which only two peaks corresponding to 2-aminobenzoic acid and N,N′-dimethyl-p-phenylenediamine were products of MR decolorization. The presence of several peaks in the HPLC of the metabolites showed that the dye was degraded to several smaller metabolites. The results of the FTIR analyses further justified the biodegradation, for instance, the disappearance of the C–H bending peaks for aromatic rings (600–800 cm−1). The removals of these peaks in the FTIR spectrum of metabolites have been reported as an indication of biodegradation . The findings suggested that this strain of P. rettgeri both decolorized and degraded the MR dye.
This strain of P. rettgeri decolorized 100 mg/L MR within 6 h and degraded the corresponding aromatic amines formed. Also, it achieved a similar fit with hydroquinone as a redox mediator in 5 h; a rate is considered significant and economically viable since only a small amount 1 mM of the mediator is required. This study, therefore, concluded that this bacterium can be used in the treatment of azo dyes in industrial effluents and that its activities can be increased by using low-cost redox mediator such as hydroquinone for the improvement of the rate of bioremediation.
Compliance with ethical standards
Conflict of interest
On behalf of all authors, the corresponding author states that there is no conflict of interest.
- 1.Somasiri W, Ruan W, Xiufen L, Jian C (2006) Decolorization of textile wastewater containing acid dyes in UASB reactor system undermixed anaerobic granular sludge. Electron J Environ Agric Food Chem 5:1224–1234Google Scholar
- 3.Olukanni OD, Osuntoki AA, Gbenle GO (2006) Textile effluent biodegradation potentials of textile effluent-adapted and non-adapted bacteria. Afr J Biotechnol 5(20):1980–1984Google Scholar
- 5.Zollinger H (1991) Syntheses, properties, and applications of organic dyes and pigments: in color chemistry. VCH Publishers Inc., New York, p 496Google Scholar
- 22.Shen J, Do T, Carwood G, Jenkins R (2002) Biotreatment of textile effluent using Pseudomonas spp. Immobilized on a polymer support. In: Hardin IR et al (eds) Advanced biotechnology for textile processes. The University of Georgia, pp 35–45. http://hdl.handle.net/2086/1777