Decolorization of Acid Red 27 and Reactive Red 2 by Enterococcus faecalis under a batch system
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- Handayani, W., Meitiniarti, V.I. & Timotius, K.H. World J Microbiol Biotechnol (2007) 23: 1239. doi:10.1007/s11274-007-9355-1
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The objectives of this study were to investigate: (1) the capacity of Enterococcus faecalis on the decolorization of the azo dyes Acid Red 27 and Reactive Red 2; and (2) the growth characteristics of E. faecalis on those dyes. E. faecalis was able to decolorize Acid Red 27 and Reactive Red 2 effectively. High decolorization efficiency (95–100%) was achieved within 3 h of incubation for Acid Red 27, and 12 h for Reactive Red 2, at room temperature, neutral pH, static and non-aerated condition. Growth characteristics of E. faecalis on azo dyes, which were indicated by cell growth rate, biomass production, and growth yield, was worse than the control. E. faecalis grew better on Acid Red 27 rather than Reactive Red 2.
KeywordsAcid Red 27Azo dyesBatch growthDecolorizationEnterococcus faecalisReactive Red 2
Azo dyes, i.e. dyes containing the azo bond (-N = N-) in their molecular structures, are the major group of synthetic dyes used for industrial purposes, such as textiles, cosmetics, foods, etc. (Rafii et al. 1990; Ramalho et al. 2004). The discharge of highly colored effluent by these industries causes problems such as environmental damage, health hazards, and aesthetic aspects (Khehra et al. 2005). Color is the first contaminant in wastewater, which should be recognized and has to be removed before it can be discharged into the environment (Miao 2005). Physicochemical methods used for color removal of the effluents are effective, but they show disadvantages in terms of operational problems, high cost, and sludge production (Kapdan et al. 2000; López et al. 2004; Kodam et al. 2005). Moreover, those methods use more energy and chemicals than biological processes (Miao 2005). Because of these disadvantages, in recent years a number of studies have focused on microbial decolorization and degradation of azo dyes (Kim et al. 1995; Miao 2005).
The human intestinal microflora is reported to contain azo dye degraders (Rafii et al. 1990). They are exposed to azo dyes both from environmental contamination and from foods, drugs, and cosmetics (Chen et al. 2004). The dyes are degraded by the intestinal microflora, in which decolorization is the first step (Rafii et al. 1990; Sweeney et al. 1994). Decolorization of azo dyes is initiated by the reduction or cleavage of the azo bond catalysed by the azoreductase of the intestinal microflora, and resulting in colorless amines (Sweeney et al. 1994; Chen et al. 2004; Ramalho et al. 2004; Kodam et al. 2005). According to Dos Santos (2004), the conversion of azo dyes into aromatic amines is caused by both the presence of microflora and the anaerobic conditions found in the human intestine. Anaerobic conditions are more favorable for decolorization than aerobic conditions. This is probably related to the electron-withdrawing nature of azo bond and the resistance of azo dyes to oxygenase attack, or because oxygen is a more effective electron acceptor than the dyes (Dos Santos 2004).
The potential of Enterococcus faecalis, a predominant species of human gastrointestinal microflora, to decolorize azo dyes has been reported (Sweeney et al. 1994; Chen et al. 2004). As industrial wastewater generally contains various dyes, azo dye degraders should exhibit decolorizing ability for a wide range of dyes (Miao 2005). In relation to that condition, the performance of E. faecalis on decolorization of chemically different dyes is an important topic to study.
The effectiveness of microbial decolorization depends on the survival, adaptability, and activity of the microorganism (Kim et al. 1995). Survival, adaptability, and activity of the microorganism are expressed by its growth in an azo dye-containing environment. This means that effectiveness of microbial decolorization depends on the growth of microorganism (Miao 2005). Furthermore, it is possible that a species of microorganism may show different responses, in terms of growth, to different dyes. As a result, microbial growth characteristics are required to describe the response of the microorganism to azo dyes and its capability to decolorize azo dyes.
This study was done, therefore, to investigate: (1) the capacity of E. faecalis on decolorization of azo dyes Acid Red 27 and Reactive Red 2, and (2) growth characteristics, i.e. growth rate, cell mass concentration, and growth yield, of E. faecalis on those dyes. In this study, we also screened for the decolorization products of the respective dyes.
Materials and Methods
Microorganism and culture condition
Enterococcus faecalis ID 6017 was obtained from Laboratory of Microbiology, Faculty of Biology, Satya Wacana Christian University. Pure culture of E. faecalis was preserved on Nutrient Agar at 4°C.
The composition of growth medium (pH 6.8–7.2) for this study was (mg/l): Glucose 1,800; MgSO4.7H2O 250; KH2PO4 2,130; K2HPO4, 5,550; (NH4)2SO4 1,980; yeast extract, 500; azo dye 100.
Decolorization of azo dyes by growing cells
Enterococcus faecalis was injected into a flask containing 250 ml dye-free growth medium, as a preculture. The preculture was incubated at 35°C until the optical density at 500 nm reached a value of 0.3. Fifty ml of preculture was injected into each of two 500-ml flasks filled with 450 ml growth media containing Acid Red 27 or Reactive Red 2. As a control, 50 ml of preculture was injected into a 500-ml flask containing 450 ml dye-free growth medium. Cultures were incubated at 27–28°C under a static and non-aerated condition. Sampling was done by taking 5 ml of each culture per hour. It started at the injection of preculture into growth media, and stopped after the bacterial growth reached stationary phase. The experiment was repeated three times.
Samples were centrifuged at 6000 rotations/min (≈ 6955 g) for 30 min followed by the separation of supernatant and cell mass. The supernatant was used for determining the dye concentration and glucose concentration. Dye concentration was determined by spectrophotometric method at 521 nm for Acid Red 27, and 519 nm for Reactive Red 2. Determination of glucose concentration was conducted by an enzymatic-colorimetric method using glucose oxidase-phenylaminophenazone (Human Glucose liquicolor, Stanbio). Cell mass concentration was determined by turbidimetric method using a standard curve of optical density at 500 nm against cell mass. All measurement of absorbance was done in a Shimadzu UV–Vis 1201 Spectrophotometer.
Screening of decolorization product of azo dyes
The decolorization product of azo dyes was analysed by spectrophotometric scanning. Detection of the product using the scanning technique was done by measuring the supernatant absorbance from 300 to 600 nm in a double beam spectrophotometer (Shimadzu mini-UV 4501).
Decolorization of Acid Red 27 and Reactive Red 2 by Enterococcus faecalis strain ID6017
Growth of E. faecalis on Acid Red 27- and Reactive Red 2-containing media
Growth characteristics of E. faecalis in azo dye containing-media
Growth rate (/h)
Cell mass concentration (mg/l)
Glucose consumption (mg/l)
Growth yield (mg cell mass/mg glucose)
Acid Red 27
0.44 ± 0.03
176 ± 24
609 ± 59
0.28 ± 0.01
Reactive Red 2
0.36 ± 0.05
115 ± 30
800 ± 204
0.14 ± 0.10
0.42 ± 0.07
118 ± 9
336 ± 64
0.35 ± 0.07
Among the media, the highest cell mass production was found in Acid Red 27. In relation to Reactive Red 2, this might be caused by the difference in cell growth rate, where the cells grew faster on Acid Red 27 rather than on Reactive Red 2. In comparison to the control, the result was possibly due to the length of the exponential phase, in which the cell exponential phase on Acid Red 27 was longer than that of the control.
The highest cell growth yield was observed in the control treatment, followed by Acid Red 27 and Reactive Red 2 (Table 1.). This suggests a difference in glucose utilization for cell mass production among the media. In relation to azo dye containing-media, glucose was possibly consumed by cell for growth and decolorization, whereas in the control glucose was probably consumed only for growth.
The relation of decolorization to growth of E. faecalis
The decolorization of Acid Red 27 by E. faecalis lasted from the beginning of incubation and reached a maximum within 3 h (Fig. 2). During that time the cell growth rate was found to be in an exponential phase, which indicates that decolorization occurred together with cell growth (Fig. 3). Moreover, during the process glucose was consumed by cells (Fig. 4), which implies glucose utilization for growth and decolorization. This was rather different from Reactive Red 2 where decolorization of the dye took place after 3 h (Fig. 2), although cell growth (Fig. 3) and glucose consumption (Fig. 4) were started at the beginning of incubation. This was possibly due to the utilization of glucose for cell mass production before starting decolorization. However, Fig. 4 shows that cells consumed glucose during the decolorization of Reactive Red 2, and this could be an indication that glucose was used for decolorization.
Screening of decolorization product of azo dyes
Under static and non-aerated conditions, Acid Red 27 and Reactive Red 2 were decolorized by E. faecalis. This result was similar to that of Supaka et al. (2004), who reported the decolorization of reactive azo dyes by mixed bacterial culture under anaerobic condition. Generally, anaerobic conditions are more favorable for decolorization than aerobic conditions. However, Chen et al (2004) have successfully cloned and identified a gene encoding aerobic azoreductase from E. faecalis ATCC 19433.
Our findings show that the capacity of E. faecalis to decolorize of Acid Red 27 was higher than that of Reactive Red 2, and this could be influenced by the structure of the dyes. Dye molecules have many different and complicated structures, and this is one of the most important factors affecting microbial decolorization (Miao 2005). The effect of dye structure on the decolorization rate has also been reported by Kim et al. (1995) and Sani and Banerjee (1999). As shown in Fig. 1, Reactive Red 2 differs from Acid Red 27 due to the presence of a triazine reactive group in its molecule. The difference in decolorization rate between Acid Red 27 and Reactive Red 2 is possibly caused by the presence of the triazine group, as the report of Van der Zee (2002) showed that dyes containing triazine group were among the dyes that reduced at slowest rates.
It was found that within 12 h decolorization of 100 mg/l Acid Red 27 and Reactive Red 2 by E. faecalis could reach 100% and 95%, respectively. As a comparison, decolorization of 50 mg/l Direct Fast Scarlet 4BS by Pseudomonas 1–10 reached an efficiency of 90%, within 36 h (He et al. 2004). Furthermore, Novotny et al. (2001) reported decolorization of 150 μg/g Methyl Red and Congo Red by Irpex lacteus reached an efficiency of 56% and 58%, respectively, within two weeks. Another study conducted by Khehra et al. (2005) showed that Pseudomonas putida, P. fluorescence, Bacillus cereus, and Stenotrophomonas acidaminiphila were able to decolorize 20 mg/l of Acid Red 88, and after 12 h the efficiencies obtained were 35%, 31%, 40%, and 50%, respectively. Based on those results, it can be seen therefore, that E. faecalis has a higher capacity for decolorization than the other microorganisms.
Our finding, which showed that decolorization of dyes occurred in the exponential growth phase, was similar to the reports of Sumathi and Manju (2000), and Ramalho et al. (2004). It is possible that the azoreductase was produced during exponential phase and this possibility could explain that phenomenon. However, Chang et al. (2001) reported decolorization of Reactive Red 22 by P. luteola at the stationary phase. It is possible therefore, that the decolorization process could be a specific response for every microorganism.
As shown from the results, glucose was consumed for decolorization. This phenomenon might be due to the role of glucose as a cosubstrate, i.e. the source of electron donors, which are needed for azo bond cleavage (Sponza and Işık 2004; Mèndez-Paz et al.2005). Glucose is one of the compounds that have been reported to be effective electron donors for dye reduction (Tan 2001).
The growth quality of E. faecalis, which was better in the control than in azo dye containing-media, might indicate the adverse effect of azo dyes to bacterial growth. This result was similar to that of Chen et al. (2004), who reported the inhibitory effect of azo dyes on the growth of E. faecalis ATCC 19433. Furthermore, it was found that cell growth yield in the control was higher compared to that in Acid Red 27, although cell growth rate in those treatments were similar. This implies that the dye affected cell mass production and/or glucose consumption rather than cell growth rate. In this case, the cells might consume more glucose for decolorization and probably detoxification of dye and/or the products.
The fact that the growth quality of E. faecalis in Reactive Red 2 was worse compared to that in Acid Red 27 might indicate a different toxic effect of the dyes toward cells, as reported by Sani and Banerjee (1999), Sumathi and Manju (2000), and Chen et al. (2004). It was reported that dye toxicity to microorganisms inhibits metabolism activity; decreasing the growth rate and/or biomass needed for decolorization, which leads to the decrease of decolorization activity (Sumathi and Manju 2000).
As shown in Fig. 5, dye concentration in decolorized treatment was reduced compared to undecolorized ones. Our observation showed that there was no new peak found in the decolorized media. This indicates that decolorization product was not detected and probably related to the stability of the compounds. Several decolorization products could be oxidized easily formation or used as further C/energy sources or as redox mediator. 1-Amino-2-naphthol, a metabolite generated during reductive cleavage of the azo bond of Orange II, is a compound that undergoes rapid autooxidation (Coughlin et al.1999). Several azo dyes could degraded and used as a sole source of carbon and nitrogen by Sphingomonas sp strain 1CX (Coughlin et al.1999). During anaerobic reduction of amaranth with strain AKE1 1,2-dihydroxynapthalene (1,2-DHN) or its autooxidation products are released. These metabolites suggested acting as redox mediator under anaerobic conditions (Keck et al.1997).
The growth characteristics and capacity of E. faecalis to decolorize Acid Red 27 was better and higher than that on Reactive Red 2 because this dye has a triazine group in its chemical structure. The more complicated structure of Reactive Red 2 was a factor that could decrease growth quality and capacity of E. faecalis to decolorize azo dye.
The authors are grateful to acknowledge Yoga Aji Handoko and Suryasatrya Trihandaru for their technical helps, and Aldi Lasso and Chris Lundry for manuscript correction.