Biodegradation of reactive dyes using soil fungal isolates and Ganoderma resinaceum

Abstract

The potential of Fusarium oxysporum, Penicillium lanosum, Penicillium chrysogenum, Aspergillus fumigatus and Ganoderma resinaceum to effect the biodegradation of five reactive textile dyes, namely Blue 21, Orange 16, Yellow 160, Blue 16 and Black 5, was evaluated. Preliminary experiments tested the decolorization ability of the tested fungi in three different media at a dye concentration of 0.02%. It was found that F. oxysporum, P. lanosum and G. resinaceum had high efficiency for decolorization of Blue 21. In the case of P. lanosum and F. oxysporum, decolorization was achieved in a basal Czapeks medium containing 0.01% reactive Blue 21 and supplemented with 3% glucose and 0.3% sodium nitrate after 5 days at pH 4.5 at 30°C. However, with G. resinaceum, optimum dye decolorization was achieved after 7 days incubation at 30°C and pH 5. Dye accumulation in the tested mycelia and changes in the outer surface of the cell wall of three studied fungi were observed. In addition, the structural integrity of Reactive Blue 21 dye untreated and treated with the tested fungi was studied by IR analysis.

Introduction

Synthetic dyes have complex aromatic molecular structures, making them very stable and difficult to biodegrade. Such dyes are used extensively in textile dyeing, paper printing, color photography, and in the pharmaceutical, food, cosmetic and other industries (Rafii et al. 1990). Over 100,000 dyes are commercially available, with a total production of over 7 × 10⁵ tons per year (Fu and Viraraghavan 2001).

It has been estimated that 10–15% of dyes are lost in effluent during dyeing processes (Zollinger 1987). Major classes of synthetic dyes, including azo, anthraquinone and triarylmethane dyes, are toxic or even carcinogenic compounds with long turnover times (Hartman et al. 1978). Azo dyes are the most important group of synthetic colorants. They represent the largest class of dyes, and more than half of the dyes produced annually (estimated for 1994 worldwide as 1 million tons) are azo dyes (Stolz 2001). Dye waste water from the textile or dye stuff industries is one of the most difficult types of wastewater to treat because dyes have various synthetic origins and contain complex aromatic molecular structures that make them very stable and hard to biodegrade (Kim et al. 2004; Abou-Okeil 2005). Many physical and chemical methods, including adsorption, coagulation, precipitation, filtration and oxidation, have been used for the treatment of dye-containing effluents. Adsorption is the method most widely used at present due to its convenience and efficiency (Churchley 1994; Nigam et al. 1996). More recent studies have demonstrated that many bacteria are able to degrade azo dyes aerobically and anaerobically (Govindaswami et al. 1993; Basibuyuk and Forster 1997). Some microorganisms, including bacteria, fungi and algae, can degrade or absorb a wide range of dyes (Robinson et al. 2001). Current interest is now focused on the microbial biodegradation of dyes as a better alternative (An et al. 2002). With the exception of some white rot fungi that can decolorize dyes, reports on decolorization by yeast or other filamentous fungi through enzymatic processes are very limited (Yang et al. 2003). The reactive dyes pose the greatest problem in terms of color because, in both ordinary and hydrolyzed form, they are not easily degradable, and thus, even after extensive treatment, color may still remain in the effluent (Santhy and Selvapathy 2006). However, reactive dyes can be removed easily and conventionally by aerobic microorganisms (Panswad and Luangdilok 2000).

The present study focused on the isolation of fungal species that can degrade Blue 21, Orange 16, Yellow 160, Blue 16 and Black 5 with high efficiency, and compared their decolorization ability with that of the higher fungus Ganoderma resinaceum. Furthermore, conditions accelerating Blue 21 dye biodegradation were optimized. The fungal morphology of the most efficient decolorizing organism was studied in order to understand the mechanism of dye degradation. The IR spectrum of reactive Blue 21 after biological treatment with the tested organisms was also studied.

Materials and methods

Dyes and chemicals

Five reactive dyes were used in this study: Yellow 4GL (basic chromophore is Yellow 160), Orange 3R (basic chromophore is Orange 16), Turquoise Blue G (basic chromophore is Blue 21) and Brilliant Blue R especial (basic chromophore is Blue 16). All these dye stuffs have only one reactive group in alkali medium that changes to vinyl sulfone to make a covalent bond between the dye and cellulose. Black B dye (basic chromophore is Black 5) has two reactive groups from vinyl sulfone (Fig. 1).

Fig. 1

Formula of reactive dye

All dyes were kindly supplied by the Cairo Dyeing Center in industrial zone A2, 10th of Ramada City, Cairo, Egypt.

Fungal isolates

Soil samples as a source of fungi were obtained from the site around a dyeing factory in Tanta, Egypt. Fungi were isolated by using the dilution plate method (Van Walbeek et al. 1968). A sample of 1 g soil was added to a 100 ml flask containing 10 ml distilled sterilized water and shaken vigorously. Serial dilutions were then prepared and 0.5 ml of an appropriate dilution was cultured on Czapeks agar plates incubated at 28°C for 7 days.

Four fungal species were isolated (Fusarium oxysporum, Penicillium lanosum, Penicillium chrysogenum, and Aspergillus fumigatus) from the developed fungal colonies, identified according to Moubasher (1993) and tested for their ability to decolorize the five reactive dyes. Strains were maintained and subcultured on potato dextrose agar (PDA) slants or plates.

Isolation of Ganoderma resinaceum

Freshly collected G. resinaceum fruiting bodies were used to produce mycelia by cutting out the fresh fruiting body longitudinally using a sterilized knife (by flame and ethyl alcohol) then cutting transversely with sterilized forceps to pick up the fresh parts carrying the basidiospores of the hymenia . These were used to inoculate sterilized malt agar plates then incubated at 28°C for 9 days (Mustafa 2002). G. resinaceum was then cultured on three different media, as described below, to study its ability to decolorize the five tested reactive dyes.

Effect of medium and incubation time on decolorization efficiency of the tested fungi on the different reactive dyes

The effect of different liquid media and different incubation time (1–7 days) on the decolorization process was studied using the following media: potato dextrose broth (PDB), M1 medium [glucose 5 g, KH₂PO₄ 1 g, (NH₄)₂SO₄ 1 g, MgSO₄·7H₂O 500 mg and yeast extract 200 mg; Yang et al. 2003] at pH 5–6, and modified Czapeks broth (Ali and Muhammad 2008) containing per litre: sucrose 30 g, NaNO₃ 3 g, KCl 0.5 g, MgSO₄·7H₂O 0.5 g, FeSO₄·7H₂O 0.01 g and KH₂PO₄ 1 g. Using a cork borer (0.5 cm), two discs for each tested fungus were inoculated in 250 ml conical flasks containing 100 ml sterilized liquid medium with 200 mg/l each tested reactive dye. The flasks were incubated at 25°C on a shaker at 140 rpm for different incubation periods (1–7 days). The experiment was repeated three times for each tested reactive dye on three media. Standard deviation was recorded for triplicate percentage decolorization readings (Yang et al. 2003).

Decolorization assays

Decolorization of dyes was detected using the culture supernatant after centrifugation at 3,000 rpm for 30 min. Control was performed using the same medium but without the tested microorganisms. Absorbance of the dyes revealed different λ max after scanning on a spectrophotometer (0–800; Table 1). All measurements were made on a Schimadzo UV visible recording Graphicord Spectrophotometer. Color removal was reported as:

$$ {\hbox{Decolorization}}\left( \% \right) = \left( {{{\hbox{A}}_{\rm{o}}} - {{\hbox{A}}_{\rm{t}}}} \right)/{{\hbox{A}}_{\rm{o}}} \times 100 $$

were A _o is the absorbance of initial dye solution, which is constant for each dye and equals 0% decolorization, and A _t is the absorbance at cultivation time, t (Yang et al. 2003). Decolorization percentage reading refers to the percentage mean of decolorization percentage of three replicas.

Table 1 Wave lengths (λ) of different tested dyes

Optimization of the decolorization process for reactive Blue 21 by the different fungi tested

Reactive Blue 21 (RB21) was selected for further investigation due to its potential for decolorization by P. lanosum, F. oxysporum and G. resinaceum. For optimization of the decolorization process, the fungi were grown on Czapeks liquid medium with added reactive Blue 21 for 5 days (P. lanosum, F. oxysporum) or 7 days (G. resinaceum). The effects of dye concentration (0.01, 0.05, 0.1, 0.15, 0.2% w/v), initial pH value (pH within the range 3–6), temperature (25, 30, 35, 40 and 45°C), different carbon sources (glucose, maltose fructose, starch and galactose were used instead of sucrose at 3% concentration), and different nitrogen sources (sodium nitrate, peptone, yeast extract and urea at 0.3% concentration) were studied.

Light microscopy examination

Morphological changes in the mycelia and spores of P. lanosum, F. oxysporum and G. resinaceum after treatment with reactive Blue 21 dye were assessed by light microscopy (magnification 40×).

IR analysis

After decolorization of Blue 21, the residual filtrate was dried. Dried reactive Blue dye was ground with KBr and analyzed (before and after treatment with the tested fungi) using Perkin-Elmer 377 at the Central Laboratory, Faculty of Science, Tanta University, Tanta, Egypt.

Results and discussion

Different physiological parameters were studied to optimize the process of dye biodegradation.

Effect of different media and incubation time periods

Three different media were used (PDB, M1 and Czapeks). The results in Tables 2, 3, 4, 5 and 6 show that P. lanosum, P. chrysogenum, A. fumigatus and F. oxysporum decolorized the five reactive dyes more efficiently on Czapeks medium than on PDB and M1 media.

Table 2 Decolorization percentage of five reactive dyes (0.02% w/v) in three different media by Penicillium lanosum. Each value is the mean ± SD of triplicate decolorization percentage. CZ Czapeks medium, PDB potato dextrose broth

Table 3 Decolorization percentage of five reactive dyes (0.02% w/v) in three different media by Fusarium oxysporum. Each value is the mean ± SD of triplicate decolorization percentage

Table 4 Decolorization percentage of five reactive dyes (0.02% w/v) in three different media by Ganoderma resinaceum. Each value is the mean ± SD of triplicate decolorization percentage

Table 5 Decolorization percentage of five reactive dyes (0.02% w/v) in three different media by Aspergillus fumigatus. Each value is the mean ± SD of triplicate decolorization percentage

Table 6 Decolorization percentage of five reactive dyes (0.02% w/v) in three different media by Penicillium chrysogenum. Each value is the mean ± SD of triplicate decolorization percentage

The dye Blue 21 was decolorized more than the other four reactive dyes. The decolorization of Blue 21 dye increased with increasing incubation periods up to 5 days in case of P. lanosum (82%), F. oxysporum (80%), A. fumigatus (86%) and P. chrysogenum (70%) and declined thereafter, while in the case of G. resinaceum, the maximum decolorization was reached after 7 days (79%). It may be concluded generally that the composition of the medium affected growth and dye decolorization. Czapeks medium was chosen for subsequent experiments.

After decolorization, the peak wavelengths of reactive Blue 21 at 330 and 658 nm disappeared, and peaks for Orange 16, Yellow 160, Blue 16 and Black 5 at 388, 274, 285 and 595 nm, respectively, disappeared. These results indicated that decolorization percentages were high.

It was clear that decolorization of reactive Blue 21 dye by P. lanosum was more efficient than decolorization by F. oxysporum and G. resinaceum. Similar results were reported by Zheng et al. (1999) for Penicillium ATCC 74414 and A. ochraceus, which had the ability to remove color from poly R-478 and reactive Blue 25, respectively. The possible mechanism of decolorization involved initial mycelial biosorption and subsequent degradation.

Husseiny (2008) found that the color reduction percentages of reactive and direct dye in the case of Aspergillus niger and Penicillium spp. were 86 and 82.66%, respectively, after 2 days, and reached a maximum reduction percentage after a 4-day incubation period.

Ali et al. (2009) also reported that decolorization of 50 µM malachite green by A. flavus and Alternaria solani increased to 96% by the 6th day in Czapeks medium. In the case of Debaryomyces polymorphus and Candida tropicalis, the decolorization percentage reached 94% and 97% of 100 mg reactive Black dye after 6 h (Yang et al. 2003). Ali and Muhammad (2008) reported that the decolorization percentage of acid violet 19 was 88.6% in the case of Alternaria solani after 4 days in modified Czapeks medium. Accordingly, Czapek’s medium was chosen for subsequent experiments.

Effect of dye concentration on decolorization percentage of reactive Blue 21 with different test fungi

Figure 2a shows the effect of different dye concentrations on the decolorization percentage of reactive Blue 21 using different test fungi. The maximum percentages of decolorization using P. lanosum, F. oxysporum and G. resinaceum were 88%, 87% and 85%, respectively, at a concentration of 0.01%. It is clear that 0.01% of reactive Blue 21 is the best concentration for high levels of decolorization. Our results are similar to results obtained by Bhatti et al. (2008) for Shizophyllum commune IBL-6, and by Zheng et al. (1999) for Penicillium strain ATTCC. Both fungi decolorized cibarcon red 2BL and poly R-478 at a concentration of 0.01%. Kapdan et al. (2000) concluded that dyestuff concentration should be lower than 0.05% for complete decolorization of Everzol turquoise Blue by Coriolus versicolor. Also, Yang et al. (2003) found that Debaromyces polymorphus, Candida tropicals and two filamentous fungi, Umbelopsis isabellina and Penicillium geastrivorus could decolorize 100 mg/L reactive Black completely within 16–48 h (Yang et al. 2003).

Fig. 2a–e

Physiological factors affecting decolorization percentage with the test fungi. a Concentration of reactive Blue 21 dye, b pH value, c temperature, d carbon source, e nitrogen source. Values are mean ± SD of triplicate decolorization percentage

Effect of different pH

The results in Fig. 2b clearly show a gradual increase in dye decolorization by P. lanosum, F. oxysporum and G. resinaceum upon increasing the pH to 4.5 in case of G. resinaceum and pH 5 in the case of F. oxysporum and P. lanosum. However, a further increase in pH caused a slowly decolorization decreasing effect.

These results clearly support the concept that best fungal growth usually occurs at low pH values (Assadi and Jahangiri 2001). At higher pH values, reactive dye solutions are more negatively charged, and dye removal efficiency of white-rot fungi is decreased (Tak et al. 2004). It was clear from data reported by Husseiny (2008) that 93.73 and 96.69% decolorization of reactive and direct dye at pH 4.5 and 7, respectively, in the case of A. niger but 89.6% and 88.4 at pH 4 and 4.5, respectively, in the case of Penicillium spp.

Effect of temperature

We next studied the effect of different incubation temperatures on decolorization percentage of reactive Blue 21 dye. The results (Fig. 2c) showed that the tested fungi reached highest percentages of dye decolorization—89, 87 and 88% for P. lanosum, F. oxysporum and G. resinaceum, respectively—in shake flasks incubated at 30°C for 5 days, but G. resinaceum required 7 days. Indeed temperature has a considerable effect on many enzymes which control the biological reactions involved in growth, biosynthesis and decolorization of dye.

A decreasing trend of dye decolorization was observed at higher temperatures (40–45°C). White rot fungi show better growth under medium temperature conditions as compared to higher temperatures (Toh et al. 2003).

Husseiny (2008) reported decolorization of a direct dye bath by A. niger of 86 and 82.66% at 28 and 35°C, respectively, with the maximum decolorization percentage recorded after 4 days at temperatures of 28 and 35°C for reactive and direct bath effluent, respectively.

Effect of different carbon sources

As shown in Fig. 2d, glucose stimulated fungal growth and enhanced decolorization percentage to 92, 88 and 99% with P. lanosum, F. oxysporum and G. resinaceum, respectively, while fructose, sucrose, maltose and starch decreased decolorization compared with that of the control.

Glucose was used by the fungus as a readily consumable carbon source and it caused a significant shortening of lag phase. Glucose provides an easily metabolizable energy source to the fungus and creates the best environment to enhance the decolorization rate of dyes. Utilization of carbon sources and decolorization of dye are dependent on the enzymatic activity of the fungal species under study.

Decolorization percentage (94%) of cibacron red 2BL increased in medium supplemented with 1% glucose in the case of Shizophyllum commune IBL-6 (Bhatti et al. 2008). Shoda et al. (2000) found that using 10 g/L molasses increased reactive Blue 5 decolorization by Geotrichum candidum Dec1, but decolorization decreased at 20 g/L. Similar results were found by Kapdan et al. (2000) with white rot fungi and Kapdan and Kargi (2002) with Coriolus versicolor—this fungus had ability to decolorize Everzol turquoise Blue G in the presence of glucose.

Effect of different nitrogen sources

Figure 2e clearly shows that the highest decolorization percentage (97%) was observed with 0.3% sodium nitrate in the case of P. lanosum and 93 and 89% with G. resinaceum and F. oxysporum, respectively.

Similar results were obtained by Ali et al. (2009), who found that Aspergillus flavus and Alternaria solani were able to decolorize (96%) of malachite green (10–50 µM) in Czapeks medium supplemented with 0.3% sodium nitrate. Lele et al. (2007) also reported that maximum decolorization percentage (96%) was recorded with 100 ppm amaranth dyes using Ganoderma sp.

Morphological changes in mycelia and spores after treatment with reactive Blue 21 dye

Penicillium lanosum absorbed the color of reactive Blue 21 after 3 days and showed irregularly shaped mycelium after 5 days (Fig. 3a). Macrospores of F. oxysporum accumulated the absorbed color of dye on septa, and the mycelium exhibited an irregular shape and wrinkled outer cell wall surface (Fig. 3b). Spores of G. resinaceum also absorbed the color of the dye and the mycelium showed an irregular shape (Fig. 3c).

Fig. 3

Morphological characteristics of mycelia and spores of Penicillium lanosum, Fusarium oxysporum and Ganoderma resinaceum after treatment with Reactive Blue 21 dye. a Mycelium with Reactive Blue 21 color, b irregularly shaped mycelium, c macrospore septa with absorbed dye, d irregularly shaped mycelium, e spore with absorbed dye, f irregularly shaped mycelium

It seemed that color removal and possible mechanism of decolorization involved initial mycelial biosorption and subsequent biodegradation.

Dye removal by a fungus occurs either through bioaccumulation or biodegradation. The color of the fungal biomass after dye removal is an indicator of the principal mechanism behind dye removal. If the color of the fungal biomass takes on the color of the dye then the process is biosorption or bioaccumulation (Chen et al. 2003). On the other hand, using fluorescence microscopy, Roncero and Duran (1985) found a change in cell wall morphology and formation of rudimentary wall material in the case of Geotrichum lactis that was promoted by calcofluor white (fluorochrome dye).

IR analysis

As shown in Fig. 4, treatment of reactive Blue 21 dye with P. lanosum gave a band at υ 3,203 cm⁻¹ that corresponds to the NH₂ group and a band at υ 3,382 cm⁻¹ that corresponds to the NH group that is not present in the spectrum of the original dye. In the case of treatment of reactive Blue 21 dye with F. oxysporum, a band appears at υ 3,262 cm⁻¹ corresponding to the NH₂ group that is not present in the spectrum of the original dye; however, a band at υ 1,111 cm⁻¹ found as υ 1,039 cm⁻¹ in the original dye corresponds to the C–O–C (ester) group, while a band at υ 1,595 cm⁻¹ that corresponds to the C = C group disappears (bands at υ 1,533 cm⁻¹ and υ 1,650 cm⁻¹ refer to C = C gp and C = O groups, respectively).

Fig. 4a–d

Infrared (IR) spectra of Reactive Blue 21. a Control, b Reactive Blue 21 treated with Penicillium lanosum, c Reactive Blue 21 treated with Fusarium oxysporum, d Reactive Blue 21 treated with Ganoderma resinaceum

Treatment of reactive Blue 21 dye with G. resinaceum gave a band at υ 3,423 cm⁻¹, that corresponds to the NH group and another at υ 1,595 cm⁻¹ that was shifted to υ 1,532 cm⁻¹ which corresponds to C = C groups. All changes in υ of the original dye of reactive Blue 21 tested with P. Lanosum, F. oxysporum and G. resinaceum indicated the ability of all three fungi to absorb the original dye.

Husseiny (2008) found the presence of an IR band at υ 3,364 cm⁻¹ and a band at υ 3,534.8 cm⁻¹ referring to a free NH₂ group that is not present in the spectrum of reactive Red 120 treated with Penicillium spp. Cao et al. (2001) and Stolz (2001) classified fungal decolorization of dye into two kinds according to their life state: living cells that biodegrade and biosorb dyes, and dead cells (fungal biomass) that adsorb dyes.

Conclusion

The current study indicates efficient dye biodegradation by the five tested fungal isolates (P. lanosum, F. oxysporum, G. resinaceum, A. fumigatus and P. chrysogenum). P. lanosum, G. resinaceum and F. oxysporum were the most efficient fungi in biodegrading the reactive Blue 21 dye under optimum conditions.