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Malachite green decolorization by the filamentous fungus Myrothecium roridum – Mechanistic study and process optimization
Bioresource Technology 194 (2015) 43–48
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Malachite green decolorization by the filamentous fungus Myrothecium roridum – Mechanistic study and process optimization Anna Jasin´ska a, Katarzyna Paraszkiewicz a, Anna Sip b, Jerzy Długon´ski a,⇑ a b
Department of Industrial Microbiology and Biotechnology, Faculty of Biology and Environmental Protection, University of Lodz, 12/16 Banacha Street, 90-237 Lodz, Poland ´ University of Life Sciences, Wojska Polskiego Street 48, 60-627 Poznan ´ , Poland Department of Biotechnology and Food Microbiology, Poznan
h i g h l i g h t s Myrothecium roridum decolorizes MG by LMWF and laccase action. Cytochrome P-450, LiP and MnP did not mediate MG decolorization. MG removal is effective in a simple medium, in both static and shaking cultures. MG is decolorized at a broad range of pH values (4–7). MG decolorization by M. roridum IM 6482 occurred in a non-toxic manner.
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Article history: Received 15 May 2015 Received in revised form 1 July 2015 Accepted 2 July 2015 Available online 8 July 2015 Keywords: Cytochrome P-450 Oxidoreductive enzymes Malachite green decolorization Myrothecium Toxicity
a b s t r a c t The filamentous fungus Myrothecium roridum isolated from a dye-contaminated area was investigated in terms of its use for the treatment of Malachite green (MG). The mechanisms involved in this process were established. Peroxidases and cytochrome P-450 do not mediate MG elimination. The laccase of M. roridum IM 6482 was found to be responsible for the decolorization of 8–11% of MG. Thermostable low-molecular-weight factors (LMWF) resistant to sodium azide were found to be largely involved in dye decomposition. In addition, MG decolorization by M. roridum IM 6482 occurred in a non-toxic manner. Data from antimicrobial tests showed that MG toxicity decreased after decolorization. To optimize the MG decolorization process, the effects of operational parameters (such as the medium pH and composition, process temperature and culture agitation) were examined. The results demonstrate that M. roridum IM 6482 may be used effectively as an alternative to traditional decolorization agents. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction Malachite green (MG), a triphenylmethane dye, is used for coloring a variety of materials, such as cotton, wood, silk, leather, jute and paper. This dye is also an antiparasitic, antifungal and antibacterial agent that is extensively applied in the aquaculture and fishery industries. However, MG is environmentally persistent and acutely toxic to a wide range of aquatic and terrestrial animals. MG and its derivatives have been reported to induce human carcinogenesis and mutagenesis. Several studies have also shown that exposure to this dye increases the risk of chromosomal fractures, reduces fertility and may act as an inhibitor of respiratory enzymes (Culp et al., 2006; Stammati et al., 2005). Due to the risk that MG imposes on fish consumers, its use in fishery applications is highly controversial. Thus, the use of MG in aquaculture has been banned ⇑ Corresponding author. Tel.: + 48 (42) 6354465. E-mail address: [email protected] (J. Długon´ski). http://dx.doi.org/10.1016/j.biortech.2015.07.008 0960-8524/Ó 2015 Elsevier Ltd. All rights reserved.
in many countries, including the United States of America, Canada and the European Union Member Countries. To date, a wide range of methods have been developed for the removal of synthetic dyes from wastewater (Verma et al., 2012). Although physical and chemical techniques have been applied to color removal, these techniques have serious disadvantages, such as high cost, low efficiency, limited versatility, interference with other wastewater constituents, formation of hazardous derivatives and/or intensive energy requirements (Shah, 2015). Due to the many drawbacks of conventional remediation methods, the development of efficient biological technologies to decrease the dye concentration in wastewater is of great importance (Sarayu and Sandhya, 2012; Singh and Singh, 2015). In recent years, many studies have focused on various microorganisms capable of MG removal, including bacteria, yeast, filamentous fungi and algae (Du et al., 2011; Karimi et al., 2012; Lv et al., 2013; Shedbalkar and Jadhav, 2011; Wang et al., 2012; Yan et al., 2014). The use of microbe-based methods in MG decomposition
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offers considerable advantages: the process is relatively inexpensive, the running costs are low, and the obtained by-products are often less toxic than the parent compounds (Forgacs et al., 2004). The most frequently mentioned factors involved in MG decolorization are the activities of cytochrome P-450, triphenylmethane reductase and extracellular lignin-modifying enzymes, particularly laccases (Fu et al., 2013; Gao et al., 2015; Jasin´ska et al., 2015). The literature data also indicate that dye decolorization may be mediated by nonenzymatic low-molecular-weight compounds that are resistant to high temperatures, which are also described as thermostable low-molecular-weight factors (LMWF) (Gomaa et al., 2008; Gomaa, 2012; Wang et al., 2012). The filamentous fungus Myrothecium roridum IM 6482 was isolated from a soil sample collected from the surroundings of a textile dyeing factory and has been proven to decolorize several industrial dyes (Jasin´ska et al., 2012, 2013). The aim of this study was to determine the usability of M. roridum IM 6482 for MG removal. The mechanisms involved in this process were also established in the present study. The participation of triphenylmethane reductase in MG elimination by M. roridum IM 6482 was previously proven (Jasin´ska et al., 2012); thus, this study focused on the involvement of cytochrome P-450, laccase, peroxidases and LMWF. The toxicities of MG and the derivatives formed during dye decolorization were also characterized. Furthermore, various process parameters, such as the carbon source, initial dye concentration, pH, culture agitation and temperature, were optimized. 2. Methods 2.1. MG, chemicals and fungal strains Technical-grade (80%) MG (4-[(4-dimethylaminophenyl)phenyl-methyl]-N,N-dimethylaniline) was kindly supplied by Boruta-Zachem Kolor Co. (Poland) and used without further purification. A stock solution of the dye (10 mg ml 1) was prepared in deionized water and autoclaved for 20 min at 121 °C before use. 2,2-Azinobis(3-ethylbenzothiazolin-6-sulfonic acid) (ABTS), veratryl alcohol, 2,6-dimethoxyphenol (DMP), sodium azide, proadifen, 1-aminobenzotriazole,metyrapone and other chemicals were purchased from Sigma–Aldrich Chemical Company (USA). The filamentous fungus tested in this study (M. roridum IM 6482) was isolated from a soil sample collected from the surroundings of a textile dyeing factory. It was deposited in the strain collection of the Department of Industrial Microbiology and Biotechnology, University of Lodz on ZT slants at 4 °C and _ transferred at 2-month intervals (Rózalska et al., 2015). 2.2. MG decolorization Decolorization studies were conducted in 100-ml Erlenmeyer flasks containing 18 ml of liquid Czapek-Dox (CzD) medium (containing glucose or sucrose at final concentrations 0.75%, 1.5% or 3%) inoculated with 2 ml of a homogenous second-step preculture (24 h-old) prepared in liquid WHI medium. Further experiments were performed using a modified CzD medium containing 0.75% glucose at various pH values (4; 5; 6 or 7), different incubation temperatures (28; 38 or 48 °C) and different agitation speeds (0; 75 or 150 rpm). At appropriate time intervals, the mycelia were separated from the culture medium by centrifugation at 15,000g for 15 min and then freeze-dried. The absorbance of the supernatant solution was analyzed using a Specord 200 spectrophotometer (Analytic Jena, Germany) and is expressed as a percentage of the removed dye. The decolorization percentage (DP) was calculated according to the following formula: DP[%] = [100 (A0 At/A0)], where A0 and At are the absorbance
values of the abiotic control and culture supernatant, respectively, measured at the maximum visible wavelength k = 615 nm. Biotic and abiotic controls without dye or mycelium supplementation, respectively, were also included. 2.3. Involvement of cytochrome P-450 in MG decolorization The homogenous preculture (2 ml) prepared as described above was transferred to 18 ml of standard Cz-D medium (in 100-ml Erlenmeyer flasks) supplemented with one of the following cytochrome P-450 inhibitors: metyrapone, 1-aminobeznotriazole or proadifen. The inhibitors were used at a concentration that had no significant effect on the growth of the fungus. The following optimal concentrations of these inhibitors (mycelial growth limitation up to 20%) were selected: 0.25 mM for metyrapone, 0.5 mM for 1-aminobenzotriazole and 0.01 mM for proadifen. Uninoculated growth media with appropriate amounts of the inhibitors were used as controls. Cultivation was performed for 30 min on a rotary shaker (150 rpm) at 28 °C. The cultures were then supplemented with 10 mg l 1 MG and incubated for 48 h under the conditions described previously. After incubation, the samples were centrifuged, and the degree of MG decolorization was determined according to the method described above. 2.4. Enzymes and LMWF involvement in MG decolorization Laccase activity in the supernatant obtained after culture centrifugation was assayed by monitoring ABTS oxidation at 420 nm. The reaction mixture contained enzyme extract, 20 mM citrate–phosphate buffer (pH 4.5) and 10 mM ABTS. Lignin peroxidise (LiP) activity was determined by monitoring the oxidation of veratryl alcohol at 310 nm. The reaction mixture contain 10 mM veratryl alcohol, 10 mM tartrate buffer (pH 3), 4 mM H2O2 and enzyme extract. The activity of manganese peroxidise (MnP) was measured at 470 nm in mixture containing 50 mM sodium malonate buffer (pH 4.5), 20 mM DMP, 20 mM MnSO4, 4 mM H2O2 and enzyme sample. The reference blanks included appropriate buffer and substrate. The enzyme activity is expressed in units, which are defined as the amount of enzyme oxidizing 1 lmol of appropriate substrate per minute. To establish the proportion of laccase involvement in MG decolorization, the liquid medium obtained after four days of culture in a modified Cz-D medium was recovered, and the following fractions were used in the decolorization experiments: extracellular liquid, supernatants in which laccase was inactivated by 0.5 mM sodium azide (chemically inactivated laccase) or by boiling for 30 min (thermally inactivated laccase), and filtrate from an ultrafiltration process using an Amicon Ultra tube (Millipore) with a cut-off of 3 kDa. To establish peroxides involvement in MG decolorization, catalase (600 U ml 1), thiourea (6 mM) and superoxide dismutase (200 U ml 1) were added to fluids before decolorization. The fluids were incubated with 10 mg l 1 MG at 28 °C for 3 h. The MG decolorization was determined as described above. 2.5. Toxicity studies These studies were performed using a modified method with serial dilutions in LB medium of the reference strains E. coli ATCC 25922, P. aeruginosa ATCC 15442 and S. aureus ATCC 61538. The optical densities of the cultures incubated with serial dilutions of the test compound were measured. LB medium, MG extracts or MG derivatives obtained after extraction (Jasin´ska et al., 2012) were placed in the wells of a microtiter plate. Samples containing extracts from fungal cultures cultivated without MG as well as the biotic (LB medium with bacteria) and abiotic controls (LB medium with MG) were simultaneously prepared. All of the samples
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(with the exception of the abiotic control) were inoculated with 100 ll of a bacterial culture (with an OD of 0.1) and incubated at 37 °C for 24 h. After this incubation period, the ODs of the cultures were measured at a wavelength of 630 nm using a Multiscan FC microplate reader (Thermo Scientific). The antimicrobial activities of MG and its derivatives were assessed after comparison with the bacterial growth obtained with the biotic control (considered 100%) and are expressed as percentages. 2.6. Statistical analysis The experiments were performed in triplicate. One-way analysis of variance was used to determine the significance of the differences between the samples. All of the statistical analyses were performed using Excel 2000 (Microsoft Corporation, USA). Readings were considered significant when p was 60.05. 3. Results and discussion 3.1. Involvement of cytochrome P-450 in MG decolorization Cytochrome P-450 constitutes a large and diverse group of enzymes that catalyze the oxidation of organic substances, drugs and other toxic chemicals (Kodam and Kolekar, 2015). To determine the contribution of cytochrome P-450 to MG decolorization by M. roridum IM 6482, inhibitors of cytochrome P-450, specifically metyrapone, proadifen and aminobenzotriazole, were used. None of the tested cytochrome P-450 inhibitors affected the MG decolorization process. A slight decrease in MG decolorization was observed only in the samples of M. roridum IM 6482 cultured with 1-aminobenzotriazole and proadifen for 24 h, but this difference was not observed after 48 h of cultivation. This result suggests that cytochrome P-450 is unlikely to be involved in the MG transformation by M. roridum IM 6482. Conversely, Cha et al. (2001) showed the complete inhibition of MG biodegradation by Cunninghamella elegans ATCC 36112 in the presence of metyrapone and a partial limitation of this process in the presence of other inhibitors. Based on these results, these researchers suggested the possible involvement of cytochrome P-450 monooxygenases in MG demethylation and the reduction of the dye to leucomalachite green by C. elegans ATCC 36112. An obvious inhibitory effect of metyrapone was also observed on MG decolorization by Mycobacterium chelonae, M. avium and Exiguobacterium sp. MG2 (Jones and Falkinham, 2003; Wang et al., 2012).
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MnP is unlikely to be involved in the MG decolorization by the strain of M. roridum. However, the addition of MG to the fungal culture changed the dynamics of laccase production as well as caused an increase in the activity of this enzyme (Fig. 1). In the control system, the highest laccase activity (44–46 U l 1) was observed in the samples derived from 48- and 72-h-old cultures. Conversely, the laccase activity of the culture containing MG incubated for 72, 96 and 120 h exceeded 68, 99 and 89 U l 1, respectively. Significant increase of laccase activity in MG containing cultures suggests the participation of the laccase in MG removal by M. roridum. Moreover, previous LC/MS/MS analyses indicated that biodegradation of MG involved reduction to leucomalachite green and conversion to N-demethylated metabolites which can by effect of laccase action (Jasin´ska et al., 2012). Thus, to better understand the extent of laccase involvement in MG decolorization, further experiments were performed using extracellular liquid and supernatants in which laccase was inactivated by 0.5 mM sodium azide or by boiling and using the liquid obtained after ultrafiltration. Despite the heat or chemical inactivation of laccase, MG decolorization was observed in these experiments. Surprisingly, MG decolorization was also detected in the nonenzymatic fraction obtained after ultrafiltration and containing compounds with masses lower than 3 kDa. In the samples in which the enzyme activity was retained, the degree of decolorization was approximately 8–11% higher than those obtained with the supernatants in which laccase was inactivated and with the compounds not retained by ultrafiltration. These results indicate the high involvement of low-molecularweight (<3 kDa) non-enzymatic factors resistant to high temperature in MG decolorization by M. roridum IM 6482. Peroxides may be responsible for the observed dye oxidation because these compounds have been proven to directly oxidize some chemical structures or promote their oxidation through hydroxyl radicals formed by the Fenton reaction. Several researchers have proposed that Fenton-like reactions are an important nonenzymatic mechanism in dye decolorization (Gomaa et al., 2008; Gomaa, 2012; Karimi et al., 2012; Moldes et al., 2012). In order to verify this hypothesis, decolorization tests with catalase, thiourea and superoxide dismutase (known scavengers of hydroxyl radical and superoxide anion radical) were designed. However, addition of mentioned chemicals did not affect the MG decolorization significantly. This result suggests that peroxides unlikely participate in the MG removal by M. roridum. Previously, Hu et al. (2006) isolated and purified from liquid culture of Phanerochaete chrysosporium new low-molecularweight peptide with phenol oxidase activity (Pc factor). Pc factor have high thermostability and remain active in weakly alkalescent pH range. Its molecular weight is about 600 Da. This
3.2. Involvement of enzymes and LMWF in MG decolorization The detoxification of synthetic dyes by various bacteria and fungi is often mediated by oxidoreductases. The oxidoreductive enzymes that play a main role in the biodegradation of dyes are LiP, MnP and laccases. The involvement of these enzymes in MG decolorization by Pycnoporus sanguineous, Phanerochaete chrysosporium and Trametes trogii classified as white rot fungi was described previously (Papinutti et al., 2006; Yan et al., 2014). These enzymes have also been shown to be involved in the decolorization of MG by Penicillium ochrochloron (Shedbalkar and Jadhav, 2011), Galactomyces geotrichum (Jadhav et al., 2008), Saccharomyces cerevisiae (Jadhav and Govindwar, 2006) and Pseudomonas sp. (Du et al., 2011). The activity of the produced laccase, MnP and LiP in a culture with the modified Cz-D medium was determined and compared with that obtained in the medium containing MG (10 mg l 1). LiP activity was not detected in M. roridum IM 6482 cultures. Activity of MnP in cultures containing MG was similar to those noticed in control cultures (about 25 U l 1). This suggests that
Fig. 1. Laccase activity in cultures of M. roridum IM 6482 in modified Cz-D medium.
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characterization is similar to those obtained for LMWF identified in M. roridum extracelluar liquid. Further works will aim to isolate and characterize LMWF of M. roridum. 3.3. Microbial toxicity of MG and its intermediates Most studies on the biodegradation of synthetic dyes have focused on the mechanisms and factors involved in the decolorization process. Only a few studies have included an evaluation of the toxicity of the obtained dye derivatives. Such data are of great importance because wastewaters decolorized by biodegradation methods should be environmentally safe. Thus, in this study, the toxicities of MG and its derivatives generated after 96 h of culture with M. roridum IM 6482 were compared. The antibacterial activity was determined using a modified method with serial dilutions in LB medium of the reference strains E. coli ATCC 25922, P. aeruginosa ATCC 15442 and S. aureus ATCC 61538. The results are shown in Fig. 2. Extracts derived from fungal cultures without MG did not
show a significant effect on the growth of the bacterial strains, whereas extracts containing MG reduced the bacterial growth. The highest growth inhibition was observed in the culture of S. aureus ATCC 61538. An addition of 2.5 mg l 1 MG resulted in more than 50% reduction in bacterial growth. In contrast, strains of E. coli ATCC 25922 and P. aeruginosa ATCC 15442 exhibited a greater tolerance to the MG extracts. Growth inhibition (more than 40%) was observed only in the P. aeruginosa ATCC 15442 samples containing 10 mg l 1 MG. In the samples containing extracts of cultures with supplemental MG, higher intensities of bacterial growth were observed. The highest tolerance to extracts of MG derivatives was observed in the S. aureus ATCC 61538 cultures. These findings suggest the non-toxic nature of the MG product generated in M. roridum IM 6482 cultures. The antimicrobial activities of MG and its degradation products obtained after culture with Micrococcus sp. BD15 were also evaluated by Du et al. (2011). These researchers showed a significant inhibition of E. coli K12 and B. subtilis B19 growth in the samples containing the MG extracts. A lower inhibition was obtained in the presence of MG degradation products, suggesting that degradation products of MG are less toxic than MG. Similarly, the results of toxicity tests have indicated that D. radiodurans R1 does not completely detoxify an MG solution but clearly reduces its toxicity (Lv et al., 2013). 3.4. Optimization of process parameters that influence MG decolorization
Fig. 2. Effect of MG extracts and extracts from M. roridum IM 6482 cultures grown for 96 h in a standard Czapek-Dox medium on the growth of the reference bacteria in the absence of MG (biotic control) or in the presence of the dye. (A) Staphylococcus aureus ATCC 61538, (B) Escherichia coli ATCC 25922 and (C) Pseudomonas aeruginosa ATCC 15442.
As shown in Fig. 3, the type and concentration of the carbon source influence the rate of MG decolorization by M. roridum, but this effect was observed only within the first 8 h after mycelium addition. During this period, the removal of MG was completely blocked in the culture containing 0.75% glucose. In turn, 8-h-old cultures in the presence of 1.5% glucose and 0.75% sucrose achieved 58% and 57% dye decolorization, respectively. The literature data indicate that glucose and sucrose promote MG decolorization by bacteria, filamentous fungi and yeast (Deivasigamani and Das, 2011; Deng et al., 2008; Jadhav and Govindwar, 2006). Similarly, Rai et al. (2005) found that addition of glucose to the growth medium is required to maintain efficient dye decolorization. This result may be due to the fact that glucose and sucrose are easily metabolized carbon sources that enhance the intensive production of biomass. These carbon sources are rapidly exhausted at low concentrations, and microorganisms therefore start using the dye as a source of carbon and energy, which contributes to the decolorization (Oranusi and Mbah, 2005) The kinetics and the efficiency of MG decolorization by M. roridum IM 6482 were also affected by the initial pH value of the medium (Table 1). In 8-h-old cultures grown in modified Cz-D medium with initial pH values of 4, 5, 6 and 7, the removal of MG reached 90%, 68%, 70% and 38%, respectively. These data may indicate that the strain of M. roridum used in this study decolorized MG with the aid of factors whose activity was at least partially dependent on the pH value. Nevertheless, after 24 h, the MG decolorization in all of the variants of the studied cultures attained a similar level of 97%. The obtained results are in agreement with the findings of many studies, which have indicated that a low pH is favorable for the removal of synthetic dyes by many filamentous fungi and yeast. According to the literature, the pH value may influence the decolorization process by impacting the transport of dye molecules through the cellular envelope. Additionally, the pH value may exert a strong influence on the activity of extracellular redox enzymes that are often involved in decolorization processes (Papinutti et al., 2006). Usually, the temperature favorable for xenobiotic removal is also optimal for microbial growth and biomass production. The impact of the incubation temperature on the decolorization
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Fig. 4. MG (10 mg l 1) decolorization by M. roridum IM 6482 mycelium cultured in modified Czapek-Dox medium at temperatures of 28, 38 or 48 °C.
rates of MG decolorization (Fig. 5). After 4, 6, 18 and 24 h of incubation, the average color removal was more than 35%, 56%, 94% and 90%, respectively. In contrast, the biomass production was strongly dependent on the oxygen availability. The biomass production was inhibited under static conditions (approximately 1.3 mg l 1 throughout the 24-h experimental period) and reached approximately 1.7 and 3.8 mg l 1 under shaking at 75 and 150 rpm, respectively. The literature data show that the Fig. 3. MG (10 mg l 1) decolorization in cultures of M. roridum IM 6482 in CzapekDox medium at pH 6.8 that contained (A) glucose and (B) saccharose at initial concentrations of 0.75%, 1.5% or 3%. The agitation rate and the incubation temperature were 150 rpm and 28 °C, respectively.
Table 1 MG (10 mg l 1) decolorization in the cultures of M. roridum IM 6482 in modified Czapek-Dox medium prepared in four variants of the initial pH (4; 5; 6 and 7). The agitation rate and the incubation temperature were 150 rpm and 28 °C, respectively. pH
4 5 6 7
MG decolorization [%] 4h
8h
24 h
57.34 ± 3.33 44.76 ± 2.63 50.43 ± 3.22 25.97 ± 2.45
90.65 ± 2.32 68.45 ± 7.65 70.33 ± 4.55 38.12 ± 3.57
97.65 ± 2.44 96.87 ± 1.28 98.75 ± 4.24 95.08 ± 2.11
capability of M. roridum IM 6482 was observed after 8 h of culture (Fig. 4). The highest difference in the rate of MG removal was observed after 24 h. The mycelium maintained at 28 °C almost completely decolorized MG (93%), whereas the cultures incubated at 38 or 48 °C exhibited a decolorization of 40%. A possible reason for this effect may be the suppression of metabolic processes involved in MG degradation. A similar dependence of the decolorization efficiency on the incubation temperature was studied by Shedbalkar and Jadhav (2011). The highest MG removal (93%) by a strain of Penicillium ochrochloron was observed at 28 °C, but an increase in the cultivation temperature to 50 °C inhibited dye decolorization. Deivasigamani and Das (2011) showed that decolorization of the other TPM dye – Basic Violet 3 by Candida krusei is also very dependent on the process temperature. The total elimination of the dye obtained only in cultures incubated at 28 °C, and a raising the temperature to 37 and 45 °C reduced the effectiveness of the process to 77% and 54%, respectively. Mycelia of M. roridum IM 6482 cultured under static conditions or under agitation at speeds of 75 and 150 rpm exhibited similar
Fig. 5. MG (10 mg l 1) decolorization (A) and biomass production (B) in cultures of M. roridum IM 6482 in modified Czapek-Dox medium under static and shaking (75 and 150 rpm) conditions.
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decolorization process that takes place in the presence of oxidative enzymes is usually more effective in cultures conducted under static conditions, whereas color removal resulting from dye sorption is promoted in shaking cultures (Torres et al., 2011). Oxygen can compete with the dye for reduced electron carriers and thereby reduce decolorization catalyzed by the TPM dyes reductase activity (Moosvi et al., 2005). Hence, MG decolorization by M. roridum IM 6482 may be based on processes involving both shakingdependent mechanisms (e.g., sorption, Fenton reaction and other oxidation reactions) and processes that do not require aeration (reactions catalyzed by reductase).
4. Conclusion The filamentous fungus M. roridum IM 6482 decolorizes MG by thermostable LMWF and laccase-induced enzymatic oxidation. Cytochrome P-450 does not mediate MG elimination. Effective decolorization (more than 90%) was achieved even in a simple, inexpensive medium over a broad range of pH values (4–7) and under both static and shaking conditions. MG derivatives were found to be less toxic to the reference bacterial strains than the original dye. M. roridum IM 6482 may be suitable for the treatment of environmental pollution caused by textile dyes. However, further large-scale experimentation is needed to assess the field applicability of this process. Acknowledgements The authors wish to thank K. Zawadzka, msc, for help in performing of toxicity tests. This work was supported by the University of Lodz – Poland (Grant No. 545/498) and co-funded by the European Union under the European Social Fund (HUMAN-BEST INVESTMENT). References Cha, Ch.-J., Doerge, D.R., Cerniglia, C.E., 2001. Biotransformation of malachite green by the fungus Cunninghamella elegans. Appl. Environ. Microbiol. 67, 4358–4360. Culp, S.J., Mellick, P.W., Trotter, R.W., Greenlees, K.J., Kodell, R.L., Beland, F.A., 2006. Carcinogenicity of malachite green chloride and leucomalachite green in B6C3F1 mice and F344 rats. Food Chem. Toxicol. 44, 1204–1212. Deivasigamani, Ch., Das, N., 2011. Biodegradation of basic violet 3 by Candida krusei isolated from textile wastewater. Biodegradation 22, 1169–1180. Deng, D., Guo, J., Zeng, G., Sun, G., 2008. Decolorization of anthraquinone, triphenylmethane and azo dyes by a new isolated Bacillus cereus strain DC11. Int. Biodeterior. Biodegrad. 62, 263–269. Du, L.-N., Wang, S., Li, G., Wang, B., Jia, X.-M., Zhao, Y.-H., Chen, Y.-L., 2011. Biodegradation of malachite green by Pseudomonas sp. strain DY1 under aerobic condition: characteristics, degradation products, enzyme analysis and phytotoxicity. Ecotoxicology 20, 438–446. Forgacs, E., Cserháti, T., Oros, G., 2004. Removal of synthetic dyes from wastewaters: a review. Environ. Int. 30, 953–971. Fu, X.-Y., Zhao, W., Xiong, A.-S., Tian, Y.-S., Zhu, B., Peng, R.-H., Yao, Q.-H., 2013. Phytoremediation of triphenylmethane dyes by overexpressing a Citrobacter sp. triphenylmethane reductase in transgenic Arabidopsis. Appl. Microbiol. Biotechnol. 97, 1799–1806. Gao, F., Ding, H., Shao, L., Xu, X., Zhao, Y., 2015. Molecular characterization of a novel thermal stable reductase capable of decoloration of both azo and triphenylmethane dyes. Appl. Microbiol. Biotechnol. 99, 255–267. Gomaa, O.M., 2012. Ethanol induced response in Phanerochaete chrysosporium and its role in the decolorization of triarylmethane dye. Ann. Microbiol. 62, 1403– 1409. Gomaa, O.M., Linz, J.E., Reddy, C.A., 2008. Decolorization of victoria blue by the white rot fungus, Phanerochaete chrysosporium. World J. Microbiol. Biotechnol. 24, 2349–2356.
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Biological decolorization of malachite green by Deinococcus radiodurans R1. Bioresour. Technol. 144, 275–280. Moldes, D., Fernández-Fernández, M., Sanromán, M.A., 2012. Role of laccase and low molecular weight metabolites from Trametes versicolor in dye decolorization. Sci. Word J. 2012, 1–9. Article ID 398725. Moosvi, S., Keharia, H., Madamwar, D., 2005. Decolourization of textile dye reactive violet 5 by a newly isolated bacterial consortium RVM 11.1. World J. Microbiol. Biotechnol. 21, 667–672. Oranusi, N.A., Mbah, A.N., 2005. Utilisation of azo and triphenylmethane dyes as sole source of carbon, energy and nitrogen by Bacillus sp. Afr. J. Appl. Zool. Environ. Biol. 7, 87–94. Papinutti, L., Mouso, N., Forchiassin, F., 2006. Removal and degradation of the fungicide dye malachite green from aqueous solution using the system wheat bran–Fomes sclerodermeus. Enzyme Microb. Technol. 39, 848–853. Rai, H.S., Bhattacharyya, M.S., Singh, J., Bansal, T.K., Vats, P., Banerjee, U.C., 2005. 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Singh, L., Singh, V.P., 2015. Textile dyes degradation: a microbial approach for biodegradation of pollutants. In: Singh, S.N. (Ed.), Microbial Degradation of Synthetic Dyes in Wastewaters. Environmental Science and Engineering. Springer International Publishing Switzerland, pp. 187–204. Stammati, A., Nebbia, C., Angelis, I.D., Albo, A.G., Carletti, M., Rebecchi, C., Zampaglioni, F., Dacasto, M., 2005. Effects of malachite green (MG) and its major metabolite, leucomalachite green (LMG), in two human cell lines. Toxicol. In Vitro 19, 853–858. Torres, J.M.O., Cardenas, C.V., Moron, L.S., Guzman, A.P.A., dela Cruz, T.E.E., 2011. Dye decolorization activities of marine-derived fungi isolated from Manila Bay and Calatagan Bay, Philippines. Philipp. J. Sci. 140, 133–143. Verma, A.K., Dash, R.R., Bhunia, P., 2012. A review on chemical coagulation/ flocculation technologies for removal of colour from textile wastewaters. J. Environ. Manage. 93, 154–168. 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Malachite green decolorization by the filamentous fungus Myrothecium roridum – Mechanistic study and process optimization Anna Jasin´ska a, Katarzyna Paraszkiewicz a, Anna Sip b, Jerzy Długon´ski a,⇑ a b
Department of Industrial Microbiology and Biotechnology, Faculty of Biology and Environmental Protection, University of Lodz, 12/16 Banacha Street, 90-237 Lodz, Poland ´ University of Life Sciences, Wojska Polskiego Street 48, 60-627 Poznan ´ , Poland Department of Biotechnology and Food Microbiology, Poznan
h i g h l i g h t s Myrothecium roridum decolorizes MG by LMWF and laccase action. Cytochrome P-450, LiP and MnP did not mediate MG decolorization. MG removal is effective in a simple medium, in both static and shaking cultures. MG is decolorized at a broad range of pH values (4–7). MG decolorization by M. roridum IM 6482 occurred in a non-toxic manner.
a r t i c l e
i n f o
Article history: Received 15 May 2015 Received in revised form 1 July 2015 Accepted 2 July 2015 Available online 8 July 2015 Keywords: Cytochrome P-450 Oxidoreductive enzymes Malachite green decolorization Myrothecium Toxicity
a b s t r a c t The filamentous fungus Myrothecium roridum isolated from a dye-contaminated area was investigated in terms of its use for the treatment of Malachite green (MG). The mechanisms involved in this process were established. Peroxidases and cytochrome P-450 do not mediate MG elimination. The laccase of M. roridum IM 6482 was found to be responsible for the decolorization of 8–11% of MG. Thermostable low-molecular-weight factors (LMWF) resistant to sodium azide were found to be largely involved in dye decomposition. In addition, MG decolorization by M. roridum IM 6482 occurred in a non-toxic manner. Data from antimicrobial tests showed that MG toxicity decreased after decolorization. To optimize the MG decolorization process, the effects of operational parameters (such as the medium pH and composition, process temperature and culture agitation) were examined. The results demonstrate that M. roridum IM 6482 may be used effectively as an alternative to traditional decolorization agents. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction Malachite green (MG), a triphenylmethane dye, is used for coloring a variety of materials, such as cotton, wood, silk, leather, jute and paper. This dye is also an antiparasitic, antifungal and antibacterial agent that is extensively applied in the aquaculture and fishery industries. However, MG is environmentally persistent and acutely toxic to a wide range of aquatic and terrestrial animals. MG and its derivatives have been reported to induce human carcinogenesis and mutagenesis. Several studies have also shown that exposure to this dye increases the risk of chromosomal fractures, reduces fertility and may act as an inhibitor of respiratory enzymes (Culp et al., 2006; Stammati et al., 2005). Due to the risk that MG imposes on fish consumers, its use in fishery applications is highly controversial. Thus, the use of MG in aquaculture has been banned ⇑ Corresponding author. Tel.: + 48 (42) 6354465. E-mail address: [email protected] (J. Długon´ski). http://dx.doi.org/10.1016/j.biortech.2015.07.008 0960-8524/Ó 2015 Elsevier Ltd. All rights reserved.
in many countries, including the United States of America, Canada and the European Union Member Countries. To date, a wide range of methods have been developed for the removal of synthetic dyes from wastewater (Verma et al., 2012). Although physical and chemical techniques have been applied to color removal, these techniques have serious disadvantages, such as high cost, low efficiency, limited versatility, interference with other wastewater constituents, formation of hazardous derivatives and/or intensive energy requirements (Shah, 2015). Due to the many drawbacks of conventional remediation methods, the development of efficient biological technologies to decrease the dye concentration in wastewater is of great importance (Sarayu and Sandhya, 2012; Singh and Singh, 2015). In recent years, many studies have focused on various microorganisms capable of MG removal, including bacteria, yeast, filamentous fungi and algae (Du et al., 2011; Karimi et al., 2012; Lv et al., 2013; Shedbalkar and Jadhav, 2011; Wang et al., 2012; Yan et al., 2014). The use of microbe-based methods in MG decomposition
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offers considerable advantages: the process is relatively inexpensive, the running costs are low, and the obtained by-products are often less toxic than the parent compounds (Forgacs et al., 2004). The most frequently mentioned factors involved in MG decolorization are the activities of cytochrome P-450, triphenylmethane reductase and extracellular lignin-modifying enzymes, particularly laccases (Fu et al., 2013; Gao et al., 2015; Jasin´ska et al., 2015). The literature data also indicate that dye decolorization may be mediated by nonenzymatic low-molecular-weight compounds that are resistant to high temperatures, which are also described as thermostable low-molecular-weight factors (LMWF) (Gomaa et al., 2008; Gomaa, 2012; Wang et al., 2012). The filamentous fungus Myrothecium roridum IM 6482 was isolated from a soil sample collected from the surroundings of a textile dyeing factory and has been proven to decolorize several industrial dyes (Jasin´ska et al., 2012, 2013). The aim of this study was to determine the usability of M. roridum IM 6482 for MG removal. The mechanisms involved in this process were also established in the present study. The participation of triphenylmethane reductase in MG elimination by M. roridum IM 6482 was previously proven (Jasin´ska et al., 2012); thus, this study focused on the involvement of cytochrome P-450, laccase, peroxidases and LMWF. The toxicities of MG and the derivatives formed during dye decolorization were also characterized. Furthermore, various process parameters, such as the carbon source, initial dye concentration, pH, culture agitation and temperature, were optimized. 2. Methods 2.1. MG, chemicals and fungal strains Technical-grade (80%) MG (4-[(4-dimethylaminophenyl)phenyl-methyl]-N,N-dimethylaniline) was kindly supplied by Boruta-Zachem Kolor Co. (Poland) and used without further purification. A stock solution of the dye (10 mg ml 1) was prepared in deionized water and autoclaved for 20 min at 121 °C before use. 2,2-Azinobis(3-ethylbenzothiazolin-6-sulfonic acid) (ABTS), veratryl alcohol, 2,6-dimethoxyphenol (DMP), sodium azide, proadifen, 1-aminobenzotriazole,metyrapone and other chemicals were purchased from Sigma–Aldrich Chemical Company (USA). The filamentous fungus tested in this study (M. roridum IM 6482) was isolated from a soil sample collected from the surroundings of a textile dyeing factory. It was deposited in the strain collection of the Department of Industrial Microbiology and Biotechnology, University of Lodz on ZT slants at 4 °C and _ transferred at 2-month intervals (Rózalska et al., 2015). 2.2. MG decolorization Decolorization studies were conducted in 100-ml Erlenmeyer flasks containing 18 ml of liquid Czapek-Dox (CzD) medium (containing glucose or sucrose at final concentrations 0.75%, 1.5% or 3%) inoculated with 2 ml of a homogenous second-step preculture (24 h-old) prepared in liquid WHI medium. Further experiments were performed using a modified CzD medium containing 0.75% glucose at various pH values (4; 5; 6 or 7), different incubation temperatures (28; 38 or 48 °C) and different agitation speeds (0; 75 or 150 rpm). At appropriate time intervals, the mycelia were separated from the culture medium by centrifugation at 15,000g for 15 min and then freeze-dried. The absorbance of the supernatant solution was analyzed using a Specord 200 spectrophotometer (Analytic Jena, Germany) and is expressed as a percentage of the removed dye. The decolorization percentage (DP) was calculated according to the following formula: DP[%] = [100 (A0 At/A0)], where A0 and At are the absorbance
values of the abiotic control and culture supernatant, respectively, measured at the maximum visible wavelength k = 615 nm. Biotic and abiotic controls without dye or mycelium supplementation, respectively, were also included. 2.3. Involvement of cytochrome P-450 in MG decolorization The homogenous preculture (2 ml) prepared as described above was transferred to 18 ml of standard Cz-D medium (in 100-ml Erlenmeyer flasks) supplemented with one of the following cytochrome P-450 inhibitors: metyrapone, 1-aminobeznotriazole or proadifen. The inhibitors were used at a concentration that had no significant effect on the growth of the fungus. The following optimal concentrations of these inhibitors (mycelial growth limitation up to 20%) were selected: 0.25 mM for metyrapone, 0.5 mM for 1-aminobenzotriazole and 0.01 mM for proadifen. Uninoculated growth media with appropriate amounts of the inhibitors were used as controls. Cultivation was performed for 30 min on a rotary shaker (150 rpm) at 28 °C. The cultures were then supplemented with 10 mg l 1 MG and incubated for 48 h under the conditions described previously. After incubation, the samples were centrifuged, and the degree of MG decolorization was determined according to the method described above. 2.4. Enzymes and LMWF involvement in MG decolorization Laccase activity in the supernatant obtained after culture centrifugation was assayed by monitoring ABTS oxidation at 420 nm. The reaction mixture contained enzyme extract, 20 mM citrate–phosphate buffer (pH 4.5) and 10 mM ABTS. Lignin peroxidise (LiP) activity was determined by monitoring the oxidation of veratryl alcohol at 310 nm. The reaction mixture contain 10 mM veratryl alcohol, 10 mM tartrate buffer (pH 3), 4 mM H2O2 and enzyme extract. The activity of manganese peroxidise (MnP) was measured at 470 nm in mixture containing 50 mM sodium malonate buffer (pH 4.5), 20 mM DMP, 20 mM MnSO4, 4 mM H2O2 and enzyme sample. The reference blanks included appropriate buffer and substrate. The enzyme activity is expressed in units, which are defined as the amount of enzyme oxidizing 1 lmol of appropriate substrate per minute. To establish the proportion of laccase involvement in MG decolorization, the liquid medium obtained after four days of culture in a modified Cz-D medium was recovered, and the following fractions were used in the decolorization experiments: extracellular liquid, supernatants in which laccase was inactivated by 0.5 mM sodium azide (chemically inactivated laccase) or by boiling for 30 min (thermally inactivated laccase), and filtrate from an ultrafiltration process using an Amicon Ultra tube (Millipore) with a cut-off of 3 kDa. To establish peroxides involvement in MG decolorization, catalase (600 U ml 1), thiourea (6 mM) and superoxide dismutase (200 U ml 1) were added to fluids before decolorization. The fluids were incubated with 10 mg l 1 MG at 28 °C for 3 h. The MG decolorization was determined as described above. 2.5. Toxicity studies These studies were performed using a modified method with serial dilutions in LB medium of the reference strains E. coli ATCC 25922, P. aeruginosa ATCC 15442 and S. aureus ATCC 61538. The optical densities of the cultures incubated with serial dilutions of the test compound were measured. LB medium, MG extracts or MG derivatives obtained after extraction (Jasin´ska et al., 2012) were placed in the wells of a microtiter plate. Samples containing extracts from fungal cultures cultivated without MG as well as the biotic (LB medium with bacteria) and abiotic controls (LB medium with MG) were simultaneously prepared. All of the samples
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(with the exception of the abiotic control) were inoculated with 100 ll of a bacterial culture (with an OD of 0.1) and incubated at 37 °C for 24 h. After this incubation period, the ODs of the cultures were measured at a wavelength of 630 nm using a Multiscan FC microplate reader (Thermo Scientific). The antimicrobial activities of MG and its derivatives were assessed after comparison with the bacterial growth obtained with the biotic control (considered 100%) and are expressed as percentages. 2.6. Statistical analysis The experiments were performed in triplicate. One-way analysis of variance was used to determine the significance of the differences between the samples. All of the statistical analyses were performed using Excel 2000 (Microsoft Corporation, USA). Readings were considered significant when p was 60.05. 3. Results and discussion 3.1. Involvement of cytochrome P-450 in MG decolorization Cytochrome P-450 constitutes a large and diverse group of enzymes that catalyze the oxidation of organic substances, drugs and other toxic chemicals (Kodam and Kolekar, 2015). To determine the contribution of cytochrome P-450 to MG decolorization by M. roridum IM 6482, inhibitors of cytochrome P-450, specifically metyrapone, proadifen and aminobenzotriazole, were used. None of the tested cytochrome P-450 inhibitors affected the MG decolorization process. A slight decrease in MG decolorization was observed only in the samples of M. roridum IM 6482 cultured with 1-aminobenzotriazole and proadifen for 24 h, but this difference was not observed after 48 h of cultivation. This result suggests that cytochrome P-450 is unlikely to be involved in the MG transformation by M. roridum IM 6482. Conversely, Cha et al. (2001) showed the complete inhibition of MG biodegradation by Cunninghamella elegans ATCC 36112 in the presence of metyrapone and a partial limitation of this process in the presence of other inhibitors. Based on these results, these researchers suggested the possible involvement of cytochrome P-450 monooxygenases in MG demethylation and the reduction of the dye to leucomalachite green by C. elegans ATCC 36112. An obvious inhibitory effect of metyrapone was also observed on MG decolorization by Mycobacterium chelonae, M. avium and Exiguobacterium sp. MG2 (Jones and Falkinham, 2003; Wang et al., 2012).
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MnP is unlikely to be involved in the MG decolorization by the strain of M. roridum. However, the addition of MG to the fungal culture changed the dynamics of laccase production as well as caused an increase in the activity of this enzyme (Fig. 1). In the control system, the highest laccase activity (44–46 U l 1) was observed in the samples derived from 48- and 72-h-old cultures. Conversely, the laccase activity of the culture containing MG incubated for 72, 96 and 120 h exceeded 68, 99 and 89 U l 1, respectively. Significant increase of laccase activity in MG containing cultures suggests the participation of the laccase in MG removal by M. roridum. Moreover, previous LC/MS/MS analyses indicated that biodegradation of MG involved reduction to leucomalachite green and conversion to N-demethylated metabolites which can by effect of laccase action (Jasin´ska et al., 2012). Thus, to better understand the extent of laccase involvement in MG decolorization, further experiments were performed using extracellular liquid and supernatants in which laccase was inactivated by 0.5 mM sodium azide or by boiling and using the liquid obtained after ultrafiltration. Despite the heat or chemical inactivation of laccase, MG decolorization was observed in these experiments. Surprisingly, MG decolorization was also detected in the nonenzymatic fraction obtained after ultrafiltration and containing compounds with masses lower than 3 kDa. In the samples in which the enzyme activity was retained, the degree of decolorization was approximately 8–11% higher than those obtained with the supernatants in which laccase was inactivated and with the compounds not retained by ultrafiltration. These results indicate the high involvement of low-molecularweight (<3 kDa) non-enzymatic factors resistant to high temperature in MG decolorization by M. roridum IM 6482. Peroxides may be responsible for the observed dye oxidation because these compounds have been proven to directly oxidize some chemical structures or promote their oxidation through hydroxyl radicals formed by the Fenton reaction. Several researchers have proposed that Fenton-like reactions are an important nonenzymatic mechanism in dye decolorization (Gomaa et al., 2008; Gomaa, 2012; Karimi et al., 2012; Moldes et al., 2012). In order to verify this hypothesis, decolorization tests with catalase, thiourea and superoxide dismutase (known scavengers of hydroxyl radical and superoxide anion radical) were designed. However, addition of mentioned chemicals did not affect the MG decolorization significantly. This result suggests that peroxides unlikely participate in the MG removal by M. roridum. Previously, Hu et al. (2006) isolated and purified from liquid culture of Phanerochaete chrysosporium new low-molecularweight peptide with phenol oxidase activity (Pc factor). Pc factor have high thermostability and remain active in weakly alkalescent pH range. Its molecular weight is about 600 Da. This
3.2. Involvement of enzymes and LMWF in MG decolorization The detoxification of synthetic dyes by various bacteria and fungi is often mediated by oxidoreductases. The oxidoreductive enzymes that play a main role in the biodegradation of dyes are LiP, MnP and laccases. The involvement of these enzymes in MG decolorization by Pycnoporus sanguineous, Phanerochaete chrysosporium and Trametes trogii classified as white rot fungi was described previously (Papinutti et al., 2006; Yan et al., 2014). These enzymes have also been shown to be involved in the decolorization of MG by Penicillium ochrochloron (Shedbalkar and Jadhav, 2011), Galactomyces geotrichum (Jadhav et al., 2008), Saccharomyces cerevisiae (Jadhav and Govindwar, 2006) and Pseudomonas sp. (Du et al., 2011). The activity of the produced laccase, MnP and LiP in a culture with the modified Cz-D medium was determined and compared with that obtained in the medium containing MG (10 mg l 1). LiP activity was not detected in M. roridum IM 6482 cultures. Activity of MnP in cultures containing MG was similar to those noticed in control cultures (about 25 U l 1). This suggests that
Fig. 1. Laccase activity in cultures of M. roridum IM 6482 in modified Cz-D medium.
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characterization is similar to those obtained for LMWF identified in M. roridum extracelluar liquid. Further works will aim to isolate and characterize LMWF of M. roridum. 3.3. Microbial toxicity of MG and its intermediates Most studies on the biodegradation of synthetic dyes have focused on the mechanisms and factors involved in the decolorization process. Only a few studies have included an evaluation of the toxicity of the obtained dye derivatives. Such data are of great importance because wastewaters decolorized by biodegradation methods should be environmentally safe. Thus, in this study, the toxicities of MG and its derivatives generated after 96 h of culture with M. roridum IM 6482 were compared. The antibacterial activity was determined using a modified method with serial dilutions in LB medium of the reference strains E. coli ATCC 25922, P. aeruginosa ATCC 15442 and S. aureus ATCC 61538. The results are shown in Fig. 2. Extracts derived from fungal cultures without MG did not
show a significant effect on the growth of the bacterial strains, whereas extracts containing MG reduced the bacterial growth. The highest growth inhibition was observed in the culture of S. aureus ATCC 61538. An addition of 2.5 mg l 1 MG resulted in more than 50% reduction in bacterial growth. In contrast, strains of E. coli ATCC 25922 and P. aeruginosa ATCC 15442 exhibited a greater tolerance to the MG extracts. Growth inhibition (more than 40%) was observed only in the P. aeruginosa ATCC 15442 samples containing 10 mg l 1 MG. In the samples containing extracts of cultures with supplemental MG, higher intensities of bacterial growth were observed. The highest tolerance to extracts of MG derivatives was observed in the S. aureus ATCC 61538 cultures. These findings suggest the non-toxic nature of the MG product generated in M. roridum IM 6482 cultures. The antimicrobial activities of MG and its degradation products obtained after culture with Micrococcus sp. BD15 were also evaluated by Du et al. (2011). These researchers showed a significant inhibition of E. coli K12 and B. subtilis B19 growth in the samples containing the MG extracts. A lower inhibition was obtained in the presence of MG degradation products, suggesting that degradation products of MG are less toxic than MG. Similarly, the results of toxicity tests have indicated that D. radiodurans R1 does not completely detoxify an MG solution but clearly reduces its toxicity (Lv et al., 2013). 3.4. Optimization of process parameters that influence MG decolorization
Fig. 2. Effect of MG extracts and extracts from M. roridum IM 6482 cultures grown for 96 h in a standard Czapek-Dox medium on the growth of the reference bacteria in the absence of MG (biotic control) or in the presence of the dye. (A) Staphylococcus aureus ATCC 61538, (B) Escherichia coli ATCC 25922 and (C) Pseudomonas aeruginosa ATCC 15442.
As shown in Fig. 3, the type and concentration of the carbon source influence the rate of MG decolorization by M. roridum, but this effect was observed only within the first 8 h after mycelium addition. During this period, the removal of MG was completely blocked in the culture containing 0.75% glucose. In turn, 8-h-old cultures in the presence of 1.5% glucose and 0.75% sucrose achieved 58% and 57% dye decolorization, respectively. The literature data indicate that glucose and sucrose promote MG decolorization by bacteria, filamentous fungi and yeast (Deivasigamani and Das, 2011; Deng et al., 2008; Jadhav and Govindwar, 2006). Similarly, Rai et al. (2005) found that addition of glucose to the growth medium is required to maintain efficient dye decolorization. This result may be due to the fact that glucose and sucrose are easily metabolized carbon sources that enhance the intensive production of biomass. These carbon sources are rapidly exhausted at low concentrations, and microorganisms therefore start using the dye as a source of carbon and energy, which contributes to the decolorization (Oranusi and Mbah, 2005) The kinetics and the efficiency of MG decolorization by M. roridum IM 6482 were also affected by the initial pH value of the medium (Table 1). In 8-h-old cultures grown in modified Cz-D medium with initial pH values of 4, 5, 6 and 7, the removal of MG reached 90%, 68%, 70% and 38%, respectively. These data may indicate that the strain of M. roridum used in this study decolorized MG with the aid of factors whose activity was at least partially dependent on the pH value. Nevertheless, after 24 h, the MG decolorization in all of the variants of the studied cultures attained a similar level of 97%. The obtained results are in agreement with the findings of many studies, which have indicated that a low pH is favorable for the removal of synthetic dyes by many filamentous fungi and yeast. According to the literature, the pH value may influence the decolorization process by impacting the transport of dye molecules through the cellular envelope. Additionally, the pH value may exert a strong influence on the activity of extracellular redox enzymes that are often involved in decolorization processes (Papinutti et al., 2006). Usually, the temperature favorable for xenobiotic removal is also optimal for microbial growth and biomass production. The impact of the incubation temperature on the decolorization
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Fig. 4. MG (10 mg l 1) decolorization by M. roridum IM 6482 mycelium cultured in modified Czapek-Dox medium at temperatures of 28, 38 or 48 °C.
rates of MG decolorization (Fig. 5). After 4, 6, 18 and 24 h of incubation, the average color removal was more than 35%, 56%, 94% and 90%, respectively. In contrast, the biomass production was strongly dependent on the oxygen availability. The biomass production was inhibited under static conditions (approximately 1.3 mg l 1 throughout the 24-h experimental period) and reached approximately 1.7 and 3.8 mg l 1 under shaking at 75 and 150 rpm, respectively. The literature data show that the Fig. 3. MG (10 mg l 1) decolorization in cultures of M. roridum IM 6482 in CzapekDox medium at pH 6.8 that contained (A) glucose and (B) saccharose at initial concentrations of 0.75%, 1.5% or 3%. The agitation rate and the incubation temperature were 150 rpm and 28 °C, respectively.
Table 1 MG (10 mg l 1) decolorization in the cultures of M. roridum IM 6482 in modified Czapek-Dox medium prepared in four variants of the initial pH (4; 5; 6 and 7). The agitation rate and the incubation temperature were 150 rpm and 28 °C, respectively. pH
4 5 6 7
MG decolorization [%] 4h
8h
24 h
57.34 ± 3.33 44.76 ± 2.63 50.43 ± 3.22 25.97 ± 2.45
90.65 ± 2.32 68.45 ± 7.65 70.33 ± 4.55 38.12 ± 3.57
97.65 ± 2.44 96.87 ± 1.28 98.75 ± 4.24 95.08 ± 2.11
capability of M. roridum IM 6482 was observed after 8 h of culture (Fig. 4). The highest difference in the rate of MG removal was observed after 24 h. The mycelium maintained at 28 °C almost completely decolorized MG (93%), whereas the cultures incubated at 38 or 48 °C exhibited a decolorization of 40%. A possible reason for this effect may be the suppression of metabolic processes involved in MG degradation. A similar dependence of the decolorization efficiency on the incubation temperature was studied by Shedbalkar and Jadhav (2011). The highest MG removal (93%) by a strain of Penicillium ochrochloron was observed at 28 °C, but an increase in the cultivation temperature to 50 °C inhibited dye decolorization. Deivasigamani and Das (2011) showed that decolorization of the other TPM dye – Basic Violet 3 by Candida krusei is also very dependent on the process temperature. The total elimination of the dye obtained only in cultures incubated at 28 °C, and a raising the temperature to 37 and 45 °C reduced the effectiveness of the process to 77% and 54%, respectively. Mycelia of M. roridum IM 6482 cultured under static conditions or under agitation at speeds of 75 and 150 rpm exhibited similar
Fig. 5. MG (10 mg l 1) decolorization (A) and biomass production (B) in cultures of M. roridum IM 6482 in modified Czapek-Dox medium under static and shaking (75 and 150 rpm) conditions.
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decolorization process that takes place in the presence of oxidative enzymes is usually more effective in cultures conducted under static conditions, whereas color removal resulting from dye sorption is promoted in shaking cultures (Torres et al., 2011). Oxygen can compete with the dye for reduced electron carriers and thereby reduce decolorization catalyzed by the TPM dyes reductase activity (Moosvi et al., 2005). Hence, MG decolorization by M. roridum IM 6482 may be based on processes involving both shakingdependent mechanisms (e.g., sorption, Fenton reaction and other oxidation reactions) and processes that do not require aeration (reactions catalyzed by reductase).
4. Conclusion The filamentous fungus M. roridum IM 6482 decolorizes MG by thermostable LMWF and laccase-induced enzymatic oxidation. Cytochrome P-450 does not mediate MG elimination. Effective decolorization (more than 90%) was achieved even in a simple, inexpensive medium over a broad range of pH values (4–7) and under both static and shaking conditions. MG derivatives were found to be less toxic to the reference bacterial strains than the original dye. M. roridum IM 6482 may be suitable for the treatment of environmental pollution caused by textile dyes. However, further large-scale experimentation is needed to assess the field applicability of this process. Acknowledgements The authors wish to thank K. Zawadzka, msc, for help in performing of toxicity tests. This work was supported by the University of Lodz – Poland (Grant No. 545/498) and co-funded by the European Union under the European Social Fund (HUMAN-BEST INVESTMENT). References Cha, Ch.-J., Doerge, D.R., Cerniglia, C.E., 2001. Biotransformation of malachite green by the fungus Cunninghamella elegans. Appl. Environ. Microbiol. 67, 4358–4360. Culp, S.J., Mellick, P.W., Trotter, R.W., Greenlees, K.J., Kodell, R.L., Beland, F.A., 2006. Carcinogenicity of malachite green chloride and leucomalachite green in B6C3F1 mice and F344 rats. Food Chem. Toxicol. 44, 1204–1212. Deivasigamani, Ch., Das, N., 2011. Biodegradation of basic violet 3 by Candida krusei isolated from textile wastewater. Biodegradation 22, 1169–1180. Deng, D., Guo, J., Zeng, G., Sun, G., 2008. Decolorization of anthraquinone, triphenylmethane and azo dyes by a new isolated Bacillus cereus strain DC11. Int. Biodeterior. Biodegrad. 62, 263–269. Du, L.-N., Wang, S., Li, G., Wang, B., Jia, X.-M., Zhao, Y.-H., Chen, Y.-L., 2011. Biodegradation of malachite green by Pseudomonas sp. strain DY1 under aerobic condition: characteristics, degradation products, enzyme analysis and phytotoxicity. Ecotoxicology 20, 438–446. Forgacs, E., Cserháti, T., Oros, G., 2004. Removal of synthetic dyes from wastewaters: a review. Environ. Int. 30, 953–971. Fu, X.-Y., Zhao, W., Xiong, A.-S., Tian, Y.-S., Zhu, B., Peng, R.-H., Yao, Q.-H., 2013. Phytoremediation of triphenylmethane dyes by overexpressing a Citrobacter sp. triphenylmethane reductase in transgenic Arabidopsis. Appl. Microbiol. Biotechnol. 97, 1799–1806. Gao, F., Ding, H., Shao, L., Xu, X., Zhao, Y., 2015. Molecular characterization of a novel thermal stable reductase capable of decoloration of both azo and triphenylmethane dyes. Appl. Microbiol. Biotechnol. 99, 255–267. Gomaa, O.M., 2012. Ethanol induced response in Phanerochaete chrysosporium and its role in the decolorization of triarylmethane dye. Ann. Microbiol. 62, 1403– 1409. Gomaa, O.M., Linz, J.E., Reddy, C.A., 2008. Decolorization of victoria blue by the white rot fungus, Phanerochaete chrysosporium. World J. Microbiol. Biotechnol. 24, 2349–2356.
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