Journal of Proteomics & EnzymologyISSN: 2470-1289

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Research Article, J Proteomics Enzymol Vol: 6 Issue: 2

Optimization of Biodegradation Process for Disperse Textile Dyes Using Brown Rot Fungi

S Sidra Batool* and Anum Hanif

Pir Mehr Ali Shah Arid Agriculture University Rawalpindi, Pakisthan

*Corresponding Author : S Sidra Batool
Student of M.phil, Pir Mehr Ali Shah Arid Agriculture University Rawalpindi, Pakisthan
Tel: +92 51 9292122
E-mail: [email protected]

Received: January 20, 2017 Accepted: July 07, 2017 Published: July 14, 2017

Citation: Batool SS, Hanif A (2017) Optimization of Biodegradation Process for Disperse Textile Dyes Using Brown Rot Fungi. J Proteomics Enzymol 6:2. doi: 10.4172/2470-1289.1000132

Abstract

The whole research was managed to study the optimized conditions which are favorable for the production of enzymes which are the active participant in degradation of dyes. For this purpose Daedalea dickinsii was collected and isolated from the indigenous environment and was subjected for further experimentation towards the decolorization of DISPERSE dyes, which are extensively practiced in textile industries. There were given optimized conditions for the dyes to be decolorized by the fungus. The maximum degradation was observed between 5 to 8 days, after this no degradation was observed, this happened so because enzyme production stopped due to the accumulation of by products that severely retarded the enzyme growth or even stopped. The enzymes produced were Laccases, Manganese peroxidases and Lignin peroxidases, were also studied and their extent of participation in biodegradation of dyes. During the whole study it was also recorded that at which parameter there was maximum degradation occurred under optimized conditions and which parameters did not support the process. There were given many nutritional sources to record the maximum degradation, degradation became at its peak when enzymes were at their peak because carbon and nitrogen sources enhanced the enzymatic growth. It was recorded that when metallic ions were given to check the effects, enzymes showed positivity, it concluded that there existed a strong relationship between enzymatic production and application of metallic ions, Ca, Mg, Zn and Fe. The extent of enzymatic activity was then recoded as a Vmax and Kmax from line- weave burk plot. From the values obtained from the above study it was concluded that Daedalea dickinsii belonging from BRF holds the ability to decolorize the dyes upto 85%, if proper nutritional needs are provided. Hence it is emphaticaly concluded that this fungus holds very active traits towards decolorization of dyes and could be a proper and active agent for the treatment of waste-water and could be useful in vanishing the environmental pollutions, thus it would possibly reduce the bad impacts arising due to environmental degradation which ultimately lead to severe disorders.

Keywords: Biodegradation;Textile Dyes; Optimization

Introduction

Artificial dyes are extensively practiced by many industries all over the world, including as textile, paper, printing, cosmetics, pharmaceuticals, color photography and petroleum [1]. Among them the biggest consumer of dyes includes only textile industry from all over the world [2]. Aqueous dye bath solution were used to apply dye on substrate like cotton wool silk and natural fibres, however due to the hydrophobic nature of the manmade dye like, Cellulose acetate created a very good phenomenon where few of the dyes have affinity for the fibre [3] On the basis of procedure and protocol of dyeing, dyes are sorted as disperse dyes, reactive dyes, vat dyes, direct dyes, acid dyes and basic dyes [4].

Textile industries are the active consumer of dyes and dispose large amount of toxic chemicals in water during dyeing process [5]. About 10-15% of the dyes are lost in the wastewater during the dyeing process [3]. Aquatic life is major sufferer when waste dyes are dispose off into waters. Coloured dyes when wasted in water includes other pollutants such as degradable organics, nutrients, pH altering agent, salts, sulphur, toxicants and refractory organics. These waste products have a very toxic impact for living organisms [6,7]. Many microorganisms, including bacteria, fungi and actinomycetes, have been reported for their ability to decolourize dyes. It has been reported that among microorganisms many fungi including white rot fungi have the ability to decolourize the dyes by producing extracellular enzymes under optimized conditions and helps in removal of industrial effluent and also have the ability to combat with undesirable environmental conditions. Brown rot is considered as the best microbe for decolourization of dyes.

Brown-rots can also play an impressive role in the bioremediation of recalcitrant aromatics like dyes. Fungal dyes removal capabilities proved to be falling into two main categories; i.e., Biosorption and Biodegradation. Fungal dyes removal is mostly considered through extra-cellular fungal peroxidases. However, further research on understanding the mechanism of dyes reduction (decolorization) after taken up onto the fungal biomass would help in the advancement of bioremediation technologies. The current study was set up for the removal or isolation of fungus D. dickinsii from multiple localities for the extraction of its extracellular enzymes following the biodegradation of disperse dyes under the optimized conditions, holding the given aims:

Evaluating the synthetic textile dyes biodegradation by Daedalea dickinsii.

Checking optimizing process for decolorization of dyes under different conditions such as pH, temperature, period of incubation (fermentation), dye concentration and inoculum size.

Analyzing various effects induced by carbon and nitrogen sources along with metal ions.

Studying the participation of enzyme system involve in biodegradation process.

Materials and Methods

D. dickinsii was collected from the trunks of pinus denisflora from various areas of Murree and Abbottabad, sterilized bags were used for sampling, properly labelled and brought to lab for further process. The isolated or collected fungi samples were taken to laboratory, there culturing was done. fungi was cultured on Malt Extract Agar Media which is shown in table .On the basis of morphological features fungus and mode of colonization fungus was identified from Department of Plant Pathology, PMAS Arid Agriculture university Rawalpindi Pakistan. Cultures of D. dickinsii were labeled and were preserved on nutrient media so that further experimentation.

Procedure

A flask of 1000 ml was taken and calculated amounts of nutrients were added. PH was adjusted at 5 by addition of HCl and NaOH. The flask was subjected to autoclave for sterilization at 121°C for 15 min at 15 pascal of pressure, after that it was brought to Laminar Air Flow and a loop full of preserved (at 4°C) microbes were transferred into flask. That was subjected to shaker at 32°C for 5 to 7 days at 120 rpm. Dye solutions of different dyes were made with varying concentrations of dye (0.01%, 0.02%, 0.03%, 0.04% and 0.05%). Parameters like tempeature, pH, inoculum size, days and dye concentration were optimized to check maximum growth rate of D. dickinsii. The production of enzymes from D. dickinsii was optimized by adopting the parameters given in Table 1 as under in order to get the highest growth of enzymes like Laccase, Mangneses peroxidase and Lignin peroxidase. Different optimized conditions are given in the Table 2.

Inoculum size (ml) Days Temp (°C) pH Dye %
10 2 32.5 6 0.03
6 5 32.5 4 0.01
6 5 40 8 0.03
10 5 32.5 4 0.03
6 5 32.5 8 0.01
10 5 32.5 6 0.05
2 5 32.5 6 0.01
2 5 32.5 4 0.03
6 2 40 6 0.03
6 2 32.5 8 0.03
6 5 25 8 0.03
6 5 40 4 0.03
10 5 32.5 6 0.01
6 5 32.5 4 0.05

Table 1: Optimized conditions for growth of Daedalea dickinsii.

Dye pH Inoculum, (mL) Incubated days Temp, °C Dye conc (%) DEGRADATION (%)
Disperse-1 6 6 5 32.5 0.03 94%
Disperse-2 6 6 5 32.5 0.03 91%
Disperse-3 6 6 5 32.5 0.03 93%
Disperse-4 6 6 5 32.5 0.03 96%

Table 2: Optimized conditions for degradation of dyes.

The filtrates were used for the centrifugation for 10 min at 10,000 rpm for performing enzyme assays. The given enzyme assays were then performed for studying D.dickinsii enzyme profile. Laccase Assay For recording enzyme assay for laccase activity was performed by the method earlier recorded by Wolfenden and Wilson.

Substrate

ABTS (Diazinobis 3-ethylbenzoline-6 sulphonate) has a molecular formula C18H18N4O6S4 was obtained from Sigma product No.A 1888 and it was used as substrate .while binding to its target enzyme that was produced this compound gives green colour and the end product is soluble.

Lignin peroxidase assay

Tien and Kirk determined the method by which we measured the enzyme activity of Lignin peroxidase by this assay.

Substrate

3, 4- dimethoxybenzylalcohol (Vataryl alcohol) is organic compound with molecular formula C9H12O3 and also called benzyl alcohol and is obtained from the reduction of veratraldehyde that was used as substrate for LiP as described by Hermann in 1963.Vataryl alcohol was oxidized to veratraldehyde while reaction with buffer of sodium acetate with pH 3 and also in the presence of H2O2. Here; ϵ 310=9300 M-1cm-1.

Manganese peroxidase assay

Wariishi and others described the method that was used for measuring the enzymatic activity of Manganese peroxidase via enzyme assay.

Substrate: Sigma product was the source of the manganese sulphate (MnSO4). It was used as substrate. Binding with the target enzymes is carried via this compound and thus it produced the product. Metal ions of Ca, Mg, Fe and Zn were used for characterizing the enzyme activity. Different concentrations of the said metal ions were made, concentrations of metal ions were used as 0.05 g, 0.1 g, 1.5 g, 0.2 g and 0.25 g. Rsm was used for the measurement and analysis of various parameters under CCD. Analysis of response with various variables were performed with the help of Rsm, for exploring maximum response it was used with series of experiments.

Results and Discussion

Collected wood samples were cultured on Malt Extract Agar Media for isolation of required fungus, pure cultures were then subjected to the Mycology Laboratory Department of Plant Pathology PMAS, Arid Agriculture University Rawalpindi for identification of the required fungus. Disperse dyes of different wavelengths and colours have been used. These dyes were collected from the textile industries of Faisalabad. DISP 1, DISP 2, DISP 3 and DISP 4 were used for biodegradation process. While conducting this study it was noticed that various growth parameters were involved in biodegradation of dyes because these parameters go side by side during decolorization process. Decolorization was maximum when enzyme production was at its peak, hence the relationship between various growth parameters can never be ignored that, at which parameter decolorization becomes maximum and at which optimized condition it could be achieved.

Relationship between inoculum size and pH for degradation of Disperse -1 by D. dickinsii (Figure 1)

Overall study during process showed that temperature range was between 30°C and 38°C gave a good degradation as the temperature was increased up to 40°C there was decline in the process because high temperature did not support the fungus to show maximum enzyme production so no healthy degradation was recorded. It was also observed that at low temperature the enzyme production was not enough to degrade the dyes, low temperature did not favour the biodegradation process.

Figure 1: Relationship between inoculum size and pH for degradation of Disperse -1 by D. dickinsii.

Relationship between temperature inoculum sizes for degradation of Disperse-2 by D.dickinsii (Figure 2)

Incubation period (days) and pH put huge impact on biodegradation of dyes. Both were studied during the decolorization process. As described earlier that different parameters are involved in the biodegradation of dyes so, we have to monitor that parameters during the whole study and must record that conditions at which the result was at its peak. In current study a very healthy relationship was recorded putting a great impact in decolorization of dye, that is incubation period and pH both parameters are very important. Dye showed maximum degradation at pH 7.5 and fermentation period was comprised between 6 to 7 days, at this parameter 77.4% degradation was recorded. It was inferred from the study on increasing the pH there was decline in enzyme production hence leading to quit the degradation process.

Figure 2: Relationship between temperature inoculum sizes for degradation of Disperse -2 by D.dickinsii.

Relationship between pH and days for degradation of Disperse-3 by D. dickinsii (Figures 3-5)

Concentration of dyes put a great impact in biodegradation process, if concentration increases the process goes positively due to production of enzymes which are involved in decolorization of dyes. In current study dye conc ranges from 0.01 g to 0.05 g were used and temperature was in range between 32.5°C to 38.5°C.The maximum degradation recorded was 80.33%.The possible degradation is shown in the Figure 6.

Figure 3: Relationship between pH and days for degradation of Disperse -3 by D. dickinsii.

Figure 4: Relationship between pH and days for degradation of Disperse -3 by D. dickinsii.

Figure 5: Nutritional sources effects.

Figure 6: Effect of magnesium and zinc ions on degradation of dyes.

Analysis of influence of varying nutritional sources on degradation of disperse dyes (1-4)

Effect of different carbon sources: Glucose and fructose were used as a carbon source to check how much decolorization of dyes occur when these sources are being applied.

Glucose and fructose effect on dye: Both are the carbon sources, carbon sources includes glucose and fructose. both were added to maximize the production of extracellular enzymes by enhancing the activity of D. dickinsii for the decolourization of dye.Glucose and fructose were added separately in duplicate experimental flasks .five different concentrations were used both for glucose and fructose, i.e, 0.5%, 1%, 1.5%, 2% and 2.5%. Then flasks were incubated at 25°C for few days. Both glucose and fructose enhanced the activity of the fungus when calculated amount added.

Effect of different nitrogen sources: Addition of nitrogen sources in different forms in growth media of D.dickinsii may change the production of extracellular enzyme., because the microbes like fungus consume the nitrogen as a source for production of proteins along with other nutritive macromolecules. ammonium sulphate, ammonium nitrate and ammonia were used as nitrogen source for boosting the activity of fungus.

Ammonium sulphate, ammonium nitrate and ammonia effects on dye: Five different concentrations of ammonium sulphate, ammonium nitrate and ammonia (0.5%, 1%, 1.5%, 2% and 2.5%) were used to check the effect of the nitrogen sources on fungus activity, for optimization of said chemicals five different concentrations were selected. each of the concentration was used in duplicate to obtain better results. The conditions of growth media for ammonium sulphate, ammonium nitrate and ammonia are shown in Table 2.

Degradation was favored by the addition of nutritional sources , including carbon and nitrogen sources. It was concluded that with the addition of nutritional sources there was maximum production of enzymes which influenced the degradation of dyes .Carbon sources like glucose and fructose enhanced enzymatic activity as it was the major energy reservoir for enzyme production, in the same way nitrogen sources like ammonium sulphate, ammonium nitrate and ammonia also put a huge effect in biodegradation of dyes by maximum production of enzymes. In current study it was seen that at conc of 1.5% maximum production was shown by fungus, when conc was increased there was decrease in degradation because of production of certain unwanted products. The effects of nutritional sources is shown in Figure 5.

Effect of carbon and nitrogen sources on degradation of dyes: Glucose and fructose were used as a carbon source to check how much decolorization of dyes occur when these sources are being applied. Addition of nitrogen sources in different forms in growth media of D.dickinsii may change the production of extracellular enzyme. Because the microbes like fungus consume the nitrogen as a source for production of proteins along with other nutritive macromolecules. Ammonium sulphate, ammonium nitrate and ammonia were used as nitrogen source for boosting the activity of fungus.

Metal ions effect: Metal ions of Ca, Mg, Fe and Zn were used for characterizing the enzyme activity. Different concentrations of the said metal ions were made, concentrations of metal ions were used as 0.05 g, 0.1 g, 1.5 g, 0.2 g and 0.25 g. In current study different concentrations of metallic ions were used, it was observed that maximum enzyme production was recorded when 2% conc of iron and calcium was applied, while 1.5% conc showed maximum production when zinc and magnesium were applied (Figure 6).

Conclusion

Enzymes kinetics study

Substrate concentration and its effects on enzymes activity: For the analysis of the influence of the effects on Kmax and Vmax for enzymatic activity , different substrate conc were used. For this purpose different solutions of varying concentrations were used. For analysis of Laccases ABTS was used, for LiP vertryl alcohol was used and for MnP MgSO4 was used. When concentration of substrate increases for every enzyme, speed of enzymes also increases, but it becomes constant after certain concentration, because of the no binding sites for the substrate on enzyme. The enzyme characterization by D.dickinsii at different concentrations of substrate for Laccases is shown in figure 4.20, for LiP in 4.21 and for MnP in 4.22.

Vmax: The concentration of substrate with maximum enzymatic speed or velocity of reaction is said to be Vmax of reaction (Graph 1).

Graph 1: Characterization of Laccases enzyme by D. dickinsii at different concentration of substrate.

Kmax: It is said that when concentration of substrate is required for an enzyme to achieve ½ of Vmax. The values from Kmax describes affinity of substrate approaches towards enzyme (Graph 2).

Graph 2: Characterization of lignin peroxidase by D. dickinsii at different concentration of substrate.

References

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