Evaluation of Lignin Degrading Efficiency of Selected Fungal Strains Isolated from Pulp and Paper Industry Wastewater

Abstract

Lignin is the second most abundant natural biopolymer on the land having complex and heterogenic chemical structures. In recent years, biodegradation of the lignin polymers and its transformation into value added products have been gaining ample scientific attraction. In present research four potent fungal strains were isolated and screened from pulp and paper industry for their possible role in biodegradation of commercial lignin and lignin in the industrial waste water. The enzymatic potential of the fungal isolates was also determined for the bio-depolymerisation of lignin and their suitability to produce peroxidises. During first phase of the research, black liquor (BL) was obtained from the pulp and paper mill effluent and its physiochemical analysis was carried out. In addition, BL was also used for the screening of lignin degrading fungi. Two step screening strategy was employed for the isolation and screening of the potent fungal strains having enzymatic capability to use and degrade lignin. For enrichment and screening of the fungi Czapek Dox agar media was made with the following composition was used (g/L); Sodium Nitrate; 2.0g/L, Dipotass ium Phosphate; 0.35g/L, Magnesium Sulfate; 0.5g/L, Potassium Chloride; 0.5g/L, Ferrous Sulfate; 0.01g/L, 200 mg/L lignin and 2% agar. Initially thirteen fungal strains showed rich growth on the aforementioned media and subjected for secondary screening in the liquid MSM using black liquor as only source of carbon. On the basis of higher growth, lignin degradation and colour reduction, four fungal strains were screened namely M-1, M-2, M-3 and M-4. These four fungal strains were then identified on the basis of 18s RNA gene sequencing as Aspergillus terrusM-1, Dipodascus autraliensisM-2, Geotrichum candidumM-3, Aspergillus fumigatus M-4. The lignin degradation efficiency was determined and it was revealed that the strain Aspergillus terrus M-1 caused biodegradation of lignin in the BL up to 73%, Dipodascus autraliensis M-2 84%, Geotrichum candidum M-3 84% and Aspergillus fumigates M-4 80%. The optimum reaction conditions for the maximum biodegradation of lignin by these strains were different. However, most of the fungi were able to perform best at 41 °C and pH above 7. The highest lignin degradation efficiency showed by stains M-2 and M-3 were recorded at 41°C and pH9; and 47°C and pH9 respectively. In the next phase, these fungi were evaluated for the biosynthesis of different peroxidises and laccases including Lignin peroxidase (LiP) and Managnese peroxidase (MnP) under submerged fermentation conditions. The DRSML QAU reaction parameters such as pH, temperatures, substrate concentration and different carbon sources were also checked for their effect on the fungal growth and enzyme production rates. The fungal strains showed varying metabolic efficiency under different reaction conditions. Maximum activity of laccase (139 U/mL) was observed from Aspergillus terrus M-1, whereas highest LiP and MnP were observed in case of Geotrichum candidum M-3, 115U/mL and 106 U/mL respectively. The other fungal strains produced relatively less amount of peroxidises and laccases as compared to the M-1 and M-3 strains. The molecular weights of the different peroxidise and laccases was determined for all four strains. The enzymes were extracted from the growing cultures of the M-1-M-4 strains and SDS Gel electrophoresis was performed. It was noted that the molecular weight of the lignin isoenzymes was found to be in the range of 35 kDa and for laccases it was in the range of 130 kDa. The effect of different types and concentrations of lignin (insoluble and soluble) on the growth of fungal strains was also studied. Our results indicated that the fungal strains showed varying capacity to use lignin variants as sole source of carbon and energy. In case of avicel, the highest growth was observed by Geotrichum candidum M-3 and Aspergillus fumigatus M-4. Whereas strain Aspergillus terrus M-1 and Dipodascus autralienses M-2, showed comparatively less growth on the avicel. The growth pattern of the fungi on Mannans indicated that all strain has the metabolic capacity of using this carbon source efficiently except Aspergillus terrus M-1. Further, Aspergillus terrus M-1was also found less competent to use pectin, starch and xylan as compared to the other fungal strains. In the next phase the analysis of various degradation products of the lignin was evaluated using H 13C HSQC Nuclear magnetic resonance. Different intermediate compounds were detected during lignin degradation by the action of our selected fungal strains. In case of Aspergillus terrus strains M-1 the chemical shift was observed at 3.5 to 4.0 1Hppm and the chemical shift at 13C, ppm was around 57 to 60. These results depicted the methoxyl group formed in the result of lignin degradation. Dipodascus australiensis M-2 and Geotrichum candidum M-3 after degradation liberated xylan from the lignin carbohydrate complex. Dipodascus australiensis M-2 showed the chemical shift of 1H, ppm at 4 – 5.1 indicated lignin transformation into carbohydrates. The strain Geotrichum candidum M-3 were more active in metabolizing residual monomers such as vanillin, acetovanillone and coniferyl alcohols. In addition, these strains DRSML QAU significantly reduced the linkage content in the lignin fraction giving the chemical shift of 1H ppm at 4.9 to 5.1 which represented the formation of carbohydrates, 6.2 to 6.7 exhibited the aromatic-H in syringyl and 6.7 to 7.1 aromatic-H guaiacyl. The chemical shift at 13C ppm by this fungus provided the evidences for a LiPhatic and aromatic groups. Relatively, both of these strains M-2 and M-3 showed cleavage not only of β-aryl-ether (β-O-4), but also of resinol (β-β) and of the less prevalent phenylcoumaran (β-5) linkages for biodegradation of lignin. Aspergillus fumigatus strain M-4 had less pronounced activity than strains. This strain showed the chemical shift of methoxy and slightly for the aliphatic groups. In the last phase of the research, the biodegradation of intermediate compounds detected by NMR results was studied. Reverse Phase High pressure liquid chromatography (RP- HPLC) was used to track down degradation of intermediate products. The guaiacol, hydroxybenzaldehyde, syringaldehyde and vaniline were used as substrate and after six days of experiments the results were analysed. HPLC analysis of control and fungal mediated degraded samples showed reduction and shifting of peaks were revealed for vanillin and hydroxybenzaldehyde for the strain Geotrichum candidum M-3 and Aspergillus fumigatus M-4. However, the fungal strains did not show any significant degradation of syringaldehyde and guaiacol since slight shift of peaks were obtained. On the basis of the results, it can be concluded that the fungi isolated and screened in the present study showed excellent metabolic efficacy to grow and degrade lignin both in the industrial waste water and pure lignin. Further, these strains produced various lignin degrading enzymes using lignin as sole source of carbon and energy. Therefore, the fungal strains Aspergillus terreus, Dipodascus australiensis, Geotrichum candidum and Aspergillis fumigatus can be used for the bioremediation of pulp and paper industrial waste and for the biotransformation of lignin containing waste into value added commercial products