PRODUCTION AND APPLICATION OF LACCASE FOR REMOVAL OF LIGNIN FROM LIGNOCELLULOSIC BIOMASS

Abstract

The increasing concern for independence from fossil fuel has led to search for novel renewable energy sources that offer sustainability, reduced cost, and environmental benefits. Lignocellulosic biomass represents one of the most abundant, cheap, and underutilized resources for fermentable sugars providing renewable energy. The major hindrance in this process occurs due to the recalcitrant nature of lignocellulosic plant material which is attributed to the presence of lignin. Lignin, a complex crosslinked organic polymer rich in aromatic subunits causes hindrance to the cellulolytic and xylanolytic enzymes during the fermentation process. There are several pre-treatment methods available for the removal of lignin from lignocellulosic biomass; however, these conventional methods are energy-intensive and require capital investment. Besides this, these also generate toxic components that further hinder the saccharification and microbial growth and have an adverse effect on the environment. This has led to an urgent need for a cost-effective and environmentally friendly pretreatment method that can remove lignin. Biological methods using oxidative enzymes from microorganisms offer advantages such as high product yield, less energy requirement, mild reaction conditions, and minimal waste generation. Fungal strains with tolerance to temperature, alkalinity, and acidity are the most attractive source for laccases. In this work, laccase producing fungal strain was isolated using a dual screening method for the laccase production in a suitable medium under submerged cultivation. Statistical optimization method Plackett–Burman design (PBD) and response surface methodology Box Behnken design (BBD) were used for the evaluation of various media components and their interactions with each other. The effect of different carbon, nitrogen and inducer sources on laccase production was studied. Statistical optimization by response surface methodology Box Behnken design (BBD) revealed that glucose contributed maximally to the overall laccase production followed by xylidin and veratryl alcohol. Laccase activity 103 IU/ml was achieved, which was more than 13.3 fold increased as compared to the unoptimized. The produced laccase was purified for the characterization using non-chromatographic (tangential flow filtration, ammonium sulfate precipitation) and chromatographic technique (anion exchange chromatography followed by size exclusion chromatography). The enzyme exhibited high stability over a wide range of temperature, pH, metal ions, and surfactants. The enzyme also displayed an elevated level of tolerance to inhibitors like sodium azide and dithiothreitol. Purified laccase showed Km and Kcat value of 59.73 μM and 1168 sec-1 respectively with ABTS and 16.29 μM, 84.3 sec-1 using syringaldazine as substrate respectively. The potential of laccase for removal of lignin-derived phenolic inhibitors was observed and the enzyme was capable of removing up to 79.28% of phenolic inhibitors even at a lower concentration in a short span of time indicating its potential role in the removal of lignin-derived inhibitors from the pre-hydrolysate. The application of laccase with mediator for delignification of jute stick biomass was tested, the change in the complete structure in the ii jute stick biomass after treatment with laccase and mediator was confirmed by analytical techniques such as XRD, BET analysis, SEM, and FTIR. The lignin compound in laccase with mediator treated jute stick was reduced by 21.8% relative to the untreated jute stick sample. Additionally, up to 19.5% increase in fermentable sugars was achieved as compared to the untreated biomass. Pyrolysis gas chromatography/mass spectrometry of the laccase -mediator treated biomass confirmed the modification in lignin polymer as a change in the syringyl-to-guaiacyl ratio. The outcome of study revealed the abundance of S lignin unit in the jute stick as compared to the H and G lignin unit. Further to improve the properties of the laccase, it was immobilized on novel, low-cost chicken feather waste material through covalent bonding and validated through model lignin compounds. The immobilization yield of 74.24 % was achieved and immobilization improved the pH and temperature optimum. The kinetics and thermodynamics of thermal inactivation of free and immobilized enzyme were studied over the temperature range from 55°C to 65°C. The apparent half-life (t1/2) and decimal reduction time (D-value) was calculated for free and immobilized enzyme. The activation energy for deactivation (Ed) was found higher for immobilized enzyme compared to free enzyme. Gibbs free energy (ΔG) and change in enthalpy (ΔH) demonstrated the stability. Overall, the laccase production from a novel fungal strain was optimized and characterized followed by its application in de-lignification of biomass as well as de-toxification of phenolic inhibitors generated during conventional pre-treatment methods. Laccase was immobilized on the novel support material and validated using model lignin compounds.