PARTHENIUM HYSTEROPHOROUS FOR BIOETHANOL PRODUCTION: PROCESS ENRICHMENT AND OPTIMIZATION

Abstract

It is predicted that fossil fuels which include oil, coal and natural gas reserves will deplete in next few decades. The world primary energy consumption fulfilled through fossil fuel is 79% and accounts 57.7% of volume used in the transport sector. The alternative fuels not only draw the attention because of depleting natural energy resources but concerns are related to environmental security also a driving factor. According to Renewable Fuel Association (RFA) ethanol industry outlook 2017, total production of bioethanol in 2016 was estimated 26.5 billion gallons. About 60% of bioethanol produce worldwide is obtained using sugarcane as feedstock rest 40% produced by other crops. Currently, two major producers of bioethanol; US and Brazil are employing corn and sugarcane respectively but any other country with agroindustrial economy can be involved in production of bioethanol. Indian government in 11th five years plan (2007-1012) under Ethanol blended Petrol Programme (EBPP), it was suggested to blend 10% in gasoline once the 5% blending target achieved across the country. It was also proposed by National Policy on Biofuels to scale up blending target 20% by 2017. Lignocellulosic materials are potential feedstocks for bioethanol production due to presence of cellulose and hemicelluloses which can be subsequently converted into sugar and then bioethanol. Biochemical route of conversion from lignocellulosic material to bioethanol principally consists of three main steps, (1) pre-treatment, (2) enzymatic hydrolysis, and (3) fermentation. Lignocelluloses can be obtained from wood, grasses, agriculture residues, and waste materials. Some weedy plants like Parthenium hysterophorous, Lantana camara, Eicchornia crassipis, Prosopis juliflora, and Saccharum spontaneum, are also potential economical lignocellulosic feedstocks and can be used in enzyme production. The “DBT-ICT Centre for Energy Biosciences” first time in the country demonstrated the production of bioethanol from lignocellulosic biomass. The first cellulosic ethanol demonstration plant in India was setup on 22nd Apr-2016 at India Glycols Limited, Kashipur, Uttarakhand with a capacity of 10 ton/day. Present study was aimed to screened potential fungal strains which were used to hydrolyse the lignocellulosic material. Parthenium hysterophorous (carrot grass) was used as a lignocellulosic material because of its abundance in nature and no significant use of its stem part. All the cultural operating parameters were optimized for optimum production of cellulase and xylanase enzyme, further partial characterization of crude enzyme was also accomplished. Crude enzymes from selected fungal isolates were analysed for their efficacy in saccharification of P. hysterophorous. A comprehensive screening was executed for the ii selection of potential fungal strains for enzymatic hydrolysis study. Total 114 fungal cultures were selected during primary screening using their potential to grow on CMC-agar media. The secondary screening was based on congo-red zone clearance assay, zone ratio of 4.0 mm or more were selected. Two fungal isolates AB-5 and AB-6 were selected based on their cellulase activity. Fungal isolates AB-5 and AB-6 were identified as Talaromyces stipitatus MTCC 12687 and Aspergillus niger NFCCI 4113 based on 16s-rRNA sequencing. Eight different lignocellulosic materials were tested for the production of enzymes by T. stipitatus AB-5, A. niger AB-6 under solid-state fermentation conditions. The fungal isolates AB-5 and AB-6 were selected on the basis of their efficient applications in hydrolysis of P. hysterophorous. Selected fungal isolates AB-5 and AB-6 were identified as T. stipitatus and A. niger respectively which were designated as T. stipitatus AB-5 and A. niger AB-6 for further study. P. hysterophorous and wheat bran were selected as the suitable carbon sources for A. niger AB-5 and T. stipitatus AB-6 respectively due to higher enzyme production level during solid state fermentation (SSF). Effect of various other parameters was also observed on enzyme production for both the fungal isolates. T. stipitatus AB-5 produced maximum endoglucanase (53.33 IU/gds), FPase (4.51 FPU/gds), xylanase (933.73) IU/gds and β-glucosidase (62.6 IU/gds) at 30°C using 5 day incubation time period; addition to this, pH 6.0 and 80%moisture content with the supplementation of 1.0%, w/v peptone as nitrogen source and 0.15% w/v Tween-80 as a surfactant were used under SSF conditions. Aspergillus niger AB-6 was found to produce maximum endoglucanase (55.26 IU/gds), FPase (4.64 IU/gds), xylanase (1175.76 IU/gds) and β-glucosidase (71.30 IU/gds) at at 30°C using 6 day incubation time period; additionally, pH 7.0 and 70% moisture content with the supplementation of 1.0%, w/v peptone as nitrogen source and 0.10%, w/v Tween-80 as a surfactant were used. The extended stability of cellulases for temperature and pH increase their suitability for enzymatic hydrolysis of lignocellulosic biomass for prolonged time period, without using extra enzymes. Effect of pH during enzyme assay it was observed that optimum temperature for FPase, endoglucanase, and glucosidase activities was 50 °C for both the fungal strains i.e. T. stipitatus AB-5 and A. niger AB-6. Endoglucanase and FPase from T. stipitatus AB-5 observed to show their maximum activity at 55 °C while a maximum β-glucosidase activity was found at 60 °C. The maximum titre of endoglucanase, FPase and β- glucosidase activities were observed at 50°C, 55°C, and 55 °C respectively for fungal strain A. niger AB-6. Although, cellulases from T. stipitatus AB-5 A. niger AB-6 were showed their stable at 50 °C upto a holding-time of 48 h. iii The percentage of cellulose, hemicelluloses, and lignin in untreated P. hysterophorous was estimated to 36.5, 27.7, and 24.5 % respectively. The hydrolysate obtained from P. hysterophorous after pre-treatment at 180 °C, ethanol to distilled water ratio 3:1, solid to liquid ratio 1:5, and reaction time 60 min with 2% of H2SO4 added as a catalyst produced maximum total reducing sugars in g/100g of oven-dried material (24.82±1.2), ethanol-organosolv lignin (20.11±1.5) and minimum acid insoluble lignin total (0.95±0.19) and pre-treated substrate produced total reducing sugars (32.34±1.2) and acid minimum insoluble lignin solids (1.13±0.12) and resulted total mass balance of 79.35 g/ 100 g of total solids for T. stipitatus AB-5. Following the same pre-treatment conditions enzyme obtained from A. niger AB-6 produced maximum total reducing sugars 32.85±1.2 g/100g of oven-dried material and total mass balance was79.86 g/ 100 g of total solids. Low level of reducing sugars into pre-treated hydrolysate having only distilled water and dilute acid as catalyst was due to conversion of hemicelluloses and cellulosic content into furans (furfural and 5-hydroxymethyl furfural) depending of pre-treatment conditions specifically high temperature. The removal of hemicelluloses and lignin during pre-treatment leads to improve enzymatic hydrolysis because of enhance accessibility of raw-material. The total reducing sugars recovery from hydrolysate and enzymatically treated substrate (P. hysterophorous) was 89.03% in case of enzymatic hydrolysis with T. stipitatus AB-5 whereas in case of enzymatic hydrolysis with A. niger AB-6 was 89.83%. The total reducing sugar and lignin recovery from hydrolysate and enzymatically treated substrate (P. hysterophorous) was 93.07% in case of enzymatic hydrolysis with T. stipitatus AB-5 whereas in case of enzymatic hydrolysis with A. niger AB- 6was 93.46%. During pre-treatment of P. hysterophorous hemicelluloses and lignin were removed significantly which was also supported by FTIR, XRD studies and SEM analysis. So due to higher reducing sugar recovery during pre-treatment at 180 °C, ethanol to distilled water ratio 3:1, solid to liquid ratio 1:5, and reaction time 60 min with 2% of H2SO4 added as a catalyst, this condition was selected for further experiments. The lesser sugar released by the untreated P. hysterophorous during enzymatic hydrolysis might be due to recalcitrance nature of raw material which leads to lower accessibility and lower surface area compared to treated P. hysterophorous. An enzyme dose of 10 FPU/g OD pre-treated raw-material was found to produce optimum reducing sugars 731±18.45 mg/g and 742±15.34 mg/g OD raw-material for T. stipitatus AB-5 and A. niger AB-6 produced enzymes after 48 h of enzymatic hydrolysis. Addition of Tween-80 dose of 0.2 g/g of OD raw-material increased the reducing sugar release and reached 785±12.66 mg/g and 798±19.54 mg/g after 48 h of enzymatic hydrolysis. The enzyme concoction (E1 (T. stipitatus):E2 (A. niger)=3:1) stood at iv the top in compared other concoctions (E1:E2= 4:0, 2:2, 1:3 and 0:4) and released maximum reducing sugars (820±12.62 mg/g OD raw-material) up to 48 h of reaction time and beyond that the increase in reducing sugars was insignificant. Fermentation efficiency with S. cerevisiae MTCC 170 for total reducing sugars obtained after enzymatic hydrolysis was about 85.12% with ethanol productivity 0.296 g/l/h, and total reducing sugar to ethanol conversion 0.426 g/g. Detoxification of pre-treatment hydrolysate was carried out using subsequent overliming and activated charcoal treatment methods, fermentation efficiency was calculated about 85.12% using P. stipitis NCIM 3497 with ethanol productivity 0.173 g/l/h, and total reducing sugar to ethanol conversion 0.408 g/g. No ethanol production was observed using undetoxified pretreatment hydrolysate. Co-fermentation was also carried out using S. cerevisiae MTCC 170 and P. stipitis NCIM 3497 using total reducing sugars obtained after enzymatic hydrolysis and presented in pre-treatment hydrolysate (ratio 1:1 v/v) , a fermentation efficiency was estimated about 86.83% with ethanol productivity 0.235 g/l/h, and total reducing sugar to ethanol conversion 0.422 g/g. Thus, it was concluded that detoxification of pretreatment hydrolysate is unavoidable to enable it produce ethanol. During fermentation process it was observed reducing sugar conversion into ethanol nearly stop after 48 h.