Comparison of submerged and solid state pretreatment of sugarcane bagasse by Pandoraea sp. ISTKB: Enzymatic and structural analysis
Introduction
India, with more than one billion people and fastest growing economy, is fourth largest primary energy consumer in the world. Domestic production of oil and gas is insufficient to meet the energy requirements. More than 75% of energy needs are fulfilled through imported crude oil and gas. Major portion of this imported crude oil is used in transportation sector. Roads are the dominant mode of transportation with 60% and 90% share of freight and passenger traffic, respectively (USDA, 2015). During 2014–15 quantity of oil imported was 189.238 MMT costing Rs. 8,64,875 crore (MPNG, 2014). To solve this issue Government of India took policy related decisions to promote biofuels. National Biofuel Policy was approved by Government of India on 24 December, 2009. The policy encourages use of biofuels (bioethanol and biodiesel) and set the target of replacing 20% of fossil motor fuel with biofuels by 2017. However at the same time National Biofuel Policy also states “Derive biofuel from inedible feedstock grown on degraded soils or wastelands unsuited to food or feed production, thus avoiding a possible conflict of fuel versus food security” (MNRE, 2009).
In India almost 70% of population depends directly or indirectly on agriculture and allied sectors. According to land use statistics 2011–12 the gross cropped area is 195.2 million hectares and cropping intensity is 138.7%. According to 4th advance estimate for the year 2013–14 estimated production (MT) for wheat is 95.91, rice 106.54, coarse cereals is 43.05 and sugarcane 50.02 (MoA, 2015). India is a world leader in agricultural production thus the waste generated is also huge in quantity. According to an estimate 686 MT gross residue is available in India annually from 26 crops. Out of this 234 MT i.e. 34% is available as surplus i.e. crop residue available for energy purpose (Hiloidhari et al., 2014). This huge quantity of agriculture waste is usually burnt in fields aggravating the problem of air pollution. Alternatively agricultural waste can also be turned into value added products. Bioethanol is one such product that can be produced from agricultural waste.
Agricultural waste also called lignocellulose biomass (LCB) is primarily composed of lignin, hemicellulose and cellulose. Cellulose is a polymer of glucose. If cellulose is depolymerised then glucose thus formed can be fermented into alcohol. The bioethanol generated from lignocellulose waste will be in accordance with National biofuel policies of India. In LCB cellulose is present as long fibers embedded in hemicellulose and lignin. Cellulose becomes inaccessible to enzymes due to the presence of lignin/hemicellulose. The complex structure needs to be disrupted before cellulose degrading microbes can act. Thus LCB requires pretreatment (Chang et al., 2014). A number of physical, chemical or physiochemical processes are used for pretreatment. Physical pretreatment involves mechanical comminution while in chemical pretreatments a variety of chemicals are used. Each treatment has its own benefits and drawbacks. Some of the common problems are production of toxic and inhibitory compounds and corrosive nature of chemicals used in the process (Carvalheiro et al., 2008, Mosier et al., 2005). Various organic solvents like methanol, acetone, ethylene glycol etc., are also used singly or in combination. The benefit of using solvents is obtaining pure lignin. However they are costly and extensive cleaning is required to remove their trace from raw material as they can inhibit the process of microbial saccharification and fermentation (Sun and Cheng, 2002). The choice of correct pretreatment method is very important for the success of the whole process. A pretreatment method that avoids the formation of inhibitors, lower enzyme dosage and shorter bioconversion time is preferable.
Apart from physical and chemical methods microbes have also been used. Many studies have reported the effectiveness of fungi in pretreatment of LCB (Pinto et al., 2012). Recently some studies have used bacteria capable of producing extracellular lignin/hemicellulose degrading enzymes for pretreatment (Priyadarshinee et al., 2015, Chang et al., 2014). Most of the studies have evaluated microbial pretreatment either in submerged fermentation (SmF) or solid state fermentation (SSF). However it is known that microbes behave differently in different culture conditions (Pinto et al., 2012). Also among the variety of crops grown in India, sugarcane produces maximum surplus residue (Hiloidhari et al., 2014). Thus this work focuses on pretreatment of sugarcane bagasse using Pandoraea sp. ISTKB under both SmF and SSF. The changes induced in sugarcane bagasse (agricultural waste) due to bacterial treatment were studied in detail. This process is enzymatic thus various lignocellulose degrading enzymes were studied. Due to the action of enzymes lignin, hemicelluloses and cellulose content changes. These changes were monitored by gravimetric measurement, SEM and FTIR. The purpose of pretreatment is to improve saccharification efficiency. Changes in saccharification efficiency were evaluated using commercial cellulases and congo red dye adsorption. Finally correlation analysis was done to help understand the relationship between saccharification efficiency and lignocellulose structure.
Section snippets
Analytical chemicals
All the chemicals used in the present study were of analytical grade purchased from Sigma–Aldrich (St. Louis, MO, USA), Merck (Darmstadt, Germany) or HiMedia unless stated otherwise. Enzymes Celluclast 1.5 L (C2730) and β-glucosidase (49,290) were purchased from Sigma–Aldrich (St. Louis, MO, USA). Sugarcane bagasse was obtained from juice vendor from local market.
Bacteria strain and culture conditions
Pandoraea sp. ISTKB was isolated from rhizospheric zone of trees growing in the backwaters of Alappuzha (9° 30′ N and 76° 23′ E),
Bacterial count
The growth of bacteria was monitored at regular intervals and in SmF and there was continuous increase in bacterial population from 7.45 × 105 CFU/g bagasse on 2nd day to 28.48 × 105 CFU/g bagasse on 9th day. In SSF the bacterial population increased to 10.37 × 105 CFU/g bagasse on 5th day and then remained almost constant. The number of bacteria was 2.7 times more in liquid culture than solid culture. Kabanova and coworkers compared the growth of Lactococcus lactis IL1403 in liquid and solid state
Conclusions
Submerged fermentation was more suitable for pretreatment of sugarcane bagasse by Pandoraea sp. ISTKB than solid state fermentation. High activity of enzymes (laccase, manganese peroxidases and xylanase) coupled with undetectable cellulases makes Pandoraea sp. ISTKB an ideal candidate for microbial pretreatment. There was 3.7-fold increase in saccharification efficiency after SmF pretreatment. High negative correlation between saccharification and lignin/hemicellulose content shows the
Acknowledgements
Madan Kumar is grateful to the Council of Scientific and Industrial Research (CSIR), New Delhi, India for SRF and Anjali Singhal thanks University Grants Commission (UGC), New Delhi, India for providing UGC-Women Post-doc fellowship. Authors thank UPOE, JNU, New Delhi for providing funds. We thank Dr Ruchita and Dr. Manoj Pratap Singh (Advanced Instrumentation Research Facility – AIRF, JNU, New Delhi) for SEM and FTIR analysis. We thank Mallika (Amity University, Noida) for her support during
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