(357a) Characterization of Feedstock Preprocessing of Energycane and Lipidcane for Effective Conversion of Cellulosic Bio-Oil into High-Quality Biodiesel

Biofuels and bio-products are green alternatives to the petro- fuels and -chemicals. Bioenergy crops like energycane, sugarcane, miscanthus and, sorghum have shown immense potential for biofuel production over recent years. These bioenergy crops primarily produce fermentable sugars which are further bio-processed to produce biofuel (especially bioethanol) and value-added bio-products. Therefore, we are still dependent on oil seeds like soybean and corn for biodiesel which, not to mention, also constitute part of human and animal food. Lignocellulosic biomass with high triacylglyceride (TAG) content and fatty acids rich in short, unbranched sand unsaturated side chains are suitable candidates for biodiesel production. Interestingly, to this end, breakthrough research is being done to genetically modify the metabolic pathway of bioenergy crops to enhance the accumulation of oilseed like energy-rich TAG molecules in their vegetative cells. Andrianov et al (2010) and Sanjaya et al (2013) have successfully reported an increase in TAG accumulation as high as 20 and 25-fold in Nicotiana tabacum and Arabidopsis thaliana, respectively, on constitutive co-expression of a cluster of genes responsible for TAG accumulation and reduction of lipid turnover (Andrianov et al., 2010; Sanjaya et al., 2013). This proof of concept was challenging to apply in high biomass C4 perennial grasses. C4 plants have high photosynthetic efficiency and hence, have more capacity to convert solar energy into chemical energy in the form of storage lipids. However, various research groups are working to apply the above-mentioned concept in different bioenergy crops i.e., sugarcane, energycane, miscanthus, and sorghum. Recently, Zale et al (2016) reported a 1.5-9.5 fold increase in TAG accumulation in vegetative tissues of sugarcane (Zale et al., 2016). The lipid producing lines of sugarcane are designated as lipidcane. A total lipid content of 0.9-1.3% was extracted from the juice and bagasse of the lipidcane lines. An analysis of the lipid composition showed that TAG constituted approximately 31 to 33% of total lipid (Huang et al., 2017). TAG being the platform compound for the production of high-quality biodiesel, lipidcane certainly exhibited potential as a cellulosic feedstock for biodiesel production. However, sugarcane is a seasonal crop and it cannot be grown around the year. Therefore, energycane, a specially designed high fiber hybrid of sugarcane is the next promising bioenergy crop for such purpose as it is more tolerant to extreme weather and drought conditions. Also, the United States is farm and factory ready for growing, transporting and processing sugars and oil from energycane (Long et al., 2015; Matsuoka, Kennedy, Santos, Tomazela, & Rubio, 2014). Since energycane is phylogenetically close to sugarcane, it would be relatively straightforward to use the proof of concept in energycane. Research efforts are underway for energycane to convert solar energy into energy-rich storage chemicals in form of TAGs to produce cellulosic bio-oil like lipidcane.

Indeed, the development of appropriate feedstock is the foremost step in the process. The second important step is the use of efficient feedstock preprocessing methods to enable minimal deterioration of the end product. For biodiesel production, the composition of TAG and free fatty acids play a deciding role in the final quality (Knothe, 2009). Thus, biomass pretreatment needs chemical-free and low severity physical methods to prevent the degeneration of oil. In the present study, we have performed a comprehensive analysis of two lignocellulosic biomasses namely, lipidcane and non- transgenic energycane (considering the unavailability of oilcane) for their suitability towards three most prevalent preprocessing i.e., Dilute acid, alkaline, and two-stage hydrothermal and mechanical pretreatment to design a fitting feedstock preprocessing. As mentioned, oilcane is still under greenhouse trials, so we tried to mimic oilcane by soaking grounded non-transgenic energycane in crude corn oil. Oil soaked biomass was incubated at 32 ºC for 2 months. Absorption of oil by energycane was pre-requisite for it to qualify as representative biomass. We used one dimensional time-domain¹H-Nuclear Magnetic Resonance (NMR) relaxometry to examine the absorption of oil. The analysis showed 31.35% absorption of oil by the biomass. Furthermore, we have established a solid‑state 1D ¹H-NMR method for precise, non-invasive and quick quantification of cellulosic oil. The concentrations of cellulosic oil measured using the NMR method were validated using hexane extraction. Both values were within a 10% deviation. The feedstocks were quantitatively and qualitatively analyzed for fermentable sugars and bio-oil.

We show categorically that each feedstock preprocessing has a different significance. Composition analysis showed that alkaline pretreated biomass has 3-fold and 5-fold less recalcitrant lignin than hydrothermal and dilute acid pretreated biomass, respectively. Alkaline pretreatment yielded 92.4% structural glucan and 81.1% structural xylan, two-stage hydrothermal and mechanical methods yielded 88.1% of structural glucan and 25.1% structural xylan. In contrast, dilute acid pretreatment performed with 2 % H₂SO₄ for 60 minutes at 121 degree Celsius could yield 10.6% of structural glucan and 9.7% of structural xylan after 72 hours of saccharification. However, dilute acid pretreatment yielded 0.291 g sugar per g dry biomass, predominantly xylose, a 3.34-fold higher total free sugar than liquid hot water pretreatment (0.067 g free sugar per g dry biomass). Dilute acid pretreatment generates 2.6-fold less inhibitors per g of dry biomass as compared to liquid hot water pretreatment. Both dilute acid and, two-staged hydrothermal and mechanical pretreatment had equivalent oil yield. However, oil and TAG composition obtained from each pretreatment varied. Alkaline pretreatment on biomass containing oil resulted in poor yields. It yielded 60% less oil as compared to the other two pretreatments. We assume that the saponification reaction between oil and alkali is the reason for the reduction in oil yield. We conclude that alkaline pretreatment solubilizes lignin and releases fermentable sugars to a great extent but is not suitable for oil containing biomass. Dilute acid pretreatment is suitable for obtaining xylose from hemicelluloses. It generates very little inhibitors. However, the two-staged hydrothermal and mechanical method is a low severity chemical-free feedstock preprocessing. It yields both fermentable sugars (glucose and xylose) and oil without much deterioration.