(519a) Characterization of Transgenic Sugarcane (lipidcane 1566) and Its Potential As a Raw Material for Co-Production of Ethanol and Biodiesel

Biofuels and bio-products are green alternatives to the petro- fuels and -chemicals. Bioethanol and biodiesel are two major biofuels used in the transportation sector. The last decade has seen a 44% increase in fuel ethanol production to 15.8 billion gallons (Energy, 2021). 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 bioethanol. Plant oils have high energy density and are catalytically upgraded to biodiesel and a wide range of products, including chemicals (Lligadas et al., 2010; Salimon et al., 2012).

Considering the growing plant oil market to make biodiesel, challenging agricultural land use and high production costs make biodiesel production from current oil crops (soybean) unfeasible (Huang et al., 2016; Huang et al., 2017). Accumulation of TAG in the vegetative tissues such as leaves and stems of bioenergy grasses discounts major drawbacks of traditional oil resources (Long et al., 2015). TAG functions as a storage lipid in plants and recent achievements show that plants can be genetically modified to increase TAG content (James et al., 2010). Lignocellulosic biomass with high triacylglyceride (TAG) content and fatty acids rich in short, unbranched, and 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. Investigators 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. 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 and then investigated the feasibility of utilizing plant oils as a platform chemical to make biobased products (Huang et al., 2017). The economic analysis of biodiesel and ethanol co-production from lipid-cane showed that transgenic lipid-cane can produce 6700 liters of biodiesel from a hectare of land while soybeans can produce 500 liters. 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.

In this work, we characterized lipidcane (1566) grown in Illinois (IL) and Florida (FL) and compared it to the wildtype sugarcane grown under the same conditions. The composition of stem and leaves were determined using the National Renewable Energy Laboratory’s analytical procedure – NREL/ TP-510-42618. The stem and leaves of IL-grown lipidcane contained 16.87% and 27.37% extractives respectively. The stems of FL lipidcane contained approximately 10% more extractives and the leaves had nearly 13% less, compared to IL lipidcane leaves. The IL lipidcane leaves recorded the lowest glucan concentration at 25.80%, while FL lipidcane leaves contained 32.61% glucan. The lipidcane stems of FL and IL contained 29.72% and 33.03% glucan and xylan concentration at 17.49% and 16.78% respectively.

For biodiesel production, the composition of TAG and free fatty acids plays 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. Lipidcane and wildtype were pretreated at 180°C for 10 min, using a chemical-free hydrothermal pretreatment technique. The pretreated sample was disk milled and then enzymatically hydrolyzed to yield monomeric sugars. Subsamples from fresh stem and leaves, pretreated and enzymatically hydrolyzed material were centrifuged. The supernatant and solids were freeze-dried and then analyzed for fatty acids and TAG distribution. The lipid content was quantified by gas chromatographic (GC) analysis of fatty acid methyl esters (FAME). The gas chromatographic retention times relative to those of FAMEs of known structures were used to identify FAMEs. The identities were verified by gas chromatography-mass spectrometry. The free fatty acids and TAG got concentrated in the pretreated biomass and the solids remaining after hydrolysis. For instance, pretreated Illinois grown lipidcane contained 35.94 µg/mg dry weight (DW), which concentrated to 47.53 µg/mg DW in the leftover solids after hydrolysis. The stems from the same plant contained 35% less concentrated free fatty acids. TAG was present at 10.65 µg/mg in pretreated leaves and 3.21 µg/mg in pretreated stems of Illinois grown lipidcane. FL lipidcane contained 8.14 µg/mg of fatty acids and 3.12 µg/mg of TAG in leaves and 5.19 µg/mg of fatty acids and 4.23 µg/mg of TAG in stems.

The composition of cellulosic biomass along with the accumulation of TAG molecules is a pleiotropic effect, hence is expected to be controlled by different growth conditions. Total lipid and sugar recovery from transgenic Lipidcane 1566 grown in Illinois and Florida will be compared to investigate the favorable growth conditions that support the maximum accumulation of energy-rich TAG molecules. The data from enzymatic hydrolysis, composition, and lipid analysis will be combined, reflecting a broad overview of sugar, lipid, and other components (lignin, extractives, ash) distribution in a FL and IL lipidcane plant. Total biodiesel and bioethanol production per hectare of IL and FL lipidcane cultivation will be discussed. The total hectares of lipidcane cultivation necessary to replace soybean biodiesel obtained per hectare of agricultural land will be addressed using the lipid yield of IL and FL lipidcane. Finally, the improvements to in-situ cellulosic lipid production will be proposed to substitute a hectare of soybean cultivation and produce same quantity of biodiesel.

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[+] Correspondence to: Vijay Singh, Department of Agricultural and Biological Engineering, University of Illinois at Urbana-Champaign, Urbana, IL, 360 G Agricultural Engineering Science Building 1304 W. Pennsylvania Urbana, IL 61801. E-mail: vsingh@illinois.edu