1. Use of Amaranth as Feedstock for Bioethanol Production Energy Postgraduate Conference 2013 Nqobile Xaba MSc student North-West University

2. Background Biomass is considered one of the viable renewable energy resource. Due to implications brought by the use of fossil derived fuels (elevation of greenhouse gases) Biomass based fuels include bioethanol and biodiesel Advantages of biofuels: wide availability, less impact on the environment, biodegradable. Concerns: food vs. fuel, Use of protected land for biomass production, Depleting local water supplies, Cost of technology manufacturing and maintenance. Lignocellulose as a "perfect" feedstock to address the above concerns 1. Balat, M. 2011. Production of Bioethanol from Lignocellulose Materials via the Biochemical Pathway: A Review. Energy Conversion and Management, 52:858-875. 2. Srirangan, K., Akawi, L., Moo-Young, M. & Chou, C.P. 2012. Towards Sustainable Production of Clean Energy Carriers from Biomass Resource. Applied Energy. Pretreatment enzymes yeast bacteria 2

3. Aims and Objectives Aim The aim of the project is to show the viability of amaranth as a sustainable feedstock for large scale bioethanol production in South Africa Objectives Develop method to convert cellulose and hemicellulose from amaranth lignocellulose into fermentable sugars Investigate the effect of parameters such as time, power and concentration of base on the pentose and hexose sugar yield during microwave pretreatment and hydrolysis Investigate the conversion of fermentable pentose and hexose sugars liberated from amaranth lignocellulose to ethanol using suitable micro- organisms Develop method to remove lignin from amaranth lignocellulose to be used for other bio-energy applications Compare the use of a conventional microwave to that of an industrial microwave when used for pretreatment and hydrolysis of lignocellulose material from amaranth 3

4. Grain Small ( 0.9 – 1.7 mm diameter White, gold, brown and pink Composition: protein (13.1 – 21%), fat (5.6 -10.9%), starch (48-69%), fibre (3.1-5.0%), ash (2.5-4.4%) Feedstock Leaves Composition: protein (15 %), Fat (7%), total carbohydrates(63%), Fiber (2.9 %), ash (2.6 %) Contains vitamins A, K, B6, C, riboflavin and folate, and also high in minerals (Ca, Fe, Mg, P, K, Zn, Cu, Mn) Description Grain amaranth; C 4 plant; Drought tolerant; Colour: maroon or crimson; Height: 1.5 m – 3 m Classification Order: Caryophyllales Family: Amaranthaceae Sub-family: Amaranthoideae Genus: Amaranthus Species: Amaranthus Cruentus Composition Moisture (6.23-6.71 %), protein (13.2-17.6 %dmN × 6.25), fats (6.3- 8.1%), fibre (3.4-5.3 %), ash (2.8- 3.6 %) 1. Teutonico, R.A. & Knorr, D. 1985. AMARANTH: Composition, properties, and applications of a rediscovered food crop. Ecological Agriculture Projects. 2. Mlakar, S.G., Turinek, M., Jakop, M., Bavec, M. & Bavec, F. 2010. Grain Amaranth as an Alternative and Perspective Crop in Temperate Climate. Journal of Geography, 5 (1):135-145. 3. South Africa Department of Agriculture forestry & fisheries. 2010. Amaranthus Production Guideline. Retrieved 17 March 2012. from http://www.nda.agric.za/docs/Brochures/Amaranthus.pdf 4

5. Base: KOH, NaOH, Ca(OH)2 Concentration: 1, 2, 3, 5 % (w/v) Power: 100, 180, 300 W Time: 5-25 minutes Biomass loading (5%, 3%, 1% (w/v) ) Cellulase enzymes: Celluclast (0.24 mL/g) Novozyme (0.25 mL/g) Tween 80 (1.25 g/L) 0.05 M trisodium citrate Buffer in 10 g/L sodium azide pH 4.8, 50 O C, 150 rpm, 48 h Saccharomyces Cerevisiae 30 O C, 120 rpm, 24-48 h Liquid fraction HPLC: Sugar monomers and total reduced sugars; Bioethanol UV: lignin, cell growth Solid Residue: FTIR, SEM Reduced sugars Solid residue Bioethanol Amaranth Acid hydrolysis 1.70% H 2 SO 4 (3mL), digest for 2 h 2.Dilute to 87 mL and auctoclave at 121 o C for 1 h Microwave Pretreatment Enzymatic hydrolysis Fermentation Analysis Separated into stem and roots, washed, dried, milled (<1 cm) Multiwave PRO microwave Power: 100-1000 W Time: 5 – 20 min Pressure: 60 bar vs Domestic microwave Method 5

8. Conclusion Composition analysis of amaranthus cruentus showed that amaranth is a viable feedstock for bioethanol production Alkaline pretreatment showed that Ca(OH) 2 is a proper base to use for high total sugar yields Increasing the power of the microwave increases total sugar yields and time does not affect concentration of sugars at low power (100W) The structural analysis (FTIR) of the biomass residue showed that these bases have an effect on removal of lignin 8

9. Acknowledgements My supervisor Prof. S. Marx for support and guidance Dr I. Chiyandzu and Mr C. Schabort for their assistance Mr G. van Rensburg for assistance in the laboratory Mrs E. De Koker for administrative assistance Dr A. Jordan for SEM analysis The biofuels group for support The National Research Foundation and North West University for funding. 9

10. References 1.Balat, M. 2011. Production of Bioethanol from Lignocellulose Materials via the Biochemical Pathway: A Review. Energy Conversion and Management, 52:858-875. 2.Srirangan, K., Akawi, L., Moo-Young, M. & Chou, C.P. 2012. Towards Sustainable Production of Clean Energy Carriers from Biomass Resource. Applied Energy. 3.Teutonico, R.A. & Knorr, D. 1985. AMARANTH: Composition, properties, and applications of a rediscovered food crop. Ecological Agriculture Projects. 4.Mlakar, S.G., Turinek, M., Jakop, M., Bavec, M. & Bavec, F. 2010. Grain Amaranth as an Alternative and Perspective Crop in Temperate Climate. Journal of Geography, 5 (1):135-145. 5.South Africa Department of Agriculture forestry & fisheries. 2010. Amaranthus Production Guideline. Retrieved 17 March 2012. from http://www.nda.agric.za/docs/Brochures/Amaranthus.pdf 10

11. 11 Thank you