Comparative evaluation of some brown midrib sorghum mutants for the production of food grain and 2,-butanediol Yadhu N Guragain 1, K.S. Vinutha 2, G.S. Anil Kumar 2, Reggeany Barrios 1, P.V. Vara Prasad, P. Srinivasa Rao 2, Praveen V. Vadlani 1 1 Bioprocessing and Renewable Energy Laboratory, Grain Science and Industry, Kansas State University, USA 2 International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), India Department of Agronomy, Kansas State University (KSU), USA Pacific Rim Summit on Industrial Biotechnology and Bioenergy December 9, 2014
Bioprocessing and Renewable Energy Laboratory, KSU Dr. Praveen Vadlani: -Director Two Post-doc Four Ph D students One Master Student Undergraduate students Research Scientists Summer REU students Research Areas Biofuels cellulosic ethanol, biobutanol Biochemicals 2,-butanediol, lactic acid, succinic acid Co-product utilization animal feed, bioproducts Specialty chemicals flavor, pre and probiotics, fragrances, neutraceuticals Parr Reactor UV-VIS 5 L fermenter GC HPLC GC-MS 2
Bioprocessing of Lignocellulosic Biomass Lignin High Value aromatic compounds and macromolecules Pretreatment Source: - http://crf.sandia.gov/index.php/ Cellulose and Hemicellulose (Holocellulose) Hydrolysis (Enzymes) Monomer sugars Fermentation Product recovery A lot of challenges in each steps Fuels and Chemicals
Major Challenges for lignocellulosic biorefineries 1. Pretreatment of biomass Cost Production of inhibitors Phenolics, furan compounds, aliphatic acids Environmental issues 2. Cost of enzymes. Valorization of lignin 4. Handling and storage of bulky biomass Source: - http://crf.sandia.gov/index.php/ 4
In general, alkali pretreatment efficiency lignin content; however, this is not always true. Among five biomass here, douglas fir the most difficult and corn stover is the easiest for delignification Switchgrass is little more difficult than sorghum, and poplar is much more difficult than sorghum Each biomass must be separately evaluated! Douglas Fir (Softwood) 12 7 Switchgrass (Grass) 20 5 Corn stover (Grass) 4 1 Poplar (Hardwood) 17 40 21 22 Glucan Xylan Lignin Others Sorghum stalk (Grass) 2 5 29 22 17 29 16 19 20 21 5
Sorghum is a model energy crop because of its high photosynthetic efficiency, abiotic stress tolerance, and wide applications as food, feed, and fuels. bmr mutation of sorghum leads to change in composition of its stover bmr sorghum as a potential feedstock for bioenergy and biochemicals production needs investigation Brown midrib White midrib 6
Bioprocessing and Renewable Energy Laboratory, KSU Bacillus licheniformis 7
Yield (t/ha) 50% Flowering time (days) 7 6 5 Stover yield Grain yield 50% Flowering time Agronomy data 90 80 70 60 4 50 40 2 1 0 20 10 0 Early Hegari (EH) bmr6 (EH) bmr12 (EH) Atlas (AT) bmr6 (AT) bmr12 (AT) Kansas Collier (KC) bmr6 (KC) bmr12 (KC) 0 Genotype Both bmr mutations had longer flowering time and lower yields than their background, except stover yield of Kansas Collier 8
Composition (%, dry weight basis) 50 40 Composition of biomass Early Hegari (EH) bmr 6 (EH) bmr 12 (EH) Atlas (AT) bmr 6 (AT) bmr 12 (AT) Kansas Collier (KC) bmr 6 (KC) bmr 12 (KC) 0 20 10 0 Cellulose + Hemicellulose Lignin Extractives Biomass Component bmr mutation led to decrease in lignin (10 to 25%) and carbohydrate polymers (2 to 9%), and increase in extractives (6 to 4% )for Atlas and Kansas collier This was not true for bmr 12 of Early Hegari. 9
Amount (%, W/W)) 70 60 Total extractives Vs sugars Total extractives Sugars in water extraction Sugar proportion 50 40 0 20 10 0 Early Hegari (EH) bmr 6 (EH) bmr 12 (EH) Atlas (AT) bmr 6 (AT) bmr 12 (AT( Kansas Collier (KC) Genotype bmr 6 (KC) bmr 12 (KC) Significant proportion of extractives in soluble sugars (sucrose, glucose and fructose) Sugar proportion significantly higher in bmr mutants in all wild types, except,bmr 12 of Kansas Collier (consistent with composition data) 10
Sugar yield (%, g/g pretreated biomass) 90 80 70 60 50 40 0 20 10 0 Hydrolysis yield for monomer sugars from pretreated biomass Early Hegari (EH) bmr 6 (EH) bmr 12 (EH) Atlas (AT) bmr 6 (AT) bmr 12 (AT) Kansas Collier (KC) bmr 6 (KC) bmr 12 (KC) 0 10 20 0 40 50 Hydrolysis time (h) Both bmr mutats had higher sugar yield than wild type in all biomass, except bmr 12 of Kansas Collier 11
2,-butanediol yield (g/g sugar consumed) Shake flask fermentation using Bacillus licheniformis 0. 0.2 0.27 0.26 0.27 0.26 0.27 0.26 0.28 0. 0.1 0.27 0.1 0.0 Early Hageri (EH) bmr6 (EH) bmr12 (EH) Atlas (AT) bmr6 (AT) bmr12 (AT) Kansas Collier (KC) Axis Title bmr6 (KC) bmr12 (KC) Control 2,-butanediol yield was around 0. g/g sugars or lower (theoretical maximum 0.5) Significant byproduct formation: glycerol, acetic acid, ethanol 12
Overall mass balance: Stover to fermentable sugars Wild bmr 6 bmr 12 EH 17 15 EH 4 4 AT 7 14 10 Sugars AT Phenolics EH KC 8 17 16 KC AT KC Liquid stream Ground Biomass Pretreatment % increment in sugar yield EH AT KC bmr 6 6-1 5 bmr 12 19 16 14 Pretreated biomass Hydrolysis Released sugars EH 45 45 55 EH 4 7 41 Sugar extraction (in water) AT 45 4 45 AT 1 0 5 KC 44 9 4 KC 29 1 4 EH 15 19 EH 50 55 42 AT 11 21 21 AT 41 51 57 KC 17 28 26 KC 44 59 59 % increment in sugar yield EH AT KC bmr 6 10 25 bmr 12-16 8 1
Conclusion: Grain and stover yield significantly vary among bmr sorghum mutants 1.25% (W/V) NaOH is optimum for pretreatment of bmr sorghum bmr mutation alters the biomass composition, and improves hydrolysis efficiency of stalk. However, the effects significantly vary among sorghum varieties indicating background effect. bmr mutation leads to increase in non-structural sugar content in sorghum, which must be extracted before pretreatment Biomass derived sugar can be used for 2,-butanediol production using Bacillus licheniformis. 14
Acknowledgement: ICRISAT-KSU bmr sorghum project from USAID- CGIAR-US varsity linkage program The Small Business Innovation Research (SBIR), Navy, and Department of Defense, USA 15
Thank you for your attention 16