Abstract
Lignin is known to impede conversion of lignocellulose into ethanol. In this study, forage sorghum plants carrying brown midrib (bmr) mutations, which reduce lignin contents, were evaluated as bioenergy feedstocks. The near-isogenic lines evaluated were: wild type, bmr-6, bmr-12, and bmr-6 bmr-12 double mutant. The bmr-6 and bmr-12 mutations were equally efficient at reducing lignin contents (by 13% and 15%, respectively), and the effects were additive (27%) for the double mutant. Reducing lignin content was highly beneficial for improving biomass conversion yields. Sorghum biomass samples were pretreated with dilute acid and recovered solids washed and hydrolyzed with cellulase to liberate glucose. Glucose yields for the sorghum biomass were improved by 27%, 23%, and 34% for bmr-6, bmr-12, and the double mutant, respectively, compared to wild type. Sorghum biomass was also pretreated with dilute acid followed by co-treatment with cellulases and Saccharomyces cerevisiae for simultaneous saccharification and fermentation (SSF) into ethanol. Conversion of cellulose to ethanol for dilute-acid pretreated sorghum biomass was improved by 22%, 21%, and 43% for bmr-6, bmr-12, and the double mutant compared to wild type, respectively. Electron microscopy of dilute-acid treated samples showed an increased number of lignin globules in double-mutant tissues as compared to the wild-type, suggesting the lignin had become more pliable. The mutations were also effective for improving ethanol yields when the (degrained) sorghum was pretreated with dilute alkali instead of dilute acid. Following pretreatment with dilute ammonium hydroxide and SSF, ethanol conversion yields were 116 and 130 mg ethanol/g dry biomass for the double-mutant samples and 98 and 113 mg/g for the wild-type samples.
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Barrière Y, Ralph J, Méchin V, Guillaumie S, Grabber JH, Argillier O et al (2004) Genetic and molecular basis of grass cell wall biosynthesis and degradability. II. Lessons from brown-midrib mutants. Comptes Rendus Biologies 327:847–860
Bout S, Vermerris W (2003) A candidate-gene approach to clone the sorghum brown midrib gene encoding caffeic acid O-methyltransferase. Mol Genet Genomics 269:205–214
Chang VS, Holtzapple MT (2000) Fundamental factors affecting biomass enzymatic reactivity. Appl Biochem Biotechnol - Part A Enzyme Eng Biotechnol 84–86:5–37
Chen F, Dixon RA (2007) Lignin modification improves fermentable sugar yields for biofuel production. Nat Biotechnol 25:759–761
Corredor DY, Salazar JM, Hohn KL, Bean S, Bean B, Wang D (2008) Evaluation and characterization of forage sorghum as feedstock for fermentable sugar production. Appl Biochem Biotechnol, 1–16
Dien BS, Iten LB, Skory CD (2005) Converting herbaceous energy crops to bioethanol: a review with emphasis on pretreatment processes. In: Hou CT (ed) Handbook of industrial biocatalysis. CRC Press LLC, Boca Raton, FL pp 1–11
Dien BS, Jung HJG, Vogel KP, Casler MD, Lamb JFS, Iten L et al (2006) Chemical composition and response to dilute-acid pretreatment and enzymatic saccharification of alfalfa, reed canarygrass, and switchgrass. Biomass Bioenergy 30:880–891
Dien BS, Nagle N, Hicks KB, Singh V, Moreau RA, Tucker MP et al (2004) Fermentation of “Quick Fiber” produced from a modified corn-milling process into ethanol and recovery of corn fiber oil. Appl Biochem Biotechnol-Part A Enzyme Eng Biotechnol 115:937–949
Dien BS, Ximenes EA, O’Bryan PJ, Moniruzzaman M, Li XL, Balan V et al (2008) Enzyme characterization for hydrolysis of AFEX and liquid hot-water pretreated distillers’ grains and their conversion to ethanol. Bioresour Technol 99:5216–5225
Donohoe BS, Decker SR, Tucker MP, Himmel ME, Vinzant TB (2008) Visualizing lignin coalescence and migration through maize cell walls following thermochemical pretreatment. Biotechnol Bioeng 101:913–925
Dowe N, McMillan J (2001) SSF experimental protocols—lignocellulosic biomass hydrolysis and fermentation laboratory analytical procedure (LAP). In: DOE (ed) National renewable energy laboratory, p 19
Farrell AE, Plevin RJ, Turner BT, Jones AD, O’Hare M, Kammen DM (2006) Ethanol can contribute to energy and environmental goals. Science 311:506–508
Grabber JH (2005) How do lignin composition, structure, and cross-linking affect degradability? A review of cell wall model studies. Crop Sci 45:820–831
Hall MB (2001) Factors affecting starch analysis of feeds. Cooperative extension service. Institute of Food and Agricultural Sciences. University of Florida, Gainesville, FL
Hennessey S, Friend J, Dunson J, Tucker MP, Elander R, Hames B (2006) Integration of alternative feedstreams in biomass treatment utilization. In: E.I.D.d.N.a. Company (ed) U.S.
Jeoh T, Ishizawa CI, Davis MF, Himmel ME, Adney WS, Johnson DK (2007) Cellulase digestibility of pretreated biomass is limited by cellulose accessibility. Biotechnol Bioeng 98:112–122
Jung H-JG, Varel VH, Weimer PJ, Ralph J (1999) Accuracy of klason lignin and acid detergent lignin methods as assessed by bomb calorimetry, pp 2005–2008
Li L, Popko JL, Umezawa T, Chiang VL (2000) 5-Hydroxyconiferyl aldehyde modulates enzymatic methylation for syringyl monolignol formation, a new view of monolignol biosynthesis in angiosperms, pp 6537–6545
Mosier N, Wyman C, Dale B, Elander R, Lee YY, Holtzapple M (2005) Features of promising technologies for pretreatment of lignocellulosic biomass. Bioresour Technol 96:673–686
Oliver AL, Pedersen JF, Grant RJ, Klopfenstein TJ (2005) Comparative effects of the sorghum bmr-6 and bmr-12 genes: I. Forage sorghum yield and quality. Crop Sci 45:2234–2239
Oliver AL, Pedersen JF, Grant RJ, Klopfenstein TJ, Jose HD (2005) Comparative effects of the sorghum bmr-6 and bmr-12 genes: II. Grain yield, stover yield, and stover quality in grain sorghum. Crop Sci 45:2240–2245
Padmore JM (1990) Protein (crude) in animal feed - dumas method, method no. 968.06. In: Herlich K (ed) Official methods of analysis of the association of official analytical chemists, 15th edn. AOAC Inc., Arlington, VA, pp 71–72
Palmer N, Sattler S, Saathoff A, Funnell D, Pedersen J, Sarath G (2008) Genetic background impacts soluble and cell wall-bound aromatics in brown midrib mutants of sorghum. Planta 229:115–127
Pedersen JF, Toy JJ, Funnell DL, Sattler SE, Oliver AL, Grant RA (2008) Registration of BN611, AN612, BN612, and RN613 sorghum genetic stocks with stacked bmr-6 and bmr-12 genes. J Plant Reg 2:258–262
Perlack R, Wright L, Turhollow A, Graham R, Stokes B, Erbach D (2005) Biomass as feedstock for a bioenergy and bioproducts industry: the technical feasibility of a billion-ton annual supply. In: DOE (ed). Oak Ridge National Laboratory, pp 78
Porter KS, Axtell JD, Lechtenberg VL, Colenbrander VF (1978) Phenotype, fiber composition, and in vitro dry matter disappearance of chemically induced brown midrib (bmr) mutants of sorghum. Crop Sci 18:205–208
Saballos A, Vermerris W, Rivera L, Ejeta G (2008) Allelic association, chemical characterization and saccharification properties of brown midrib mutants of sorghum (Sorghum bicolor (L.) Moench). BioEnergy Res 1:193–204
Sarath G, Mitchell RB, Sattler SE, Funnell D, Pedersen JF, Graybosch RA (2008) Opportunities and roadblocks in utilizing forages and small grains for liquid fuels. J Ind Microbiol Biotechnol 35:343–354
Sattler SE, Saathoff AJ, Haas EJ, Palmer NA, Funnell-Harris DL, Sarath G et al (2009) A nonsense mutation in a cinnamyl alcohol dehydrogenase gene is responsible for the sorghum brown midrib 6 phenotype. Plant Physiol 150(2):584–595
Selig M, Weiss N, Ji Y (2008) Enzymatic saccharification of lignocellulosic biomass; laboratory analytical procedure. In: DOE (ed) National Renewable Research Laboratory, p 8
Sluiter A, Hames B, Ruiz R, Scarlata C, Sluiter J, Templeton D et al (2008) Determination of structural carbohydrates and lignin in biomass; laboratory analytical procedure. In: DOE (ed) National Renewable Energy Laboratory, p 16
van Soest PJ, Robertson JB, Lewis BA (1991) Methods for dietary fiber, neutral detergent fiber, and nonstarch polysaccharides in relation to animal nutrition, pp 3583–3597
Vermerris W, Saballos A, Ejeta G, Mosier NS, Ladisch MR, Carpita NC (2007) Molecular breeding to enhance ethanol production from corn and sorghum stover. Crop Sci 47:S-142-153
Vogel J (2008) Unique aspects of the grass cell wall. Curr Opinion Plant Biol 11:301–307
Vogel KP, Jung H-JG (2001) Genetic modification of herbaceous plants for feed and fuel. Critical Rev Plant Sci 20:15–49
Weng J-K, Li X, Bonawitz ND, Chapple C (2008) Emerging strategies of lignin engineering and degradation for cellulosic biofuel production. Curr Opin Biotechnol 19:166–172
Wescott PC (2007) Ethanol expansion in the United States. How will the agricultural sector adjust? Economic Research Service of the U.S.D.A., pp 1–18
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The authors would like to thank Ms. Patricia J. O’Bryan, Mr. Loren Iten, and John J. Toy for their fine technical assistance.
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Dien, B.S., Sarath, G., Pedersen, J.F. et al. Improved Sugar Conversion and Ethanol Yield for Forage Sorghum (Sorghum bicolor L. Moench) Lines with Reduced Lignin Contents. Bioenerg. Res. 2, 153–164 (2009). https://doi.org/10.1007/s12155-009-9041-2
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DOI: https://doi.org/10.1007/s12155-009-9041-2