BioEnergy Research

, 2:179 | Cite as

High-Throughput Screening Techniques for Biomass Conversion

  • Stephen R. Decker
  • Roman Brunecky
  • Melvin P. Tucker
  • Michael E. Himmel
  • Michael J. Selig
Article

Abstract

High-throughput (HTP) screening of biomass or biomass-degrading enzymes, regardless of the desired outcome, is fraught with obstacles and challenges not typically faced in more traditional biotechnology. The enzyme systems are complex and synergistic and the substrate is highly heterogeneous, insoluble, and difficult to dispense. Digestions are often carried out for days at temperatures of 50°C or higher, leading to significant challenges regarding evaporation control in small well volumes. Furthermore, it is often desirable to condition or “pretreat” the biomass at extreme temperatures and/or pH to enhance enzyme digestibility. Once the substrate has been saccharified, evaluation of the extent and efficiency of conversion is made more difficult by time-consuming and tedious techniques used to measure the sugar products. Over the past decade or so, biomass researchers have creatively addressed these challenges by developing techniques to reduce biomass heterogeneity, uniformly distribute biomass samples at the small scale, pretreat the biomass at the small scale, quantitatively load these samples with enzymes, control evaporation of small reaction volumes for multiday incubations, and rapidly quantify the products. Other aspects of these measurements remain problematic and are being addressed. This review will address some of these challenges in detail, but more importantly, we will endeavor to educate the reader about the trials, tribulations, and pitfalls of carrying out HTP screening in biomass conversion research.

Keywords

Biomass conversion Biomass pretreatment Cellulase assay Lignocellulosic biomass High-throughput screening 

Abbreviations

BESC

BioEnergy Science Center

NREL

National Renewable Energy Laboratory

HTP

high throughput

US DOE

United States Department of Energy

MS

mass spectrometry

GC

gas chromatography

HPLC

high-performance liquid chromatography

UPLC

ultra-performance liquid chromatography

SBS

Society for Biomolecular Screening

AFEX

ammonium fiber expansion

DNS

dinitrosalicylic Acid

BCA

bicinchoninic Acid

MBTH

3-methyl-2-benzothiazolinonehydrazone

RI

refractive index

PAD

pulsed amperometric detection

Notes

Acknowledgments

The authors wish to thank Charles Wyman and his coworkers at the University of California-Riverside for the description and images of their high-throughput pretreatment reactor system. This work was supported by the DOE Office of Science, Office of Biological and Environmental Research through the BioEnergy Science Center (BESC), a DOE Bioenergy Research Center.

References

  1. 1.
    Aden A, Foust T (2009) Technoeconomic analysis of the dilute sulfuric acid and enzymatic hydrolysis process for the conversion of biomass to ethanol. Cellulose 16:535–545CrossRefGoogle Scholar
  2. 2.
    Yang B, Wyman CE (2008) Pretreatment: the key to unlocking low-cost cellulosic ethanol. Biofuels, Bioproducts, and Biorefining 2:26–40CrossRefGoogle Scholar
  3. 3.
    Dasari RK, Berson RE (2007) The effect of particle size on hydrolysis reaction rates and rheological properties in cellulosic slurries. Appl Biochem Biotechnol 137:289–299CrossRefPubMedGoogle Scholar
  4. 4.
    Chundawat SPS, Venkatesh B, Dale BE (2007) Effect of particle size based separation of milled corn stover on AFEX pretreatment and enzymatic digestibility. Biotechnol Bioeng 96:219–231CrossRefPubMedGoogle Scholar
  5. 5.
    Zhu JY, Wang GS, Pan XJ, Gleisner R (2009) Specific surface to evaluate the efficiencies of milling and pretreatment of wood for enzymatic saccharification. Chem Eng Sci 64:474–485CrossRefGoogle Scholar
  6. 6.
    Mais U, Esteghlalian AR, Saddler JN, Mansfield SD (2002) Enhancing the enzymatic hydrolysis of cellulosic materials using simultaneous ball milling. Appl Biochem Biotechnol 98:815–832CrossRefPubMedGoogle Scholar
  7. 7.
    Jung HJG, Jorgensen MA, Linn JG, Engels FM (2000) Impact of accessibility and chemical composition on cell wall polysaccharide degradability of maize and lucerne stems. J Sci Food Agric 80:419–427CrossRefGoogle Scholar
  8. 8.
    Selig MJ, Adney WS, Himmel ME, Decker SR (2009) The impact of cell wall acetylation on corn stover hydrolysis by cellulolytic and xylanolytic enzymes. Cellulose 16:711–722CrossRefGoogle Scholar
  9. 9.
    Berlin A, Maximenko V, Bura R, Kang KY, Gilkes N, Saddler J (2006) A rapid microassay to evaluate enzymatic hydrolysis of lignocellulosic substrates. Biotechnol Bioeng 93:880–886CrossRefPubMedGoogle Scholar
  10. 10.
    Esteghlalian AR, Bilodeau M, Mansfield SD, Saddler JN (2001) Do enzymatic hydrolyzability and Simons’ stain reflect the changes in the accessibility of lignocellulosic substrates to cellulase enzymes? Biotechnol Prog 17:1049–1054CrossRefPubMedGoogle Scholar
  11. 11.
    Sun FB, Chen HZ (2008) Enhanced enzymatic hydrolysis of wheat straw by aqueous glycerol pretreatment. Bioresour Technol 99:6156–6161CrossRefPubMedGoogle Scholar
  12. 12.
    Chundawat SP, Balan V, Dale BE (2008) High-throughput microplate technique for enzymatic hydrolysis of lignocellulosic biomass. Biotechnol Bioeng 99:1281–1294CrossRefPubMedGoogle Scholar
  13. 13.
    Decker SR, Adney WS, Jennings E, Vinzant TB, Himmel ME (2003) Automated filter paper assay for determination of cellulase activity. Appl Biochem Biotechnol 105–108:689–703CrossRefPubMedGoogle Scholar
  14. 14.
    Saeman JF (1945) Kinetics of wood saccharification: hydrolysis of cellulose and decomposition of sugars in dilute acid at high temperature. Ind Eng Chem 37:43–52CrossRefGoogle Scholar
  15. 15.
    Baugh KD, Mccarty PL (1988) Thermochemical pretreatment of lignocellulose to enhance methane fermentation.1. Monosaccharide and furfurals hydrothermal decomposition and product formation rates. Biotechnol Bioeng 31:50–61CrossRefPubMedGoogle Scholar
  16. 16.
    Chen RF, Lee YY, Torget R (1996) Kinetic and modeling investigation on two-stage reverse-flow reactor as applied to dilute-acid pretreatment of agricultural residues. Appl Biochem Biotechnol 57–8:133–146CrossRefGoogle Scholar
  17. 17.
    Selig MJ, Viamajala S, Decker SR, Tucker MP, Himmel ME, Vinzant TB (2007) Deposition of lignin droplets produced during dilute acid pretreatment of maize stems retards enzymatic hydrolysis of cellulose. Biotechnol Prog 23:1333–1339CrossRefPubMedGoogle Scholar
  18. 18.
    Montane D, Salvado J, Farriol X, Chornet E (1993) The fractionation of almond shells by thermomechanical aqueous-phase (TM-AV) pretreatment. Biomass Bioenergy 4:427–437CrossRefGoogle Scholar
  19. 19.
    Grohmann K, Torget R, Himmel ME (1986) Dilute acid pretreatment of biomass at high solids concentration. Biotechnol Bioeng Symp 17:135–151Google Scholar
  20. 20.
    Lloyd T, Wyman CE (2003) Application of a depolymerization model for predicting thermochemical hydrolysis of hemicellulose. Appl Biochem Biotechnol 105:53–67CrossRefPubMedGoogle Scholar
  21. 21.
    Michel FC, Nagle NJ, Weiss N, Elander RT (2006) Pretreatment screening protocol for bioethanol production from lignocellulosics. Paper presented at the Asabe Annual International Meeting, Portland, Oregon, July 9–12Google Scholar
  22. 22.
    Ximenes, EA, Kim, Y, Li, X, Meilan, R, Ladish, M, and Chapple, C. (2009) New method for fast detection of improved biodegradability in genetically modified plants. Paper presented at the 31st Symposium on Biotechnology for Fuels and Chemicals, San Francisco, CA, May 3–6Google Scholar
  23. 23.
    Zavrel M, Bross D, Funke M, Buchs J, Spiess AC (2009) High-throughput screening for ionic liquids dissolving (ligno-)cellulose. Bioresour Technol 100:2580–2587CrossRefPubMedGoogle Scholar
  24. 24.
    Studer M, DeMartini JD, McKenzie HL, Wyman CE (2008) Integrated high throughput pretreatment and enzymatic hydrolysis in 96 well plates. Paper presented at the AIChE Annual Meeting, Philadelphia, PAGoogle Scholar
  25. 25.
    Selig MJ, Tucker MP, Brunecky R, Himmel ME, Decker SR (2009) Parallel plate processing for high-throughput pretreatment and enzymatic saccharification of lignocellulosic materials. Paper presented at the 31st Symposium on Biotechnology for Fuels and Chemicals, San Francisco, CA, May 3–6Google Scholar
  26. 26.
    Hodge DB, Karim MN, Schell DJ, McMillan JD (2009) Model-based fed-batch for high-solids enzymatic cellulose hydrolysis. Appl Biochem Biotechnol 152:88–107CrossRefPubMedGoogle Scholar
  27. 27.
    Wyman CE, Dale BE, Elander RT, Holtzapple M, Ladisch MR, Lee YY (2005) Comparative sugar recovery data from laboratory scale application of leading pretreatment technologies to corn stover. Bioresour Technol 96:2026–2032CrossRefPubMedGoogle Scholar
  28. 28.
    Berlin A, Maximenko V, Gilkes N, Saddler J (2007) Optimization of enzyme complexes for lignocellulose hydrolysis. Biotechnol Bioeng 97:287–296CrossRefPubMedGoogle Scholar
  29. 29.
    Selig MJ, Knoshaug EP, Adney WS, Himmel ME, Decker SR (2008) Synergistic enhancement of cellobiohydrolase performance on pretreated corn stover by addition of xylanase and esterase activities. Bioresour Technol 99:4997–5005CrossRefPubMedGoogle Scholar
  30. 30.
    Selig MJ, Weiss N, Ji Y (2008) Enzymatic saccharification of lignocelluosic biomass. Technical Report NREL/TP-510-42629. Available at http://www.nrel.gov/biomass/pdfs/42629.pdf
  31. 31.
    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 98:112–122CrossRefPubMedGoogle Scholar
  32. 32.
    Selig M, Vinzant T, Himmel M, Decker S (2009) The effect of lignin removal by alkaline peroxide pretreatment on the susceptibility of corn stover to purified cellulolytic and xylanolytic enzymes. Appl Biochem Biotechnol 155:94–103CrossRefGoogle Scholar
  33. 33.
    Ghose TK (1987) Measurement of cellulase activities. Pure Appl Chem 59:257–268CrossRefGoogle Scholar
  34. 34.
    Xiao Z, Storms R, Tsang A (2004) Microplate-based filter paper assay to measure total cellulase activity. Biotechnol Bioeng 88:832–837CrossRefPubMedGoogle Scholar
  35. 35.
    Hodge DB, Karim MN, Schell DJ, McMillan JD (2008) Soluble and insoluble solids contributions to high-solids enzymatic hydrolysis of lignocellulose. Bioresour Technol 99:8940–8948CrossRefPubMedGoogle Scholar
  36. 36.
    Jorgensen H, Vibe-Pedersen J, Larsen J, Felby C (2007) Liquefaction of lignocellulose at high-solids concentrations. Biotechnol Bioeng 96:862–870CrossRefPubMedGoogle Scholar
  37. 37.
    Kristensen JB, Felby C, Jorgensen H (2009) Determining yields in high solids enzymatic hydrolysis of biomass. Appl Biochem Biotechnol 156:557–562CrossRefGoogle Scholar
  38. 38.
    Mohagheghi A, Tucker M, Grohmann K, Wyman C (1992) High solids simultaneous saccharification and fermentation of pretreated wheat straw to ethanol. Appl Biochem Biotechnol 33:67–81CrossRefGoogle Scholar
  39. 39.
    Bernfeld P (1955) Amylases, α and β. In: Colowick SP, Kaplan NO (eds) Methods in enzymology. Academic Press, New YorkGoogle Scholar
  40. 40.
    Miller GL (1959) Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 31:426–428CrossRefGoogle Scholar
  41. 41.
    Bailey MJ (1988) A note on the use of dinitrosalicylic acid for determining the products of enzymatic reactions. Appl Microbiol Biotechnol 29:494–496CrossRefGoogle Scholar
  42. 42.
    Redinbaugh MG, Turley RB (1986) Adaptation of the bicinchoninic acid protein assay for use with microtiter plates and sucrose gradient fractions. Anal Biochem 153:267–271CrossRefPubMedGoogle Scholar
  43. 43.
    Walker JM (1996) The bicinchoninic acid (bca) assay for protein quantitation. In: Walker JM (ed) The protein protocols handbook. Humana Press, Totowa, NJGoogle Scholar
  44. 44.
    Nelson N (1944) A photometric adaptation of the Somogyi method for the determination of glucose. J Biol Chem 153:375–380Google Scholar
  45. 45.
    Anthon GE, Barrett DM (2002) Determination of reducing sugars with 3-methyl-2-benzothiazolinonehydrazone. Anal Biochem 305:287–289CrossRefPubMedGoogle Scholar
  46. 46.
    Bergmeyer HU, Bernt E (1974) Determination with glucose oxidase and peroxidase. In: Bergmeyer HU (ed) Methods of enzymatic analysis, 2nd edn. Academic Press, New YorkGoogle Scholar
  47. 47.
    Kanauchi M, Bamforth CW (2003) Use of xylose dehydrogenase from trichoderma viride in an enzymic method for the measurement of pentosan in barley. J Inst Brew 109:203–207Google Scholar
  48. 48.
    Garber CC, Feldbruegge D, Miller RC, Carey RN (1978) Evaluation of the co-immobilized hexokinase/glucose-6-phosphate dehydrogenase method for glucose, as adapted to the Technicon SMAC. Clin Chem 24:1186–1190PubMedGoogle Scholar
  49. 49.
    Kunst A, Draeger B, Zeigenhorn J (1983) UV-methods with hexokinase and glucose-6-phosphate dehydrogenase. In: Bergermeyer HU (ed) Methods of enzymatic analysis, 3rd edn. Verlag Chemie, WeinheimGoogle Scholar
  50. 50.
    Iijima S, Mizutani F, Yabuki S, Tanaka Y, Asai M, Katsura T et al (1993) Ferrocene-attached-lysine polymers as mediators for glucose-sensing electrodes. Anal Chim Acta 281:483–487CrossRefGoogle Scholar
  51. 51.
    Hou S-F, Yang K-S, Fang H-Q, Chen H-Y (1998) Amperometric glucose enzyme electrode by immobilizing glucose oxidase in multilayers on self-assembled monolayers surface. Talanta 47:561–567CrossRefPubMedGoogle Scholar
  52. 52.
    Hale PD, Boguslavsky LI, Inagaki T, Karan HI, Lee HS, Skotheim TA et al (2002) Amperometric glucose biosensors based on redox polymer-mediated electron transfer. Anal Chem 63:677–682CrossRefGoogle Scholar
  53. 53.
    Smolander M, Livio H-L, Rasanen L (1992) Mediated amperometric determination of xylose and glucose with an immobilized aldose dehydrogenase electrode. Biosens Bioelectron 7:637–643CrossRefPubMedGoogle Scholar
  54. 54.
    Feng J, Himmel ME, Decker SR (2005) Electrochemical oxidation of water by a cellobiose dehydrogenase from Phanerochaete chrysosporium. Biotechnol Lett 27:555–560CrossRefPubMedGoogle Scholar
  55. 55.
    Hilden L, Eng L, Johansson G, Lindqvist SE, Pettersson G (2001) An amperometric cellobiose dehydrogenase-based biosensor can be used for measurement of cellulase activity. Anal Biochem 290:245–250CrossRefPubMedGoogle Scholar
  56. 56.
    Larsson T, Lindgren A, Ruzgas T, Lindquist SE, Gorton L (2000) Bioelectrochemical characterisation of cellobiose dehydrogenase modified graphite electrodes: ionic strength and ph dependences. J Electroanal Chem 482:1–10CrossRefGoogle Scholar
  57. 57.
    Slavin JL, Marlett JA (1983) Evaluation of high-performance liquid chromatography for measurement of the neutral saccharides in neutral detergent fiber. J Agric Food Chem 31:467–471CrossRefPubMedGoogle Scholar
  58. 58.
    Windham WR, Barton FE, Himmelsbach DS (1983) High-pressure liquid chromatographic analysis of component sugars in neutral detergent fiber for representative warm- and cool-season grasses. J Agric Food Chem 31:471–475CrossRefGoogle Scholar
  59. 59.
    Agblevor FA, Murden A, Hames BR (2004) Improved method of analysis of biomass sugars using high-performance liquid chromatography. Biotechnol Lett 26:1207–1211CrossRefPubMedGoogle Scholar
  60. 60.
    Slimestad R, Vagen IM (2006) Thermal stability of glucose and other sugar aldoses in normal phase high performance liquid chromatography. J Chromatogr A 1118:281–284CrossRefPubMedGoogle Scholar
  61. 61.
    Fox A, Morgan SL, Gilbart J (1989) Preparation of alditol acetates and their analysis by gas chromatography (gc) and mass spectrometry (ms). In: Biermann CJ, McGinnis GD (eds) Analysis of carbohydrates by glc and ms. CRC Press, Boca RatonGoogle Scholar
  62. 62.
    Kakehi K, Honda S (1989) Silyl ethers of carbohydrates. In: Biermann CJ, McGinnis GD (eds) Analysis of carbohydrates by glc and ms. CRC Press, Boca RatonGoogle Scholar
  63. 63.
    Englmaier P (1989) Carbohydrate triflouroacetates. In: Biermann CJ, McGinnis GD (eds) Analysis of carbohydrates by glc and ms. CRC Press, Boca RatonGoogle Scholar
  64. 64.
    Black GE, Fox A (1996) Recent progress in the analysis of sugar monomers from complex matrices using chromatography in conjunction with mass spectrometry or stand-alone tandem mass spectrometry. J Chromatogr A 720:51–60CrossRefGoogle Scholar
  65. 65.
    Jiannong Y, Baldwin RP (1994) Determination of carbohydrates, sugar acids and alditols by capillary electrophoresis and electrochemical detection at a copper electrode. J Chromatogr A 687:141–148CrossRefGoogle Scholar
  66. 66.
    Khandurina J, Blum DL, Stege JT, Guttman A (2004) Automated carbohydrate profiling by capillary electrophoresis: a bioindustrial approach. Electrophoresis 25:2326–2331CrossRefPubMedGoogle Scholar
  67. 67.
    Khandurina J, Olson NA, Anderson AA, Gray KA, Guttman A (2004) Large-scale carbohydrate analysis by capillary array electrophoresis: part 1. Separation and scale-up. Electrophoresis 25:3117–3121CrossRefPubMedGoogle Scholar

Copyright information

© US Government 2009

Authors and Affiliations

  • Stephen R. Decker
    • 1
  • Roman Brunecky
    • 1
  • Melvin P. Tucker
    • 2
  • Michael E. Himmel
    • 1
  • Michael J. Selig
    • 1
  1. 1.Biosciences CenterNational Renewable Energy LaboratoryGoldenUSA
  2. 2.National Bioenergy CenterNational Renewable Energy LaboratoryGoldenUSA

Personalised recommendations