The specificity of the Simons' staining technique for determining the specific surface area (SSA) of cellulose—the amount of cellulose in a pretreated substrate that is accessible to cellulases—was assessed over a range of cellulosic substrates. Both the Direct Blue (DB) dye and the high molecular weight (HMW) fraction (>100 kda) of the Direct Orange (DO) dye were shown to bind with high specificity to the cellulose rather than hemicellulose and/or lignin. The use of the DO dye by itself could also indicate differences in the accessibility/SSA of various types of cellulose resulting in an R2 value of 0.87 when plotted against enzymatic hydrolysis yields. The previously reported fluctuations in the ratio of DO:DB when the dyes were adsorbed to similar cellulosic substrates were shown to be primarily due to differences in the method used for the isolation of the high molecular weight fraction of the DO dye. A procedure for the enrichment of the >100 kDa HMW fraction of the DO using a minimum of two rounds of ultrafiltration is described. The ability of the modified assays to assess the specific surface area of pretreated and enzymatically hydrolyzed substrates showed good reproducibility.
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References
1.
MansfieldSD, MooneyC, SaddlerJN. Substrate and enzyme characteristics that limit cellulose hydrolysis. Biotechnol Prog, 1999; 15:804–816.
2.
ZhangP, LyndLR. Toward an aggregated understanding of enzymatic hydrolysis of cellulose: Noncomplexed cellulase systems. Biotechnol Bioeng, 2004; 88:797–824.
3.
ChandraRP, EsteghlalianA, SaddlerJN. Assessing substrate accessibility to enzymatic hydrolysis by cellulases. HuT. Characterization of Lignocellulosic Materials. Oxford, UK: Blackwell, 2008; 60–80.
4.
PuriV. Effect of crystallinity and degree of polymerization of cellulose on enzymatic saccharification. Biotechnol Bioeng, 1984; 26:1219–1222.
5.
HallM, BansalP, LeeJHet al.Cellulose crystallinity—a key predictor of the enzymatic hydrolysis rate. FEBS J, 2009; 277:1571–1582.
6.
GrethleinHE, ConverseAO. Common aspects of acid prehydrolysis and steam explosion for pretreating wood. Bioresour Technol, 1991; 36:77–82.
7.
PanX, XieD, YuRWet al.Pretreatment of lodgepole pine killed by mountain pine beetle using the ethanol organosolv process: Fractionation and process optimization. Ind Eng Chem Res, 2007; 46:2609–2617.
8.
SinitsynAP, GusakovAV, VlasenkoE. Effect of crystallinity and degree of polymerization of cellulose on enzymatic saccharification. Appl Biochem Biotech, 1991; 30:43–59.
9.
ChandraR, EwanickS, HsiehCet al.The characterization of pretreated lignocellulosic substrates prior to enzymatic hydrolysis, part 1: A modified Simons' staining technique. Biotechnol Prog, 2008; 24,5:1178–1185.
10.
StoneJ E, ScallanAM, DoneferEet al.Digestibility as a simple function of a molecule of a similar size to a cellulose enzyme. Adv Chem Ser, 1969; 95:219–241.
11.
GrethleinHE. The effect of pore size distribution on the rate of enzymatic hydrolysis of cellulosic substrates. Nat Biotechnol, 1985; 3:155–160.
12.
WangQQ, HeZ, ZhuZet al.Evaluations of cellulose accessibilities of lignocelluloses by solute exclusion and protein adsorption techniques. Biotechnol Bioeng, 2012; 109:381–389.
13.
HongJ, YeX, ZhangYH. Quantitative determination of cellulose accessibility to cellulase based on adsorption of a nonhydrolytic fusion protein containing CBM and GFP with its applications. Langmuir., 2007; 23:12535–12540.
14.
LaivinsGV, ScallanAM. The mechanism of hornification of wood pulps. BakerCF. Products of Papermaking. Transactions of the 10th Fundamental Research Symposium. Oxford UK: Pira International, 1993; 1235–1260.
15.
Del RioLF, ChandraRP, SaddlerJN. Fibre size does not appear to influence the ease of enzymatic hydrolysis of organosolv-pretreated softwoods. Bioresour Technol, 2012; 107:235–242.
16.
ChandraRP, EwanickSM, ChungPAet al.Comparison of methods to assess the enzyme accessibility and hydrolysis of pretreated lignocellulosic substrates. Biotechnol Lett, 2009; 8:1217–1222.
17.
ChandraRP, Au-YeungK, ChanisCet al.The influence of pretreatment and enzyme loading on the effectiveness of batch and fed-batch hydrolysis of corn stover. Biotechnol Prog, 2011; 27,1:77–85.
18.
ArantesV, SaddlerJN. Cellulose accessibility limits the effectiveness of minimum cellulase loading on the efficient hydrolysis of pretreated lignocellulosic substrate. Biotechnol Biofuels, 2011; 4:3.
19.
HuJ, ArantesV, SaddlerJN. The enhancement of enzymatic hydrolysis of lignocellulosic substrates by the addition of accessory enzymes such as xylanase: Is it an additive or synergistic effect?Biotechnol Biofuels, 2011; 4:36.
20.
KumarL, ChandraR, ChungPAet al.Can the same steam pretreatment conditions be used for most softwoods to achieve good, enzymatic hydrolysis and sugar yields?Bioresour Technol, 2010; 101,20:7827–7833.
21.
Del RioLF, ChandraRP, SaddlerJN. The effects of increasing swelling and anionic charges on the enzymatic hydrolysis of organosolv-pretreated softwoods at low enzyme loadings. Biotechnol Bioeng, 2011; 108,7:1549–1558.
22.
ChandraR, SaddlerJN. What are the key substrate factors which limit the hydrolysis of biomass by cellulases?32nd Symposium for Biotechnology for Fuels and Chemicals (presentation)Clearwater: FL, April19–22, 2010.
23.
YuX, MinorJL, AtallaRH. Mechanism of action of Simons' stain. Tappi J, 1995; 78,6:175–180.
24.
CowlingEB, KirkTK. Properties of cellulose and lignocellulosic materials as substrates for enzymatic conversion processes. Biotechnol Bioeng Symp, 1976; 6:95–123.
25.
YuX, MinorJL, AtallaRH. Mechanism of action of Simons' stain. Tappi J, 1995; 78,6:175–180.
26.
YuX, AtallaRH. A staining technique for evaluating the pore structure variations of microcrystalline cellulose powders. Powder Technol, 1998; 98,2:135–138.
27.
LuoX, ZhuJY. Effects of drying-induced fiber hornification on enzymatic saccharification of lignocelluloses. Enzyme Microb Tech, 2011; 48,1:92–99.
28.
KeshwaniDR, ChengJJ. Microwave-based alkali pretreatment of switchgrass and coastal bermudagrass for bioethanol production. Biotechnol Prog, 2010; 26,3:644–645.
29.
KooBW, TreasureTH, JameelHet al.Reduction of enzyme dosage by oxygen delignification and mechanical refining for enzymatic hydrolysis of green liquor-pretreated hardwood. Appl Biochem Biotechnol, 2011; 165,3-4:832–844.
30.
Del RioLF, ChandraRP, SaddlerJN. The effect of varying organosolv pretreatment chemicals on the physicochemical properties and cellulolytic hydrolysis of mountain pine beetle-killed lodgepole pine. Appl Biochem Biotech, 2010; 161,1–8:1–21.
31.
EsteghlalianAR, BilodeauM, MansfieldSDet al.Do enzymatic hydrolyzability and Simons' stain reflect the changes in the accessibility of lignocellulosic substrates to cellulase enzymes?Biotechnol Prog, 2001; 17,6:1049–1054.
32.
BerlinA, BalakshinM, GilkesNet al.Inhibition of cellulase, xylanase and beta-glucosidase activities by softwood lignin preparations. J Biotechnol, 2006; 125,2:198–209.
33.
EwanickSM, BuraR, SaddlerJN. Acid-catalyzed steam pretreatment of lodgepole pine and subsequent enzymatic hydrolysis and fermentation to ethanol. Biotechnol Bioeng, 2007; 98,4:737–746.
34.
NakagameS, ChandraRP, SaddlerJN. The effect of isolated lignins, obtained from a range of pretreated lignocellulosic substrates on enzymatic hydrolysis. Biotechnol Bioeng, 2010; 105,5:871–879.
35.
SluiterA, HamesB, RuizRet al.Determination of structural carbohydrates, lignin in biomass. National Renewable Energy Laboratory, 2008. www.nrel.gov/biomass/pdfs/42618.pdf. 2012June.
36.
RahikainenJ, MikanderS, MarjamaaKet al.Inhibition of enzymatic hydrolysis by residual lignins from softwood—study of enzyme binding and inactivation on lignin-rich surface. Biotechnol Bioeng, 2011; 108,12:2823–2834.
37.
BuraR, ChandraR, SaddlerJ. Influence of xylan on the enzymatic hydrolysis of steam-pretreated corn stover and hybrid poplar. Biotechnol Prog, 2009; 25,2:315–322.
38.
JeohT, IshizawaCI, DavisMFet al.Cellulase digestibility of pretreated biomass is limited by cellulose accessibility. Biotechnol Bioeng, 2007; 98:112–122.
39.
ChandraRP, BuraR, MabeeWEet al.Substrate pretreatment: The key to effective enzymatic hydrolysis of lignocellulosics?Adv Biochem Eng Biot, 2007; 108:67–93.
40.
GongaR, SunaY, ChenJet al.Effect of chemical modification on dye adsorption capacity of peanut hull. Dyes Pigments, 2005; 67,3:175–181.
41.
ShoreJ. Developments in direct dyes for cellulosic materials. Rev Prog Color Relat Top, 1991; 21,1:23–42.
42.
SannigrahiP, RagauskasAJ, MillerSJ. Lignin structural modifications resulting from ethanol organosolv treatment of loblolly pine. Energ Fuel, 2010; 24,1:683–689.
43.
SuteuaD, MalutanT, BilbaD. Removal of reactive dye Brilliant Red HE-3B from aqueous solutions by industrial lignin: Equilibrium and kinetics modeling. Desalination, 2010; 255,1–3:84–90.
44.
DrnovšekaT, PerdihA. Selective staining as a tool for wood fibre characterization. Dyes Pigments, 2005; 67,3:197–206.
45.
OverendRP, ChornetE. Fractionation of lignocellulosics by steam-aqueous pretreatments [and discussion]Philos T R Soc Lon A, 1987; 321:523–536.
