Bioresource Technology 152 (2014) 38–45

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Bioresource Technology

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Probing the nature of AFEX-pretreated corn stover deriveddecomposition products that inhibit cellulase activity q

0960-8524/$ - see front matter � 2013 Published by Elsevier Ltd.http://dx.doi.org/10.1016/j.biortech.2013.10.082

Abbreviations: UC, ultra-centrifugation; UF, ultra-filtration; SPE, solid phaseextraction; AFEX-CS, AFEX-treated corn stover; AFEX-CS-E, aqueous extract ofAFEX-treated corn stover; UC-AFEX-CS-E, ultra-centrifuged extract of AFEX treatedcorn stover; UF-UC-AFEX-CS-E, ultra-centrifuged and ultra-filtered aqueous extractof AFEX-treated corn stover; MX, Multifect Xylanase; MP, Multifect Pectinase.

q AFEX (Ammonia Fiber Expansion) process is a trademark of MBI Incorporated,Lansing, MI, USA.⇑ Corresponding author. Tel.: +1 202 885 1772; fax: +1 202 885 1752.

E-mail address: acheh@american.edu (A.M. Cheh).

James F. Humpula a,b, Nirmal Uppugundla a,b, Ramin Vismeh b,c,d, Leonardo Sousa a,b,Shishir P.S. Chundawat a,b,e, A. Daniel Jones b,c,d, Venkatesh Balan a,b, Bruce E. Dale a,b, Albert M. Cheh f,⇑a Biomass Conversion Research Laboratory, Chemical Engineering and Materials Science, Michigan State University, Lansing, MI 48824, USAb DOE-Great Lakes Bioenergy Research Center (GLBRC), Michigan State University, East Lansing, MI 48824, USAc Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824, USAd Department of Chemistry, Michigan State University, East Lansing, MI 48824, USAe Department of Biochemistry, University of Wisconsin–Madison, Madison, WI 53706, USAf Department of Environmental Science, American University, Washington, DC 20016, USA

h i g h l i g h t s

� Cellulase inhibitors in AFEX-treated corn stover include lignin, Maillard products.� Fractionation by centrifugation, filtration, and solid phase extraction was done.� The largest materials were not inhibitory; most inhibitors were 3–10 kDa or <3 kDa.� A substantial fraction of inhibitors were hydrophobic and were removed by SPE.� Mass spectrometry saw many phenolic compounds in the hydrophobic fraction.

a r t i c l e i n f o

Article history:Received 7 August 2013Received in revised form 22 October 2013Accepted 25 October 2013Available online 1 November 2013

Keywords:AFEXUltra-filtrationFractionationCellulaseInhibition

a b s t r a c t

Sequential fractionation of AFEX-pretreated corn stover extracts was carried out using ultra-centrifuga-tion, ultra-filtration, and solid phase extraction to isolate various classes of pretreatment products toevaluate their inhibitory effect on cellulases. Ultra-centrifugation removed dark brown precipitates thatcaused no appreciable enzyme inhibition. Ultra-filtration of ultra-centrifuged AFEX-pretreated corn sto-ver extractives using a 10 kDa molecular weight cutoff (MWCO) membrane removed additional highmolecular weight components that accounted for 24–28% of the total observed enzyme inhibition whilea 3 kDa MWCO membrane removed 60–65%, suggesting significant inhibition is caused by oligomericmaterials. Solid phase extraction (SPE) of AFEX-pretreated corn stover extractives after ultra-centrifuga-tion removed 34–43% of the inhibition; ultra-filtration with a 5 kDa membrane removed 44–56% of theinhibition and when this ultra-filtrate was subjected to SPE a total of 69–70% of the inhibition wereremoved. Mass spectrometry found several phenolic compounds among the hydrophobic inhibitionremoved by SPE adsorption.

� 2013 Published by Elsevier Ltd.

1. Introduction using waste biomass, including corn stover or switchgrass grown

Cellulosic ethanol is a promising next generation liquid biofuelthat can avoid the food versus fuel and indirect land use debate by

on marginal lands unsuitable for farming (Dale et al., 2010). To pro-duce cellulosic ethanol, plant cell walls must first be hydrolyzed tomonomeric sugars (glucose, xylose, arabinose and mannose) viaenzymatic hydrolysis. However, plant cell walls are recalcitrantto enzymatic and microbial catalyzed deconstruction (Himmel,2008). Recalcitrance can be overcome by disrupting the plant cellwall by thermochemical pretreatments, e.g., steam, dilute acid, io-nic liquids, lime, and ammonia, which increases enzyme access tocellulose (Chundawat et al., 2011a). Currently, processing improve-ments are necessary to make cellulosic ethanol cost-effective;these improvements include optimizing pretreatment methods,reducing enzyme use, and improving fermentation methods/

J.F. Humpula et al. / Bioresource Technology 152 (2014) 38–45 39

strains (Alvira et al., 2010; Gao et al., 2011; Clarke, 2010; Lau andDale, 2009).

AFEX pretreatment cleaves lignin–carbohydrate ester linkages,and partially solubilizes and relocates lignin, phenolics, and hemi-cellulose fragments (oligosaccharides) to cell wall surfaces and in-creases porosity and access to cellulose, thereby increasingcellulose digestion rates (Chundawat et al., 2011b). However,pretreatments can also form degradation products that inhibit bio-mass hydrolyzing enzymes and fermentation microbes. To miti-gate this inhibition solvent-washing, nutrient supplementation,or detoxification may be needed, increasing processing costs(Wyman et al., 2011; Palmqvist and Hahn-Hagerdal, 2000; Kaziet al., 2010). Although AFEX produces fewer inhibitors than otherpretreatment methods, it can still inhibit enzymatic hydrolysisby releasing phenolics, lignin fragments and a number of othercompounds that have not yet been identified (Chundawat et al.,2010, 2011a; Humpula et al., 2011; Sharma et al., 2009).

The chemistry of AFEX pretreatment involves cleaving linkagesbetween lignin and hemicellulose and mobilizing lignin, while atthe same time creating products of Maillard and Amadori reactionsbetween nitrogen nucleophiles and sugar carbonyls, and manycomplex rearrangements that ensue. See Ledl and Schleicher(1990) for a comprehensive review of Maillard and Amadori reac-tions, which generate a host of products, including furans. Thecomplex mixture of water extractable inhibitors of Avicel digestionin an aqueous extract of AFEX-treated corn stover (AFEX-CS-E)could contain large and small molecules derived from cellulose,hemicellulose and lignin. Given this complex composition,AFEX-CS-E was fractionated with a simple batch processes usingdifferent separation principles to shed light on the nature of theinhibitors. Volumes were kept constant through each process toavoid dilution artifacts.

In the present study ultra-centrifugation (UC), ultra-filtration(UF) with 10, 5 or 3 kDa molecular weight cutoff (MWCO) filters,and solid phase extraction (SPE) were used to separate the inhibi-tors present in aqueous extracts of AFEX-treated corn stover(AFEX-CS); these were combined with cellulase activity assaysand liquid chromatography/mass spectrometry (LC/MS) to shedlight upon the physical and chemical properties of the inhibitors.The results indicate that materials with nominal molecular weights610 kDa account for a majority of the inhibition. Both hydrophobicmolecules (likely derived from lignin and Maillard reactions) andnon-hydrophobic (non-SPE binding) molecules could significantlycontribute to enzyme inhibition. Solid phase extraction could offera reusable means of removing and recovering inhibitors.

2. Methods

2.1. Biomass and AFEX pretreatment

Corn stover was obtained from Michigan State University Farms(East Lansing, MI, USA). The corn hybrid was NK 49-E3 (Syngenta,Basel, Switzerland). Hammer milled corn stover was dried at roomtemperature until the moisture content was <10% (dry weight ba-sis). Further fine milling was done using a Thomas Model 4 Wiley�

Mill (Swedesboro, NJ) to 4 mesh size (0.5 cm). The fine milled cornstover was stored at 4 �C in Zip-lock bags. AFEX pretreatment wasperformed as described previously (Balan et al., 2009): 60% bio-mass moisture content (dry weight basis; dwb), 130 �C, 15 min res-idence time, 1:1 ammonia to biomass loading (w/w).

2.2. Aqueous extraction

All extractions were performed using a Dionex Accelerated Sol-vent Extractor (ASE 200, Dionex Corporation, Sunnyvale, CA). The

moisture content of AFEX treated corn stover (AFEX-CS) prior toextraction was determined using a MA 35 gravimetric moistureanalyzer (Sartorius, Edgewood, NY). Three grams of AFEX-CS wastransferred into a 22 mL extraction cell. The biomass was extractedwith distilled water at 70 �C and 1500 psi for 7 min, yielding about30 mL extract and a final 10% solids loading equivalent extract(w/v). The extract was stored at 4 �C. The extracted biomass wasallowed to dry completely in an incubator oven at 50 �C for 24 hand the final dry weight was used to calculate percent mass loss.

2.3. Fractionation of water-soluble extractives

AFEX-CS extract (AFEX-CS-E) was ultra-centrifuged at 205,000gfor 16 h at 4 �C. Aliquots of the supernatant were ultra-filteredusing a 3 kDa, 5 kDa, or 10 kDa MWCO Vivaspin 20 centrifugal fil-ters (GE Healthcare, Waukesha, WI) spun at 5000g for 6 h at 4 �C.The retentate was re-suspended in a volume of distilled waterequal to that of the original supernatant. The filtrates and reten-tates were stored at 4 �C.

Analytical scale removal of inhibitors from AFEX-CS-E or itsfractions was done by elution through SPE cartridges (WatersCorp., Milford, MA): C-18 Sep-Pak (3 mL bed volume, 820 mg sor-bent) and Oasis HLB, MAX and MCX (6 mL bed volume, 500 mg sor-bent) cartridges. Three-cc C-18 Sep-Pak cartridges were wettedwith 4 mL HPLC grade methanol and 4 mL of water. About 16 mLof AFEX-CS-E was applied to each cartridge, 4 mL at a time,followed by washing with 4 mL water, and in succession, 5 mL vol-umes of methanol, isopropanol and DMSO; the first 2 mL of eluatewas discarded and the next 4 � 4 mL volumes were collected fortriplicate enzyme assay (one column per assay). The organic sol-vent eluates were also collected. One gram amounts of Bio-BeadsSM-2 (XAD-2 polystyrene–divinylbenzene resin), employed with-out initial washing (Bio-Rad Laboratories, Hercules, CA) wereplaced in fine mesh nylon bags and added to 10 mL volumes ofAFEX-CS-E in 50 mL plastic tubes. After shaking at 50 �C and200 rpm for 1 h the extract was tested for reduced enzymeinhibition.

Preparative scale SPE adsorption was done with UC-AFEX-CS-Eand with aliquots of 5 kDa MWCO filtrate, using Sep-Pak Vac35cc C-18 cartridges (Waters Corp., Milford, MA). The cartridgewas flushed with 34 mL of HPLC grade methanol and rinsed with34 mL of water. Up to 40 mL of AFEX-CS-E was applied and the first34 mL was discarded before collecting the rest for enzyme assaysand mass spectrometric analysis. All the fractionation steps, theirabbreviations and the enzymatic assays done with them are shownin the flow chart (Fig. 1).

2.4. Enzyme assays

The enzyme assays measured glucose release from Avicel PH-101, (Fluka, St. Louis MO) at 1% glucan loading in either 15 mL vials(5 mL reaction volume) or in micro-plates (0.5 mL reaction vol-ume) (Gao et al., 2010; Chundawat et al., 2008). The assays used6 mg protein/g of glucan of a commercial enzyme mixture, Accel-lerase 1000 (Genencor, Palo Alto, CA), with and without an addi-tional 3 mg/g glucan of Multifect Xylanase (MX) and 3 mg/gglucan Multifect Pectinase (MP) (Genencor, Palo Alto, CA). ThepH was maintained at 4.8 with 50 mM citrate buffer. When pres-ent, AFEX extract or fraction comprised 40% of the assay volume.Assay vials or micro-plates were incubated at 50 �C for 24 h at250 rpm. After hydrolysis, the micro-plates or aliquots from theassay vials were centrifuged and the supernatant was collected.Glucose release was assayed with glucose oxidase kits (R-Biop-harm, Marshall, MI) (Gao et al., 2010). Paired t-tests were used todetermine the significance of inhibition removal by the variousbatch processes.

Fig. 1. Flow chart of steps performed in inhibitor separation.

40 J.F. Humpula et al. / Bioresource Technology 152 (2014) 38–45

2.5. Acid hydrolysis and sugar analysis by HPLC

Sugar concentrations in AFEX-CS-E and fractions were mea-sured by HPLC. Monomeric sugar concentrations were measuredinitially, while oligosaccharide concentrations were calculated bytaking sugar concentrations measured after acid hydrolysis andsubtracting the monomeric sugar concentrations. For acid hydroly-sis, 500 lL of sample and 17.6 lL of 72% sulfuric acid were vor-texed in tightly capped 10 mL culture tubes that were placed in apreheated dry-block heater at 121 �C for 1 h and cooled on ice toroom temperature. Samples were filtered through 0.22 lm PES syr-inge filters (Whatman Inc., Piscataway, NJ), and were analyzedusing a Prominence HPLC system (Shimadzu, Columbia, MD) withan Aminex HPX-87H HPLC carbohydrate analysis column, a CationH guard column (Bio-Rad, Hercules, CA) and a RID-10A refractiveindex detector (Shimadzu, Kyoto, Japan). 5 mM H2SO4 was the mo-bile phase, run at a flow rate of 0.6 mL/min and 50 �C. The injectionvolume was 10 lL and the sample run time was 20 min.

2.6. LC/MS analysis

Degradation products from AFEX-CS-E and its fractions wereseparated on an Ascentis Express C-18 column (2.1 � 50 mm,2.7 lm, Sigma–Aldrich, St. Louis MO) using a 20-min solvent gradi-ent based on 0.15% aqueous formic acid (88%, J.T. Baker, USA) (sol-vent A) and methanol (solvent B). Eluted analytes were analyzedusing a LCT Premier TOF mass spectrometer for accurate massmeasurement (Waters Corporation, Milford, MA) and electro-sprayionization (ESI) in negative ion mode. Gradient conditions were asfollows: initial 99% solvent A/1% solvent B, held for 1 min; lineargradient to 1% solvent A/99% solvent B at 15 min and held until17 min; returning to the initial condition and held there until20 min. Injection volume, column temperature and flow rate were10 lL, 50 �C and 0.25 mL/min, respectively. Spectra were acquiredover m/z 50–1000.

Negative mode electrospray ionization was performed alongwith multiplexed collision induced dissociation (CID) by switchingamong two different AP1 voltages (15 and 65 V). Switching AP1voltages allows for quasi-simultaneous acquisition of molecularand fragment masses in a single analysis (Vismeh et al., 2013a).

Coumaroyl amide (m/z 162.0569), coumaric acid (m/z163.0410), feruloyl amide (192.0673) and three diferulates (m/z384.1096, 384.1080 and 384.1099) were confirmed via analysisof authentic standards.

3. Results and discussion

3.1. Inhibition of Avicel digestion by aqueous extracts of AFEX-CS

Fig. 2 shows Accellerase at 6 mg/g glucan released an average of44% of the glucose from 1% Avicel in 24 h, and adding 3 mg protein/g glucan each of MX and MP to Accellerase increased this to asignificantly larger average of 55% released (P < 0.001). Recentproteomic analysis has shown that MX and MP have significantendo-glucanase and exo-glucanase activity that may further com-plement cellulase activity present in Accellerase (Chundawat et al.,2011c). Avicel has also been reported to contain some residual xy-lan and the addition of xylanase causes glucose release to increasewell beyond what would be predicted from removal of just a few %of xylan and mannan (Várnai et al., 2011; Sloneker, 1971). The de-gree of increase we observed with added MX was virtually identi-cal to the increase observed by Várnai et al. (2011) when theyadded xylanase.

AFEX pretreatment cleaves linkages between lignin and hemi-cellulose and mobilizes lignin, while at the same time creatingproducts of Maillard and Amadori reactions. Maillard and Amadoritype reactions between ammonia and reducing sugar ends couldinclude both end products and intermediates consisting of sugarchains with altered reducing ends. Interference with exoglucanaseactivity operating at reducing ends of these intermediate productswould be one likely inhibitory mechanism (Chundawat et al.,2011a). The 130 �C reaction temperature was lower than what isneeded for caramelization to occur (Hodge, 1967).

The complex mixture of water extractable AFEX-CS-E inhibitorswas fractionated with a simple batch processes using different sep-aration principles to shed light on the nature of the inhibitors. Add-ing AFEX–CS-E reduced the 24 h glucan conversion of Avicel byAccellerase from 44% to 30%, for a (44–30)/44 = 32% inhibition(Fig. 2, P < 0.001). When MX and MP were added to Accellerase,glucan conversion of Avicel in the presence of AFEX-CS-E was re-duced from 55% to 42% (P < 0.001), for a lower 24% inhibition(Fig. 2); this lower inhibition would be consistent with either theadditional digestion achieved with the extra enzymes being insen-sitive to the inhibitors, or the enzymes destroyed some of theinhibitors.

AFEX-CS-E contained about 1.5 g dry weight/100 mL extract, or15% of the original weight of the biomass. Monomeric sugars to-taled 0.028 g/100 mL of AFEX-CS-E (0.12, 0.09 and 0.07 g/L for glu-cose, xylose and arabinose, respectively, data not shown) andoligosaccharides were 0.307 g/100 mL (mostly xylose residues,Table 1), making sugars 22% of the extracted dry weight in AFEX-CS.The small amounts of monomeric sugar concentrations remainedconstant throughout the inhibitor removing fractionation steps be-low (data not shown), so they are not associated with any inhibitorremoval in the fractionations. On the other hand, removal of oligo-saccharides, which include gluco-oligomers, xylo-oligomers, arabi-no-xylans, xylo-glucans and phenolic glucosides (Vismeh et al.,2013a; Chen et al., 2007), by some fractionation steps did coincidewith removal of inhibition.

Fig. 2. Percent glucan conversion of 1% (w/v) Avicel after 24 h with no additions, with added AFEX-CS-E and with added UC-AFEX-CS-E. Each point represents triplicateassays; error bars depict 95% confidence intervals digestion with Accellerase is on the left, with Accellerase supplemented with Multifect Xylanase and Multifect Pectinase ison the right.

J.F. Humpula et al. / Bioresource Technology 152 (2014) 38–45 41

3.2. Effect of UC on inhibition by AFEX-CS-E

Prior to embarking on these experiments AFEX-CS-E wasroutinely filtered through 0.22 lm filters immediately after itwas produced, but would still show slow aggregation and precipi-tation of particulate matter upon storage at 4 �C. It was hypothe-sized that these particles might be lignocellulose fragments ofcell walls that could bind and inhibit enzymes (Petridis et al.,2011; Várnai et al., 2010) and they might be removed by UC.

UC created three layers; a pellet, a dark brown lower layer withconsiderable cloudiness above it, and a yellow supernatant. Thesupernatant made up about 90% of the volume. The supernatantfrom UC-AFEX-CS-E showed no significant change in inhibitorconcentration with either enzyme mix despite removal of the UCpellet and material in the dark brown cloudy layer (Fig. 2). In a sin-gle complementary experiment, the dark brown cloudy layercaused the same amount of inhibition as the supernatant, so therewas no change in inhibitor concentration in this layer. Lack ofinhibitory activity associated with the materials that sedimentedwas unexpected.

In AFEX-CS-E, high molecular weight oligosaccharides com-posed primarily of hemicellulose and hemicellulose that is linked,either covalently or non-covalently, to lignin constituted up to aquarter of the dry mass of the extract, with the remainderconsisting largely of lignin and ash (Chundawat et al., 2010). The

Table 1Oligosaccharide concentrations in AFEX-CS-E and its ultra-centrifuged (UC) and ultra-filterepresent the differences in sugar concentrations before and after acid hydrolysis.

Sample Oligosaccharide

Glucose (g/L)

AFEX-CS-E 0.54 ± 0.05AFEX-CS-E: UC 0.39 ± 0.03AFEX-CS-E; UC; 10 kDa filtrate 0.30 ± 0.02AFEX-CS-E; UC; 5 kDa filtrate 0.30 ± 0.02AFEX-CS-E; UC; 3 kDa filtrate 0.20 ± 0.02AFEX-CS-E; UC; 10 kDa retentate 0.11 ± 0.02AFEX-CS-E; UC; 5 kDa retentate 0.19 ± 0.03AFEX-CS-E; UC; 3 kDa retentate 0.28 ± 0.03AFEX-CS-E; UC; SPE eluate 0.50 ± 0.03AFEX-CS-E; UC; 10 kDa filtrate; SPE eluate 0.32 ± 0.03AFEX-CS-E; UC; 5 kDa filtrate; SPE eluate 0.28 ± 0.02AFEX-CS-E; UC; 3 kDa filtrate; SPE eluate 0.21 ± 0.02

sedimenting pellet and cloudy layer contained about 25–35% ofthe total oligomeric glucose, xylose and arabinose in the originalAFEX-CS-E (Table 1), so an appreciable fraction of the sugarssedimented, with or without prior aggregation. Three types ofmaterial, lignin based components, Maillard intermediates withintact sugar chain moieties, and Maillard end products are likelyto be responsible for the brown color in AFEX-CS-E. Much ofthe color in the extract also appeared in the cloudy layer andpellet, with the UC supernatant becoming much less intenselycolored.

Sedimentation velocity rates are shown by the Svedberg equa-tion to depend on molecular weight, density (partial specific vol-ume) and geometry (friction). Cellulose and hemicellulose havedensities of 1.5–1.6 g/mL while lignin is much lower at 1.1–1.3 g/mL(Sun, 2005; Terashima et al., 2009). Linear oligosaccharides couldexhibit higher friction than lignin, so it is probably the highestmolecular weights of all three types of colored material thatsedimented and are not inhibitory.

3.3. Fractionation of AFEX-CS-E inhibitors by UF

The filtrates of UF-UC-AFEX-CS-E obtained with 10, 5 or 3 kDaMWCO ultra-filters caused significantly less inhibition than non-ultra-filtered UC-AFEX-CS-E. After ultra-filtration of UC-AFEX-CS-E with 10 kDa, or 5 kDa or 3 kDa MWCO filters, the remaining

red fractions. Oligosaccharide concentrations are measured after acid hydrolysis and

sugar concentrations

Xylose (g/L) Arabinose (g/L)

2.04 ± 0.16 0.49 ± 0.001.52 ± 0.11 0.31 ± 0.010.23 ± 0.05 0.07 ± 0.000.17 ± 0.05 0.06 ± 0.010.09 ± 0.04 0.03 ± 0.010.60 ± 0.03 0.21 ± 0.010.82 ± 0.05 0.28 ± 0.021.03 ± 0.08 0.34 ± 0.031.64 ± 0.04 0.46 ± 0.010.25 ± 0.06 0.08 ± 0.010.16 ± 0.05 0.06 ± 0.010.08 ± 0.03 0.03 ± 0.01

42 J.F. Humpula et al. / Bioresource Technology 152 (2014) 38–45

Accellerase inhibition was 72% (P < 0.001), 43% (P < 0.001) and 36%(P < 0.003), respectively, of the inhibition seen before UF (Fig. 3a–c). When Accellerase was supplemented with MX and MP, theremaining inhibition after UF of UC-AFEX-CS-E using 10 kDa,5 kDa or 3 kDa MWCO filters was 77% (P = 0.01), 54% (P = 0.002)and 40% (P < 0.001), respectively, of the inhibition before UF(Fig. 3a–c). As noted above, AFEX-CS-E inhibited digestion byAccellerase alone by 32%, while it inhibited digestion by Acceller-ase supplemented with MX and MP by a lesser 24%. Therefore, withUF removing more Accellerase inhibition than inhibition of Accel-lerase supplemented with MX and MP, the difference betweenthe degree of inhibition of Accellerase and Accellerase supple-mented with MX and MP appeared to diminish.

With only 15% of xylose residues and 23% of arabinose residuesremaining after UC followed by UF with a 10 kDa MWCO ultra-fil-ter (Table 1), while 72–77% inhibition remained, most xylan spe-cies (including arabino-xylans) with nominal molecular weightgreater than 10 kDa, including the presumably still higher MWspecies previously removed by UC, can be responsible for only asmall part of the overall inhibition. The same would be true ofany lignin-derived species removed by the 10 kDa ultra-filter.

Smaller MWCO ultra-filters removed more inhibition. The 5 kDaultra-filter removed 57% of the inhibition of Accellerase (P < 0.01)and 46% of the inhibition of Accellerase supplemented with MXand MP (P < 0.001), while the 3 kDa ultra-filter removed 64%(P < 0.003) and 60% (P < 0.003) of the inhibition of the two enzymesets, respectively. Greater inhibition removal by 5 kDa and 3 kDaultra-filters compared to the 10 kDa ultra-filter was also statisti-cally significant (P < 0.05) for both enzyme mixes. Significantlygreater removal of inhibition of Accellerase + MX and MP by3 kDa ultra-filtration compared to 5 kDa ultrafiltration wasobserved (P < 0.03), but the difference between 5 kDa and 3 kDaultra-filtration in reducing inhibition of Accellerase alone was notstatistically significant. The conclusion is that an appreciablefraction of the inhibition is due to intermediate-sized species

Fig. 3. Glucan conversion (Accellerase, left; Accellerase supplemented with Multifect Xy5 kDa and (c) 3 kDa molecular weight cut-off ultra-filters. Each point represents triplica

between 10 and 3 kDa nominal molecular weight. Table 1 showsthat the 5 and 3 kDa ultra-filters removed the large majority ofthe residual xylo-oligomers and arabino-xylans remaining afterultra-centrifugation and the 10 kDa ultra-filter and the 3 kDa ul-tra-filter in particular removed some oligomeric glucose. Theseintermediate-sized species could comprise part of the extra inhibi-tion removed by ultra-filters with MWCO less than 10 kDa. Ligninderived species with nominal molecular weights between 3 and10 kDa could also be responsible for a significant part of the inhi-bition caused by AFEX-CS-E.

The remaining 35–40% of the inhibition had nominal molecularweights below 3 kDa (Fig. 3c), and includes small molecules. Only4–6% of the xylose or arabinose containing oligosaccharides but37% of those containing glucose in AFEX-CS-E had nominal molec-ular weights 63 kDa (Table 1). Although fewer are produced byAFEX than by conventional acid pretreatment (Chundawat et al.,2010), furans produced during thermochemical pretreatment ofbiomass are also known to be inhibitory to various cellulase activ-ities (Jing et al., 2009).

The sums of the glucose and arabinose concentrations in the10 kDa UF filtrate and retentate approximated their concentra-tion before UF, but much of the xylose concentration could notbe accounted for in the sum of the filtrate and the retentate, indi-cating either sorption or mechanical trapping of xylose contain-ing species within the pores or sorption to the surface of thefilter. During UF, the membrane turned the same color as AFEX-CS-E, indicating lignin-derived or Maillard intermediate materialattached to the filter. Some of the missing xylose species couldhave been lost on the filter through attached lignin or as Maillardintermediates. The sums of the xylose concentrations in the UFfiltrates and retentates rose as the MWCO decreased, suggestingmore xylose containing species entered the larger pores and be-come adsorbed or mechanically trapped, with less of this occur-ring as the pore size diminished. The different MWCO filterswere made of the same material, so differential sorption to the

lanase and Multifect Pectinase, right) after UF-UC-AFEX-CS-E using (a) 10 kDa, (b)te assays; error bars depict 95% confidence intervals.

J.F. Humpula et al. / Bioresource Technology 152 (2014) 38–45 43

surface of the filter would be less of a factor than mechanicaltrapping or sorption in the pore.

It should be noted that the nominal MWCOs are only approxi-mate. Linear molecules larger than the MWCO can pass throughif they enter a pore end-on, while those smaller than the MWCOwill not if there is a charge interaction or an interaction with high-er MW materials that are trapped (Revchuk and Suffet, 2009).Retention of the vast majority of oligomeric xylose and much ofthe oligomeric glucose by the highest 10 kDa MWCO filter suggeststhat the oligomeric sugar fragments, including Maillard intermedi-ates, present in AFEX-CS-E are rather large (P10 kDa/DP 55). Thisagrees with published data suggesting that AFEX-CS has glucanchains of average length DP 6600 (Kumar et al., 2009).

3.4. Removal of AFEX-CS-E inhibitors by SPE

Passing UC-AFEX-CS-E through C-18 columns removed 41% ofthe inhibition of Accellerase (57% remaining, P < 0.004) and 35%of the inhibition of Accellerase supplemented with MX and MP(66% remaining, P < 0.04) (Fig. 4a). As with UF, SPE somewhat re-duced the difference in inhibition between the two enzyme sets.SPE adsorption removed relatively little of the glucose or arabinosespecies present in UC-AFEX-CS-E, so most of the inhibition re-moved by SPE are not associated with these two sugar residues.

The 5 kDa MWCO eluate from UF-UC-AFEX-CS-E was also sub-jected to C-18 SPE. After UF with a 5 kDa MWCO filter, 44% ofUC-AFEX-CS-E inhibition of Accellerase (Fig. 4b; not significant atthe P < 0.05 level) and 56% of UC-AFEX-CS-E inhibition of Acceller-ase with added MX and MP remained (Fig. 4b; significant,P < 0.007). When SPE adsorption followed UF, the remainingamounts of inhibition were nearly identical (31% and 30% respec-tively, Fig. 4b). Very little sugar was removed from the 5 kDa ul-tra-filtrate by SPE adsorption, so the inhibitors removed havelimited association with sugars.

Un-centrifuged AFEX-CS-E was also eluted through varioustypes of SPE media. Waters Oasis HLB Cartridges with mixedhydrophobic and polar adsorption, Oasis MAX cartridges with qua-ternary amine groups added to HLB, and Oasis MCX cartridges withsulfonate groups added to HLB all reduced the inhibition by a com-parable percentage to the C-18 silica-bonded cartridges. SPE re-moval appeared to rely mostly on hydrophobic rather than ionicsorption. A substantial amount of the brown-colored extractivescame through starting with the solvent front and the eluate stillcontained about 60% of the total inhibition (data not shown).Bio-Rad Biobeads SM-2 (purified XAD-2 hydrophobic styrene–divi-nyl-benzene resin) placed in nylon bags immersed in un-centri-fuged AFEX-CS-E also reduced inhibition, but with about 10% less

Fig. 4. Glucan conversion (Accellerase, left; Accellerase supplemented with Multifect Xsupernatant of UC-AFEX-CS-E and (b) the eluate from 5 kDa UF-UC-AFEX-CS-E. Each po

removal than when AFEX-CS-E passed through an SPE cartridgedirectly.

3.5. LC/MS analysis of AFEX-CS-E and fractions

LC/MS profiles for AFEX-CS-E and fractions appear in Supple-mentary Fig. S1. UF with 5 kDa MWCO filters removed approxi-mately 50% of the inhibition and hence the 5 kDa MWCO filtratewas chosen for comparative mass spectrometric analysis againstAFEX-CS-E. AFEX-CS-E is a complex mixture, but the profiles afterUC and UC followed by UF through a 5 kDa MWCO filter, were lar-gely identical to the profile for the original extract. The greatestchange occurred after subsequent C-18 SPE adsorption, whichmarkedly simplified the profile by removing a large number ofphenolic materials that eluted between 3 and 10 min.

Coumaric acid (m/z 163.0410, [M-H]� ion) was detected in allfour LC/MS chromatograms from the initial extract and after UC,UF and SPE adsorption. AFEX-CS-E itself is mildly basic (pH 7.5–7.8) due to residual ammonia, and most carboxylic acids in thismixture (with pKa’s of 4–5) would be negatively charged duringUC, UF and SPE adsorption. The coumaric acid anion is insuffi-ciently hydrophobic to be strongly retained on a C-18 cartridge.

Coumaroyl amide (m/z 162.0569, [M-H]�) was removed by SPEadsorption (Fig. S2) as were feruloyl amide (m/z 192.0673, [M-H]�),diferulates (with m/z values of 385.0941, 384.1096, 384.1080,384.1099 and 383.1265 for negatively-charged di-acids and acid-amides, uncharged di-amides) and many other phenolics deter-mined by integration of selected extracted ion chromatograms(e.g., m/z 237.0765, 329.0669, 415.1567, 517.1169, 593.1671,637.1796) (Figs. S3 and S4). In contrast to coumaric acid, any ofthese species containing negatively charged carboxylates werehydrophobic enough to be retained on the SPE column. The degreeof SPE retention of lignin monomers and dimers, reflecting thecountervailing effects of charged, polar and hydrophobic moietiesis consistent with the observed partial binding to SPE materialsof natural organic matter such as the more highly oxidized,polar and charged (compared to lignin) humic and fulvic acids(McElmurry et al., 2010), and would be relevant to the binding oflignin or Maillard materials that are larger than monomeric ordimeric small molecules.

Ions with m/z 415.1567, 517.1169, 593.1671, 329.0669 andmany other ions eluted in the retention time window of 3–10 min are considered phenolics for a couple of reasons. Accuratemass measurements of these ions show relative mass defects(RMD) values of 200–400 ppm which is the expected value for phe-nolic compounds owing to their relatively low hydrogen content(Chundawat et al., 2010; Stagliano et al., 2010). RMD (in ppm) is

ylanase and Multifect Pectinase, right) after solid phase extraction (SPE) of (a) theint represents triplicate assays; error bars depict 95% confidence intervals.

44 J.F. Humpula et al. / Bioresource Technology 152 (2014) 38–45

calculated as follows: [(exact mass-nominal mass)/(exactmass)] � 106 (Table S1). Another confirmation is the presence offerulate or coumarate as fragment ions in the MS/MS spectra ofsome of these phenolics (Fig. S5), which means the ions have aferulic or coumaric acid core with other moieties attached. Someof these ions were also identified as flavonoids, such as m/z637.1796 which is determined as methylated rutin. More detailedanalyses of AFEX-CS-E, including detailed analysis of nitrogen con-taining and Maillard reaction products and a complete character-ization of diferulates, have been recently published (Vismehet al., 2013b; Chundawat et al., 2010).

The brown color in the SPE eluate of AFEX-CS-E was probably amixture of lignin-derived material and Maillard products. Metha-nol and DMSO eluted deeply brown colored fractions that wouldcontain small phenolic compounds, such as those identified bymass spectrometry, somewhat larger lignin-derived materialsand most of the Maillard end products, all of which are hydropho-bic. After DMSO elution, the SPE cartridges still retained somebrown color, particularly around the top frit which was likely tobe large lignin derived or Maillard materials, trapped in the fritor strongly attached to the sorbent by multi-site attachment.

3.6. Process removal of enzyme inhibitors by SPE

Inhibitors produced by necessary pretreatments in turn need tobe removed but many removal methods are expensive (e.g. overliming or adsorption on activated carbon). C-18 SPE cartridgesare not meant to be reused or used with very large volumes, butXAD-type resins lend themselves to both types of use. Resinsplaced in mesh bags can be placed in large volumes to adsorbhydrophobic inhibitors if mixing and stirring is possible and theresins may be regenerated by washing with recoverable organicsolvents. Hydrophobic resins are especially effective at removingcolored Maillard intermediates and phenolic amides (Fig. S1).Small, charged species like phenolic acids were not removed bySPE, but if the extract pH were lowered to 4.0–4.5 for optimum cel-lulase activity, better removal of these species would be expected.

4. Conclusion

Inhibition of cellulose digestion is caused by AFEX productsfrom lignin, cellulose and hemicellulose, formed via Maillard andother reactions. About one quarter of inhibition is due to speciesP10 kDa, with an appreciable amount nominally between 3 and10 kDa. Much of the remaining inhibition is due to <3 kDa nominalmolecular weight phenolics, Maillard reaction products and possi-bly small lignin and oligosaccharide fragments. Removal of theseinhibitors will reduce enzyme costs. Aqueous ammonia extractionpretreatment (Liu et al., 2013) or reusable SPE adsorption could bealternatives to ultra-filtration.

Acknowledgements

This work was funded by Great Lakes Bioenergy ResearchCenter (http://www.greatlakesbioenergy.org/) supported by theU.S. Department of Energy, Office of Science, Office of Biologicaland Environmental Research, through Cooperative AgreementDEFC02-07ER64494 between the Board of Regents of theUniversity of Wisconsin System and the U. S. Department ofEnergy. The authors thank Dr. David Culver of the Departmentof Environmental Science, American University for his help withthe statistical analyses used in this manuscript.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.biortech.2013.10.082.

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