Bioresource Technology 170 (2014) 53–61

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Bioresource Technology

journal homepage: www.elsevier .com/locate /bior tech

Investigations on a wheat bran biorefinery involving organosolvfractionation and enzymatic treatment

http://dx.doi.org/10.1016/j.biortech.2014.07.0680960-8524/� 2014 Elsevier Ltd. All rights reserved.

⇑ Corresponding author at: Department of Food Science and Technology,University of Natural Resources and Life Sciences, Muthgasse 18, 1190 Vienna,Austria. Tel.: +43 1 47654 6162; fax: +43 1 47654 6629.

E-mail address: michael.reisinger@boku.ac.at (M. Reisinger).

Michael Reisinger ⇑, Özge Tirpanalan, Florian Huber, Wolfgang Kneifel, Senad NovalinDepartment of Food Science and Technology, University of Natural Resources and Life Sciences, Muthgasse 18, 1190 Vienna, AustriaChristian Doppler Laboratory for Innovative Bran Biorefinery, Austria

h i g h l i g h t s

� Ethanol organosolv fractionation of wheat bran was investigated in detail.� Enzymatic hydrolysis of the obtained fractions gave a glucose yield of 75%.� Proteins could be extracted almost quantitatively.� During the lignin precipitation step, proteins partially coprecipitated.

a r t i c l e i n f o

Article history:Received 10 June 2014Received in revised form 16 July 2014Accepted 17 July 2014Available online 24 July 2014

Keywords:Wheat branBiorefineryOrganosolvEnzymatic hydrolysis

a b s t r a c t

The present study elucidates the organosolv treatment of wheat bran, the major by-product of the millingindustry. The influence of temperature (160–200 �C) and ethanol concentration (30–60% (w/w)) at agiven process time of 30 min was investigated. Enzymatic treatments of the organosolv extracts includingsolid residues led to an overall glucose yield of 75%. The conversion of hemicelluloses into xylose andarabinose was approximately 60% and 45%, respectively. Proteins could be almost completely dissolved,however, practically no free amino acids were obtained. Surprisingly, only around 30% of lignin and 65%of minerals were dissolved. Severe treatment conditions induced the disintegration of fat into glyceroland fatty acids as well as the formation of sugar degradation products. During the lignin precipitationstep, proteins partially coprecipitated.

� 2014 Elsevier Ltd. All rights reserved.

1. Introduction

As the world faces the progressive depletion of fossil resources,the conversion of biomass within biorefineries is seen as a possibil-ity to essentially contribute in particular to the continuous supplyof materials in the future. In order to avoid the competition withfood production, the utilization of raw materials of high lignocellu-losic content is an interesting option. Besides wood and non-foodcrops, agricultural residues like straw and corn stover as well asother by-products of various origins are of high interest as feed-stocks. Wheat bran represents such a by-product, which accruesin enormous quantities during the production of white wheat flour.It is estimated that 150 million tons are produced per yearworldwide (Prückler et al., 2014). Currently wheat bran is mainlyused as a low value ingredient in animal feed.

As biomass generally shows a complex composition, a separa-tion into certain fractions can be advantageous for subsequent pro-cessing (Kamm and Kamm, 2004). In the case of wheat bran, itscomposition (Table 1) with relatively high levels of protein andstarch as well as smaller contents of lignocellulose differs from thatof typical lignocellulosic raw materials (Yu et al., 2008). While theglucose extraction from the starch of wheat bran by means of com-mercially available amylolytic enzymes is performed ratheruncomplicated, the recalcitrance of the dense lignocellulosic frac-tion hinders its easy conversion into free sugars. In order toincrease the accessibility of the cellulose for hydrolytic enzymes,a pretreatment seems to be inevitable. In this context, hydrother-mal and acidic pretreatments of wheat bran were already investi-gated in a number of studies (Aguedo et al., 2013; Choteborskaet al., 2004; Favaro et al., 2012; Kabel et al., 2002; Kataoka et al.,2008; Palmarola-Adrados et al., 2005; Reisinger et al., 2013; Roseand Inglett, 2010; Tirpanalan et al., 2014; van den Borne et al.,2012). The present work deals with the so-called ethanol organo-solv treatment of wheat bran. This process offers the opportunityto separate the biomass into the fractions cellulose, hemicellulose

Table 1Chemical composition of the untreated wheat bran.

Analyte g per 100 gdry mass

Amino acid g per 100 gdry mass

Total carbohydrates 56.9 Aspartic acid 1.84Starch 9.1 Threonine 0.64Glucose (total) 25.4 Serine 0.78Xylose (total) 20.3 Glutamic acid 3.62Arabinose (total) 9.3 Proline 1.05Galactose (total) 1.9 Glycine 1.08

Crude protein 13.2 Alanine 0.96Klason lignin 8.0 Valine 0.80Ash 7.0 Isoleucine 0.52Crude fat 4.3 Leucine 1.10

Tyrosine 0.52Phenylalanine 0.67Histidine 0.51Lysine 0.78Arginine 1.52

54 M. Reisinger et al. / Bioresource Technology 170 (2014) 53–61

and lignin due to differences in their solubility in a mixture ofwater and an organic solvent, commonly ethanol (Zhao et al.,2009). In the case of wheat bran, this technique could also leadto an extraction of ethanol soluble proteins, in particular prolamin(Idris et al., 2003). By reducing the ethanol concentration after thepretreatment, lignin can be precipitated, which represents an addi-tional advantage of this process. Based on a detailed characteriza-tion of the obtained fractions, in the end, the present study enablesa comparison between the hydrothermal (Reisinger et al., 2013)and the organosolv treatment of wheat bran.

2. Methods

2.1. Raw material

Wheat bran was kindly provided by the VonWiller mill (Schwe-chat, Austria) and directly used without further comminution.

2.2. Organosolv fractionation

The organosolv pretreatment was performed in a 2 L pressure-tight agitator vessel with a double jacket heated by thermal oil(Kiloclave type 3, Büchiglasuster, Switzerland). For each trial,1160 mL ethanol/water-solvent (30%, 45% and 60% (w/w) ethanol)were added to 290 g wheat bran. The slurries were heated to thetarget temperatures (160–200 �C) and held for a reaction time of30 min. During the whole procedure, the slurries were continu-ously stirred at 400 rpm. The obtained mixtures were centrifugedat 7000 rpm equal to an rcf of 10,722g (Sorvall Evolution RC),and the lignin was precipitated from the supernatants by dilutionwith deionized water to a final ethanol concentration of 10% (w/w).The lignin precipitate was separated by centrifugation under abovedescribed conditions.

2.3. Enzymatic hydrolysis

In order to gain monomeric sugars, pretreated lignocellulosicbiomasses have to undergo an additional hydrolysis step, in thiswork by application of amylolytic, cellulolytic and hemicellulolyticenzymes. Cellulase (0.017 units/mg), endo-1,3(4)-ß-glucanase(0.017 units/mg) and endo-1,4-ß-xylanase (1 unit/mg) from Trich-oderma longibrachiatum were obtained from Sigma Aldrich. Thestarch degrading commercial enzymes Termamyl 120 L and AMG300 L were received from Novozymes. An enzyme cocktail display-ing concentrations of 0.04 g/mL cellulase, 0.04 g/mL ß-glucanase,

0.01 g/mL xylanase, 5 lL/mL Termamyl and 5 lL/mL AMG, dis-solved in sodium citrate buffer (0.1 mol/L, pH 4.8), was used.

Both the solid residue of the organosolv pretreatment and thesupernatant of the lignin precipitation step were used for enzy-matic hydrolysis. The former was first washed four times with eth-anol at concentrations equivalent to those used for the organosolvpretreatment itself and then once with deionized water. To 1 gwashed residue, 1 mL of the enzyme cocktail and 9 mL sodium cit-rate buffer was added. This equals a cellulase activity of around17 units per g cellulose. To 5 mL supernatant of the lignin precipi-tation, 1 mL of the enzyme cocktail and 4 mL sodium citrate bufferwas added. Supplementation with 10 mg/mL of NaN3 preventedmicrobial growth. The mixtures were prepared in duplicate andincubated for 72 h at 40 �C and shaken regularly. Since in the pres-ent study emphasis was paid on the organosolv fractionation, theenzymatic process was not further optimized. However, to ensurea high degree of carbohydrate hydrolysis, the enzymes wereapplied in excess.

2.4. Analytical methods

All measurements were performed with the supernatants of thecentrifuged organosolv-slurries before and after the lignin precip-itation step as well as with the enzymatically hydrolyzed samples.The mentioned fractions were used for analysis of free and totalsugars, degradation- and by-products, free and total amino acids,dry matter and ash content, ion chromatography as well as fornitrogen determination using the Kjeldahl method. The proteincontent was calculated using a nitrogen-to-protein-factor of 5.26(Tkachuk, 1969). Dry matter and the ash content were determinedaccording to the Standard Laboratory Analytical Procedures (LAP)for biomass analysis provided by the National Renewable EnergyLaboratory (Sluiter and Sluiter, 2011a,b). Unless stated otherwise,all analytical procedures were performed at least in duplicate.The accuracy of the obtained data was ±10%.

2.4.1. Carbohydrate and degradation product analysisThe sugar composition of the original bran, free and total sugar

concentrations (glucose, xylose, arabinose and galactose) as well asthe levels of degradation products (furfural, hydroxymethylfurfur-al, glycerol, acetic and levulinic acid) in solution were measuredaccording to the Standard Laboratory Analytical Procedures (LAP)for biomass analysis provided by the National Renewable EnergyLaboratory (Sluiter and Sluiter, 2011a,b). The sugars were sepa-rated using a Phenomenex Rezex RPM-Monosaccharide column,the degradation products using a Phenomenex Rezex ROA-OrganicAcid column, each equipped with the appropriate precolumn. Thestarch content of the original bran was determined using theMegazyme Total Starch (AA/AMG) enzyme assay kit, according toAOAC 996.11.

2.4.2. Amino acid analysisThe amino acids were analyzed via HPLC using pre-column

derivatization with ortho-phthalaldehyde (OPA) and 9-fluorenylm-ethyl chloroformate (FMOC) and fluorescence detection (Cigicet al., 2008; Henderson and Brooks, 2010).

The mobile phases were prepared as follows: mobile phase A:1.4 g anhydrous Na2HPO4, 3.8 g Na2B4O7�10H2O and 32 mg NaN3

were dissolved in 1 L demineralized water, and the pH wasadjusted to 8.2 with concentrated HCl; mobile phase B: a mixtureof acetonitrile:methanol:water was prepared equal to a volumetricratio of 45:45:10.

The following reagents were used for the derivatization proce-dure: 0.1 M borate buffer in water with pH 9.9; OPA-reagent:10 g/L OPA dissolved in 0.02 M borate buffer (pH 9.9) with 0.8%of 3-mercaptopropionic acid; FMOC-reagent: 5 mg/mL FMOC

M. Reisinger et al. / Bioresource Technology 170 (2014) 53–61 55

dissolved in acetonitrile; injection diluent reagent: 100 mL ofmobile phase A mixed with 0.4 mL concentrated H3PO4.

The pre-column derivatization was performed directly in theautosampler and accomplished by the programmable injector unit:2.5 lL of 0.1 M borate buffer were mixed with 1 lL sample bydrawing air for 5 times. After 0.2 min, 0.5 lL OPA reagent, 0.4 lLFMOC reagent and 32 lL injection diluent reagent were added,mixed 8 times by drawing air, and finally injected after a waitingtime of 0.1 min. The separation was carried out using an AgilentZORBAX Eclipse Plus C18 column and the appropriate precolumn.The gradient program started with 98% phase A at a flow rate of0.64 mL/min and changed linearly between 0.5 min and 20 minto 43% phase A. Then, from 20.1 to 23.5 min, 100% phase B wasapplied before changing back to the initial concentration of 98%phase A until the end of the program at 25 min. The detectionwas done via fluorescence using different parameters for OPA-(ex230 nm, em 450 nm) and FMOC-derivatives (ex 266 nm, em305 nm). Amino acids were quantified using norvaline andsarcosine as internal standards, which were added prior toderivatization.

For the measurement of the total amino acids, a protein hydro-lysis step was performed according to the community methods ofanalysis for the determination of amino acids in feedingstuffs(European Communities, 1998). Liquid samples were concentratedprior to hydrolysis in a drying oven at 60 �C in order to preventadditional dilution of the acid.

2.4.3. Ion chromatographyThe determination of cations was executed as follows: 25 lL of

the diluted and filtered (0.2 lm) sample was injected into theHPLC. The separation was performed on a Dionex RFIC IonPacCS12A column with the appropriate precolumn. The elution wasisocratic with 20 mmol/L methanesulfonic acid at a flow rate of1 mL/min (DIONEX, 2005).

Prior to the determination of anions, protein precipitation wasperformed using Carrez reagents. For this purpose, a sample ali-quot of 500 lL was mixed thoroughly with 40 lL Carrez A solution(2.7 g potassium hexacyanoferrate per 100 mL demineralizedwater). Then, 40 lL Carrez B solution (5.5 g zinc acetate per100 mL of 90 mmol/L acetic acid) were added, mixed again andallowed to stand for 10 min. Subsequently, after adding 420 lLdemineralized water, the solution was filtered through a 0.2 lmsyringe filter. An aliquot of 25 lL of the diluted sample was thenused for HPLC separation using a Dionex RFIC IonPac AS14A col-umn with the appropriate precolumn. The elution was isocraticwith 8 mmol/L Na2CO3 and 1 mmol/L NaHCO3 in demineralizedwater at a flow rate of 1 ml/min (DIONEX, 1996).

The anion and cation detection was performed based on inte-grated conductivity measurement.

2.4.4. Klason ligninThe determination of Klason lignin of the unprocessed wheat

bran and of the solid residues of the organosolv pretreatmentswas performed in accordance to Sluiter and Sluiter, 2011a. The lat-ter was first washed four times with ethanol of concentrationsequivalent to those used for the organosolv pretreatment itselfand then one time with deionized water. In order to remove inter-fering extractives, samples were first extracted for 24 h with deion-ized water and then again 24 h with ethanol (99.8%) under refluxusing a soxhlet apparatus. After drying at 40 �C, 3 mL sulfuric acid(72%, w/w) was added to 0.3 g sample and incubated for 60 min ina water bath at 30 �C. Then 84 mL deionized water were added andthe sealed samples were autoclaved for 1 h at 121 �C. The auto-claved hydrolysis solutions were then filtered through previouslyannealed filtering crucibles and extensively rinsed with hot deion-ized water. After drying for 24 h at 105 �C they were finally

incinerated at 575 �C until constant weight. The mass differencebetween the dried and the incinerated samples was consideredas Klason lignin. As acid insoluble protein is known to falsify thein this way measured lignin content, it was corrected by an addi-tional Kjeldahl N-analysis of the dried samples prior toincineration.

3. Results and discussion

The compositional details of the original wheat bran are shownin Table 1. Compared to literature (Choteborska et al., 2004; vanden Borne et al., 2012), the relatively low starch content indicatessome high degree of comminution during the milling process.

In the following sub-chapters, the results are presented in rela-tion to the main bran fractions (glucose from starch and cellulose,hemicellulose, protein, minerals, lignin and fat). The formation ofby-products (furfural, hydroxymethylfurfural, glycerol and levu-linic acid) is discussed separately.

3.1. Glucose

As a universal substrate for microbial conversion into productsof higher value, the extraction of glucose as a monomer is of highimportance within a biorefinery processing chain (Kamm andKamm, 2004). In this respect, the organosolv fractionation led tothe results shown in Fig. 1a. About 30% of the glucose could be dis-solved, however, mostly in oligomeric form. Free monomers werefound only at levels of 1.5–3%. In general, the glucose extractionyields were decreased with increasing ethanol concentrations. Asexpected, higher temperatures led to elevated solubilization.Despite certain limitations regarding the accuracy of the measure-ment, no coprecipitation of polymeric glucose was observed duringthe lignin precipitation step.

In order to increase the yield and to obtain the glucose in itsmonomeric form, both the solid residue of the organosolv pretreat-ment and the supernatant of the lignin precipitation step weresubjected to subsequent enzymatic hydrolysis. Correspondingresults are shown in Fig. 2a. A maximum yield of free glucose ofapprox. 75% could be achieved after an organosolv treatment with30% ethanol (180 �C, 30 min). In comparison, hydrothermal pre-treatment (180 �C, 10 min) of wheat bran followed by enzymatichydrolysis led to a maximum monomeric glucose yield of 90%(Reisinger et al., 2013).

3.2. Hemicellulose

In the case of wheat bran, the hemicellulose is mainly com-posed of xylose and arabinose together with galactose at low levelsand other sugar species in trace amounts. By organosolv treatment,the highest yields of total xylose and arabinose (in oligomeric andmonomeric form) account for 45% and 43%, respectively (Fig. 1band c). These values were achieved under conditions of 30% etha-nol, 180 �C and 30 min, where also the highest enzymatic glucoseyield was obtained (3.1). These results also indicate to a certainextent the correlation between the removal of hemicellulose andthe elevated access of cellulolytic enzymes to the cellulosic struc-ture (Mosier et al., 2005; Sun and Cheng, 2002). In comparison,hydrothermal treatment (180 �C, 20 min) of wheat bran led to amore efficient removal of hemicellulose, as total xylose and arabi-nose yields of 65% and 82% could be achieved (Reisinger et al.,2013). By organosolv treatment, the yield of monomeric xylosewas found to be negligible (below 3%), whereas the free arabinosereached about 10%. Galactose was almost completely solubilized,however mostly appearing in its oligomeric form (Fig. 1d). Similar

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Fig. 1. Organosolv fractionation of wheat bran. Effect of process conditions (ethanol concentration and temperature) on the yield of free and total sugars after thepretreatment and of total sugars in solution after a subsequent lignin precipitation step: glucose (a), xylose (b), arabinose (c), galactose (d).

56 M. Reisinger et al. / Bioresource Technology 170 (2014) 53–61

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Fig. 2. Enzymatic hydrolysis of organosolv derived fractions of wheat bran. Effect of process conditions (ethanol concentration and temperature) on the yield of free sugars:glucose (a), xylose (b), arabinose (c), galactose (d).

M. Reisinger et al. / Bioresource Technology 170 (2014) 53–61 57

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Fig. 3. Organosolv fractionation of wheat bran. Effect of process conditions (ethanol concentration and temperature) on the solubilization of N-determined protein and totalamino acids (a). Yield of individual amino acids (protein-bound and free) dissolved after the organosolv pretreatment (b). Amino acid profiles of the dissolved protein mixturebefore and after the lignin precipitation step. Process conditions of the pretreatment: 45% ethanol, 180 �C, 30 min (c).

58 M. Reisinger et al. / Bioresource Technology 170 (2014) 53–61

to glucose, no noteworthy coprecipitation of hemicellulose carbo-hydrates could be observed during the lignin precipitation step.

Enzymatic hydrolysis of both the solid residue from the organo-solv pretreatment and the supernatant of the lignin precipitationstep partially led to an increase in hemicellulosic sugar yields.

The maximum conversion of hemicellulose into monomeric xyloseand arabinose was about 60% and 45%, respectively (Fig. 2b and3c). Polymeric galactose was almost quantitatively hydrolyzed(Fig. 2d). Separate enzymatic hydrolysis of both the solid residueand the supernatant showed that a considerable amount of

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Fig. 4. Organosolv fractionation of wheat bran. Effect of process conditions (ethanol concentration and temperature) on the solubilization of ash and individual ions duringthe pretreatment and of analytes in solution after a subsequent lignin precipitation step.

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H, 2

00°C

60%

EtO

H, 1

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60%

EtO

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80°C

60%

EtO

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00°C

g pe

r 100

g b

ran

dry

mas

s

Fig. 5. Organosolv fractionation of wheat bran. Effect of process conditions (ethanolconcentration and temperature) on the amount of Klason lignin remaining in thesolid residue after the organosolv pretreatment.

M. Reisinger et al. / Bioresource Technology 170 (2014) 53–61 59

monomeric sugars mainly originated from the solid phase. Inter-estingly, the polymers solubilized by organosolv treatment werenot completely hydrolyzed by means of the enzymes applied.The reason can be due to the type and structure of the carbohy-drate polymers (Van Craeyveld et al., 2009). However, the charac-teristic of the applied enzyme preparations can be a factor as well.

3.3. Protein

Considering the amount of protein in wheat bran (up to around15% based on dry mass), its separation and utilization within acomprehensive biorefinery processing chain is of great relevance.Additionally, proteins in bran are partially linked to carbohydratepolymers and lignin (Barberousse et al., 2009; Zhou et al., 2010).Therefore their extraction may positively influence the recalci-trance of the biomass. Regarding the behavior of wheat bran pro-teins during high temperature processing, only a few studies,focussing on acidic (van den Borne et al., 2012) and hydrothermal(Reisinger et al., 2013) treatments, are available. Concerning theeffect of organosolv treatment on protein-rich raw materials littleis known.

In the present study, the solubilized protein was characterizedboth via the Kjeldahl-method and by measurement of bound and

free amino acids, thus also allowing to assess losses of amino acids.By monitoring these parameters also during the lignin precipita-tion step, the behavior of the protein could be elucidated. Fig. 3ashows the results throughout the organosolv fractionation proce-dure. Interestingly, proteins could be dissolved almost quantita-tively under certain conditions. However, under conditions ofelevated severity, a difference between Kjeldahl protein and totaldissolved amino acids was found. This indicates some loss of cer-tain amino acids during the pretreatment. Fig 3b shows the indi-vidual yields of specific amino acids (bound and free ones). Likein the previous work (Reisinger et al., 2013), especially theamounts of lysine, arginine and aspartic acid are reduced. Theselosses can be due to the formation of Maillard reaction products(van den Borne et al., 2012). Apart from that, no release of freeamino acids occurred under the tested conditions (data notshown).

During the lignin precipitation step up to approx. 40% of thedissolved protein coprecipitated, depending on the pretreatmentconditions.

Fig. 3c exemplarily demonstrates the amino acid compositionbefore and after the lignin precipitation step concerning thedissolved protein mixture at pretreatment conditions involving45% ethanol, 180 �C and 30 min. It shows that the amino aciddistribution practically did not undergo changes during theprecipitation of proteins.

3.4. Minerals

Fig. 4 shows the behavior of ash including the relevant ionspotassium and phosphate during the organosolv fractionation.Other inorganic ionic species were also measured, but are notshown due to their marginal concentrations. As can be seen clearly,the amount of extracted ash decreased with increasing ethanolconcentrations. Irrespectively, it has to be noted that the massbalance between ash and individual ions only partially is in accor-dance. As a sum parameter, the ash content includes both individ-ual ions as well as phytate-bound phosphate. So the difference liesin dissolved phytic acid comprising six phosphate groups. Duringthe lignin precipitation step, no significant coprecipitation of ashoccurred.

0

1

2

3

4

5

6

30%

EtO

H, 1

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30%

EtO

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30%

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H, 2

00°C

45%

EtO

H, 1

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45%

EtO

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45%

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60%

EtO

H, 1

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60%

EtO

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00°C

g pe

r 100

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ran

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mas

s

Dissolved ace�c acid a�er organosolv pretreatmentAce�c acid s�ll dissolved a�er lignin precipita�onDissolved glycerol a�er organosolv pretreatmentGlycerol s�ll dissolved a�er lignin precipita�onDissolved hydrolyzed fat calculated on glycerolHydrolyzed fat (calculated on glycerol) s�ll dissolved a�er lignin precipita�on

0.0

0.5

1.0

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30%

EtO

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EtO

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EtO

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EtO

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g pe

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Dissolved furfural a�er organosolv pretreatmentFurfural s�ll dissolved a�er lignin precipita�onDissolved HMF a�er organosolv pretreatmentHMF s�ll dissolved a�er lignin precipita�onDissolved levulinic acid a�er organosolv pretreatmentLevulinic acid s�ll dissolved a�er lignin precipita�on

a

b

Fig. 6. Organosolv fractionation of wheat bran. Effect of process conditions (ethanol concentration and temperature) on the formation of by-products and hydrolyzed fat(calculated on the free glycerol level) (a) and of sugar degradation products (b) including a subsequent lignin precipitation step.

60 M. Reisinger et al. / Bioresource Technology 170 (2014) 53–61

3.5. Lignin

A primary aim of the organosolv process is to delignify a feed-stock and thus to improve the enzymatic digestibility by reducingthe recalcitrance and minimizing the irreversible binding of hydro-lytic enzymes to lignin (Hu and Ragauskas, 2012). As a majorby-product of this technique, organosolv lignin tends to be of highpurity and feature main functionalities of native lignin. This favorsthe conversion into valuable products like aromatic chemicals,resins and antioxidants (Huijgen et al., 2011). In the present study,lignin was exclusively determined as Klason lignin. Acid solublelignin generally quantified by UV-absorption (at a recommendedwavelength of 240–320 nm and absorptivity depending on thefeedstock) was not measured due to interferences with dissolvedprotein absorbing at similar wavelengths and also due to the lackof data regarding the absorptivity of wheat bran-based lignin.

Fig. 5 shows the amounts of Klason lignin remaining in the solidresidues of the organosolv pretreatment. Irrespective of the used

ethanol concentration, the highest yield of delignification (approx.28%) was achieved at 160 �C, 30 min. Interestingly, the amount ofremaining lignin in the solid residue increased with increasingseverity, and in one case (30% ethanol, 200 �C, 30 min) a highervalue than the original one was measured. Similar aspects werereported in literature and said to be owed to the formation ofso-called pseudo-lignin. This, for example, can be generated viathe repolymerization of polysaccharide degradation products suchas furfural and HMF and/or polymerization with lignin, especiallyunder severe conditions (Hu and Ragauskas, 2012; Sannigrahiet al., 2011). In the end, it seems that the organosolv process ishardly applicable for the efficient delignification of wheat bran.

3.6. Fat

Like under hydrothermal conditions, where fat hydrolysis intoglycerol and fatty acids occurs to a large extent (Holliday et al.,1997), similar effects are also expected to happen during the

M. Reisinger et al. / Bioresource Technology 170 (2014) 53–61 61

organosolv pretreatment. Therefore, to follow the fat behavior, theformation of glycerol was measured. Based on the average fattyacid distribution of wheat bran found in literature (Souci et al.,2008), a ratio (mass and amount of substance) between glyceroland triglycerides was calculated and used as a basis for estimatingthe extent of fat hydrolysis and solubilization.

Fig. 6a shows the measured values of glycerol and the resultantcalculated levels of hydrolyzed fat during the organosolv process.As expected, the levels of hydrolyzed fat generally increased withincreasing treatment severity. Both higher temperatures and etha-nol concentrations led to increased fat hydrolysis. Finally it has tobe noted that no significant coprecipitation of glycerol wasobserved during the lignin precipitation step.

3.7. Degradation and by-products

It is well known that, depending on the severity of the process,the treatment of biomass leads to reactions that yield a range ofundesired degradation products. Fig. 6b shows the behavior ofthe sugar derived products furfural, hydroxymethylfurfural(HMF) and levulinic acid during the organosolv fractionation. Asexpected, their levels increased with increasing pretreatment tem-perature, but decreased with increasing ethanol concentration. Theincrease of sugar degradation products is accompanied by drasticlosses of xylose, arabinose and galactose (Fig. 1b and c). The levelsof free acetic acid (Fig. 6a) increased with temperature, however,rather independently of the applied ethanol concentration.

Regarding the coprecipitation of degradation products duringthe lignin precipitation step, results obtained do not really allowa specific interpretation.

4. Conclusions

The organosolv process offers the opportunity to partially disin-tegrate and fractionate biomass. This work shows that in case ofwheat bran the fractionation of lignin is not satisfactory. The per-formance of the organosolv-enzyme process is similar to thehydrothermal-enzyme one. In both cases, about 85% of the feed-stock can be transferred into liquid phase. However, protein solu-bilization is higher with the organosolv treatment. The presentand previous work (hydrothermal treatment) indicate that aremaining proportion of approximately 15% of the biomass is verydifficult to disintegrate, thus processes of significantly higherseverities seem to be necessary to increase the yields.

Acknowledgements

This work was supported by GoodMills Group GmbH, Austria,and the Christian Doppler Forschungsgesellschaft, Austria.

References

Aguedo, M., Vanderghem, C., Goffin, D., Richel, A., Paquot, M., 2013. Fast and highyield recovery of arabinose from destarched wheat bran. Ind. Crop Prod. 43,318–325.

Barberousse, H., Kamoun, A., Chaabouni, M., Giet, J.M., Roiseux, O., Paquot, M.,Deroanne, C., Blecker, C., 2009. Optimization of enzymatic extraction of ferulicacid from wheat bran, using response surface methodology, andcharacterization of the resulting fractions. J. Sci. Food Agr. 89, 1634–1641.

Choteborska, P., Palmarola-Adrados, B., Galbe, M., Zacchi, G., Melzoch, K., Rychtera,M., 2004. Processing of wheat bran to sugar solution. J. Food Eng. 61, 561–565.

Cigic, I.K., Vodosek, T.V., Kosmerl, T., Strlic, M., 2008. Amino acid quantification inthe presence of sugars using HPLC and pre-column derivatization with 3-MPA/OPA and FMOC-Cl. Acta Chim. Slov. 55, 660–664.

DIONEX, 1996. Installation instruction and troubleshooting guide for the IONPACAS14 analytical column. Document No. 031199, Sunnyvale, California.

DIONEX, 2005. Product manual for the IonPac CS12A analytical column. DocumentNo. 031132-08.

European Communities (EEC), 1998. Commission directive 98/64/EC. Part A.Determination of amino acids. Off. J. Eur. Commun. OJ L 257, 14–23.

Favaro, L., Basaglia, M., Casella, S., 2012. Processing wheat bran into ethanol usingmild treatments and highly fermentative yeasts. Biomass Bioenerg. 46, 605–617.

Henderson, J.W., Brooks, A., 2010. Improved Amino Acid Methods using AgilentZORBAX Eclipse Plus C18 Columns for a Variety of Agilent LC Instrumentationand Separation Goals. Agilent Technologies Application Note.

Holliday, R.L., King, J.W., List, G.R., 1997. Hydrolysis of vegetable oils in sub- andsupercritical water. Ind. Eng. Chem. Res. 36, 932–935.

Hu, F., Ragauskas, A., 2012. Pretreatment and lignocellulosic chemistry. Bioenerg.Res. 5, 1043–1066.

Huijgen, W.J.J., Smit, A.T., Reith, J.H., den Uil, H., 2011. Catalytic organosolvfractionation of willow wood and wheat straw as pretreatment for enzymaticcellulose hydrolysis. J. Chem. Technol. Biot. 86, 1428–1438.

Idris, W.H., Babiker, E.E., El Tinay, A.H., 2003. Fractionation, solubility and functionalproperties of wheat bran proteins as influenced by pH and/or saltconcentration. Nahrung 47, 425–429.

Kabel, M.A., Carvalheiro, F., Garrote, G., Avgerinos, E., Koukios, E., Parajo, J.C., Girio,F.M., Schols, H.A., Voragen, A.G.J., 2002. Hydrothermally treated xylan rich by-products yield different classes of xylo-oligosaccharides. Carbohyd. Polym. 50,47–56.

Kamm, B., Kamm, M., 2004. Principles of biorefineries. Appl. Microbiol. Biotechnol.64, 137–145.

Kataoka, M., Wiboonsirikul, J., Kimura, Y., Adachi, S., 2008. Properties of extractsfrom wheat bran by subcritical water treatment. Food Sci. Technol. Res. 14,553–556.

Mosier, N., Wyman, C., Dale, B., Elander, R., Lee, Y.Y., Holtzapple, M., Ladisch, M.,2005. Features of promising technologies for pretreatment of lignocellulosicbiomass. Bioresour. Technol. 96, 673–686.

Palmarola-Adrados, B., Choteborska, P., Galbe, M., Zacchi, G., 2005. Ethanolproduction from non-starch carbohydrates of wheat bran. Bioresour. Technol.96, 843–850.

Prückler, M., Siebenhandl-Ehn, S., Apprich, S., Höltinger, S., Haas, C., Schmid, E.,Kneifel, W., 2014. Wheat bran-based biorefinery 1: composition of wheat branand strategies of functionalization. LWT – Food Sci. Technol. 56, 211–221.

Reisinger, M., Tirpanalan, O., Pruckler, M., Huber, F., Kneifel, W., Novalin, S., 2013.Wheat bran biorefinery – a detailed investigation on hydrothermal andenzymatic treatment. Bioresour. Technol. 144, 179–185.

Rose, D.J., Inglett, G.E., 2010. Two-Stage hydrothermal processing of wheat(Triticum aestivum) bran for the production of feruloylatedarabinoxylooligosaccharides. J. Agric. Food Chem. 58, 6427–6432.

Sannigrahi, P., Kim, D.H., Jung, S., Ragauskas, A., 2011. Pseudo-lignin andpretreatment chemistry. Energy Environ. Sci. 4, 1306–1310.

Sluiter, J., Sluiter, A., 2011a. Laboratory Analytical Procedure NREL/TP-510-48087.National Renewable Energy Laboratory.

Sluiter, J., Sluiter, A., 2011b. Laboratory Analytical Procedure NREL/TP-510-48825.National Renewable Energy Laboratory.

Souci, S.W., Fachmann, W., Kraut, H., 2008. Food Composition and Nutrition Tables,seventh ed. MedPharm Scientific Publishers, Stuttgart, Germany.

Sun, Y., Cheng, J.Y., 2002. Hydrolysis of lignocellulosic materials for ethanolproduction: a review. Bioresour. Technol. 83, 1–11.

Tirpanalan, Ö., Reisinger, M., Huber, F., Kneifel, W., Novalin, S., 2014. Wheat branbiorefinery: an investigation on the starch derived glucose extractionaccompanied by pre- and post-treatment steps. Bioresour. Technol. 163, 295–299.

Tkachuk, R., 1969. Nitrogen-to-Protein conversion factors for cereals and oilseedmeals. Cereal Chem. 46, 419–423.

Van Craeyveld, V., Holopainen, U., Selinheimo, E., Poutanen, K., Delcour, J.A., Courtin,C.M., 2009. Extensive dry ball milling of wheat and rye bran leads to in situproduction of arabinoxylan oligosaccharides through nanoscale fragmentation.J. Agric. Food Chem. 57, 8467–8473.

van den Borne, J.J.G.C., Kabel, M.A., Briens, M., van der Poel, A.F.B., Hendriks, W.H.,2012. Effects of pretreatment of wheat bran on the quality of protein-richresidue for animal feeding and on monosaccharide release for ethanolproduction. Bioresour. Technol. 124, 446–454.

Yu, Y., Lou, X., Wu, H.W., 2008. Some recent advances in hydrolysis of biomass inhot-compressed, water and its comparisons with other hydrolysis methods.Energ. Fuel 22, 46–60.

Zhao, X.B., Cheng, K.K., Liu, D.H., 2009. Organosolv pretreatment of lignocellulosicbiomass for enzymatic hydrolysis. Appl. Microbiol. Biotechnol. 82, 815–827.

Zhou, S.M., Liu, X.Z., Guo, Y., Wang, Q.A., Peng, D.Y., Cao, L., 2010. Comparison of theimmunological activities of arabinoxylans from wheat bran with alkali andxylanase-aided extraction. Carbohyd. Polym. 81, 784–789.