Bioresource Technology 101 (2010) 8526–8534

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Bioresource Technology

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Polymer induced flocculation and separation of particulates from extracts oflignocellulosic materials

G.V. Duarte, B.V. Ramarao *, T.E. AmidonEmpire State Paper Research Institute, Department of Paper and Bioprocess Engineering, State University of New York College of Environmental Science andForestry, 1 Forestry Drive, Syracuse, NY 13210, USA

a r t i c l e i n f o

Article history:Received 29 September 2009Received in revised form 10 May 2010Accepted 26 May 2010

Keywords:Particle sizeZeta potentialSugar maple extractsMass removalBiorefinery separation processes

0960-8524/$ - see front matter � 2010 Elsevier Ltd. Adoi:10.1016/j.biortech.2010.05.079

* Corresponding author. Tel.: +1 315 470 6513; faxE-mail address: bvramara@esf.edu (B.V. Ramarao)

a b s t r a c t

Biofuels from lignocellulosic materials like wood are renewable and sustainable alternatives to petroleumand other fossil fuels. Wood can be grown and harvested without adding to the carbon load of the atmo-sphere and thus can be part of the solution to the problem posed by global climate changes. Recentlymuch interest has developed on the concept of the forest product biorefinery, where wood is hydrolyzedprior to conventional pulping and papermaking processes and the hydrolyzate consisting of hemicellu-lose sugars are to be used as a feedstock for biofuels or bioplastics. The purification of the hydrolyzatestream and the separation of fermentable sugars from it thus constitutes an important step in biorefineryprocesses.

The separation of particulate material from wood hydrolyzates is considered in this paper. Sugar maplehardwood was extracted with hot water at 160 �C. The extracts contain hemicelluloses (primarily xyloo-ligomers, xylose and xylan), acetic acid and smaller amounts of lignin. The colloidal stability of theextracts plays a critical role in the separation and purification of the wood extracts. Here, we reportthe size and charge of the particles in the extract measured using standard instruments based on lightscattering and microelectrophoresis. Particles were found to be in the size range from �220 nm to270 nm. Zeta potential measurements showed them to be negatively charged. By treating the extractswith a cationic flocculating agent poly-DADMAC, it was possible to preferentially precipitate out the col-loidal fraction containing lignin and lignin derived compounds. Upon the addition of poly-DADMAC theturbidity of the suspension reduced from 920 NTU to 4 NTU in a 24 h period and particulates sedimentedfrom the extract. The lignin concentration was reduced in the supernatant, while the sugar contentremained unchanged. The addition of an indifferent electrolyte hindered the effectiveness of the poly-electrolyte. The optimum pH for the effectiveness of the polymer was found to be around 4.5. In orderto accelerate the sedimentation of the particles, Kaolin was added to attach to the flocs after addingthe polyelectrolyte. Kaolin helped accelerate the separation of the particulates by almost an order of mag-nitude. This work shows that flocculation with a cationic polyelectrolyte followed by separation by sed-imentation, centrifugation or microfiltration can be an effective technique to purify biorefinery woodextracts.

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1. Introduction

Finding alternative and renewable energy sources is a top prior-ity in today’s world. Lignocellulosic materials such as wood consti-tute an important natural resource for the production of biofuelsand biodegradable plastics and can be a component for sustainableindustrial development (Ragauskas et al., 2006). Biofuels can helpalleviate climate change by reducing greenhouse gas emissionswhile bioresources can substitute for fossil based carbon resourcesas a raw material for plastics. These bioplastics are also biodegrad-able and therefore can minimize environmental impact after use.

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The biorefinery has been projected as a facility for the sustainedprocessing of biomass into a spectrum of commercially viableproducts. Forest biorefineries are expected to process forest bio-mass feedstock such as wood into a spectrum of fuel and materialproducts, similar to the operation of conventional petroleum refin-eries (Amidon, 2006; Amidon et al., 2008). Biorefineries will exploiteconomies of scale to transform these renewable resources intouseful energy and materials. Current mills producing papermakingpulps can be converted into integrated biorefineries producing bio-fuels, acetic acid and bioplastics, while still producing pulp andpaper.

In the forest biorefinery, wood is hydrolyzed to extract some ofthe hemicellulose after which it is sent further to the conventionalpulping and bleaching process to make papermaking pulps

1 Calculated taking into account the solution normality of 0.001 N and assuming amolecular weight of 126 g per equivalent.

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(Amidon, 2006). Hardwoods are better suited for implementingpre-extraction because they contain significant fraction of xylanthat is easily hydrolyzed and removed. Extractions of differenthardwoods and softwoods have been recently reported in the liter-ature (Mittal, 2006; Tunc and van Heiningen, 2008; Alen, 2008).Sugar maple presents a particularly attractive hardwood specieson account of its abundance in the Northeastern US and its pres-ence as part of the mix of hardwoods traditionally used for pulping.Extensive investigations of extraction of sugar maple using waterand subsequent pulping of the extracted wood have been reported(Bolton, 2008). Mittal (2006) and Mittal et al. (2009) reported thekinetics of the extraction reactions treated as the production ofhemicelluloses and his dissertation provides an extensive tabula-tion of the chemical composition of extracts under a variety oftemperature and acid pretreatment conditions. NMR techniqueswere used to analyze the composition of different sugars in the ex-tracts. In general, the extracts contain hemicelluloses, woodextractives and lignin besides smaller quantities of other organics(Mittal, 2006). Biological pretreatment ahead of hydrothermaltreatments can enhance wood hydrolysis and was studied byBarber (2007).

The hemicellulose rich extract can be fermented into ethanol,butanol or polyhydroxyalkanoates. However, some of the extrac-tion products are inhibitors for downstream fermentation opera-tions and need to be removed prior to processing (Wilson et al.,1989). Acetic acid is a potent fermentation inhibitor as it is toxicto the microorganisms producing ethanol or other products. Furancompounds such as furfural, hydroxyl methyl and various phenoliccompounds formed from degradation reactions of lignin are inhib-itors too. Therefore, processes to separate such compounds fromhydrolyzates are necessary for the further fermentation. A reviewof possible separation techniques useful in biorefineries was re-cently published by Huang et al. (2008). Many methods of detoxi-fication have been reported in the literature. These includeextraction (Martinez et al., 2001), treatment with lime (called over-liming) (Eken-Saracoglu and Arslan, 2000), and adsorption usingzeolites (Gong et al., 1993) or activated carbon (Miyafuji et al.,2003). Effective adsorbents can remove not only the sugar derivedcompounds, furfural and HMF but also vanillin, vanillic acid, p-Hy-droxy benzoic acid and coniferaldehyde from hydrolyzates. Villa-real et al. (2006) used cationic and anionic resins to detoxifyeucalyptus extracts. They reported that ionic resins were signifi-cantly more efficient at detoxification as compared to activatedcarbons. Han et al. (2006) used adsorptive membranes for the re-moval of acetic acid from hydrolyzate and compared it with anion exchange resin. They concluded that membranes providedhigher capacity and separation than ion exchange resins. Recently,Mao et al. (2008) suggested filtration to separate lignin from woodextracts followed by liquid–liquid extraction for separating aceticacid and furfural in a study optimizing biorefinery processes. Liuet al. (2008) reported the application of reverse osmosis mem-branes for separating acetic acid and furfural from the extracts toyield a concentrated hemicellulose (xylooligomers) rich retentateand a dilute acetic acid permeate.

Separation processes such as sedimentation, filtration, mem-brane separation and centrifugal separations can be used for frac-tionating the wood extract. For the success of any molecular orionic separation process downstream from wood hydrolysis andextraction, the extracts must be relatively clean and particle free.This is particularly important since fouling and flux decay in nano-filtration or reverse osmosis applications can render these separa-tions unviable on large scale. In the present paper, we first reportthe colloidal characteristics of the wood extracts, specifically theparticle size and charge characteristics followed by the applicationof a commonly used polymeric flocculant to separate the particu-late phase in these extracts. Since the particles were found to be

negatively charged, we felt that a low or medium molecular weighthighly charged cationic polymer would be effective at neutralizingthe charges and cause flocculation of the particles. Long chain andmildly charged cationic polymers such as substituted polyacryla-mides tend to affect flocculation through bridging processes. Thesepolymers tend to yield bulky flocs which may bind water andtherefore prove difficult to dewater. Therefore, for a preliminaryinvestigation, we chose the former type of flocculant as opposedto the latter ones which are left for a future investigation. The poly-mer flocculant for this investigation was a short chain, high chargedensity polyelectrolyte (poly-diallyl dimethyl ammonium chloride,pDADMAC) (MW in the range of 50,000 to 100,000 Da, according tothe manufacturer) typical of a polymer that causes patching floccu-lation. Due to the high charge density, this polymer can also neu-tralize the surface charge of anionic particles and causeflocculation. We also added Kaolin, a commonly used inert fillermaterial in papermaking processes for enhancing the separationof the wood extracts.

2. Methods

In the following, we first describe our experimental techniquefor obtaining wood extracts which follow the techniques devel-oped and analyzed by earlier workers (Mittal, 2006; Bolton,2008; Mittal et al., 2009; Barber, 2007). Then we provide our meth-ods for conducting flocculation experiments on the extracts andthe methods to analyze their particle size distributions and zetapotentials. We then provide a brief description of the analysismethods for the extracts’ chemical composition – lignin and carbo-hydrates (sugars in oligomeric and monomeric forms).

Sugar maple (Acer saccharum) chips were prepared from de-barked wood logs in a Carthage chipper. The chips were screenedand air dried subsequently before extraction. Extractions were car-ried out in an MK digester. 500 g (on OD basis) of the wood chipswere placed in the digester and 2000 ml of RO water was added(to yield a water–wood ratio of 4:1). The digester temperaturewas increased linearly from the initial room temperature up to160 �C (ramp time 15 min) and then held for different periods (0,5, 40, 60, 90 and 120 min) to achieve different mass removals. Atthe end of the extraction, the digester was cooled, depressurizedand the reaction mixture was withdrawn. The extraction liquorwas separated, collected and the chips were washed, dried andweighed. Extractions were always carried out in duplicate andthe extracts were analyzed separately and the data were averagedfor the two runs.

The flocculation experiments were performed with the neat aswell as diluted solutions. The dilute solutions were used for theanalysis of particle size distributions and zeta potentials. The floc-culant used was pDADMAC (poly-dimethyl diallyl ammonium chlo-ride). The amounts of pDADMAC added were 0, 5.7, 7.9, 15.8, 23.6and 47.3 ppm (weight of polymer1 by weight of extract). Extractsfrom wood treated for 120 min was chosen for all the flocculationexperiments with polymers. This extract (representing 20.6% massremoval) was chosen as the baseline since it contained the maxi-mum amount of solids in both dissolved and particulate forms. Oncethe extract was added, the mixture was strongly agitated by handand a timer was started once the agitation ceased. The particle sizeand turbidity of the solutions were measured every minute for30 min, and the turbidity change recorded up to 48 h. For the Kaolinexperiments, the extract solutions were prepared by diluting 4 ml ofextract in 36 ml of water that already contained a pre-weighedamount of Kaolin (0.06, 0.10, 0.15, and 0.20 g of Kaolin, which corre-

Fig. 1a. Mass fraction of wood chips removed after hot water pretreatment (ovendry basis). Averages of duplicate runs are shown.

Fig. 1b. Particle size in wood extracts as a function of fractal mass removed that isproportional to processing (extraction) time as in Fig 1a. Error bars representstandard deviations of particle size distributions.

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spond to 1.5, 2.5, 3.75 and 5.0%). This mixture was agitated to ensurethat the Kaolin particulates were properly dispersed. The flocculant(pDADMAC) was then added at 47.3 ppm (of extract), followed byhand agitation for two minutes and was left to settle. The settlingtime was measured.

The flocculation of neat extracts was also measured by addingthe required dose of flocculant to 100 ml of the extract in a sampletube. This was agitated vigorously for 10 min and allowed to standfor 24 h. Samples from this solution were withdrawn and dilutedto 10X and analyzed for particle size and zeta potential.

Particle size and zeta potential measurements were carried outusing a BIC Particle Size and Zeta Potential Analyzer (90 Plus� andZetaPlus�) [Brookhaven Instruments Corporation (BIC), HoltsvilleNY]. In order to obtain the zeta potential at different pH values,the extract pH was adjusted using 0.04 M NaOH and 0.1 M HCl.All the dilutions required were performed with a 10 mM KCl solu-tion in order to ensure a good conductivity of the solution (follow-ing the experimental protocol recommended by BIC). The KCl usedwas of analytical grade and the water used was RO water, filteredusing a 100 nm Millipore� filter. The extracts were diluted to 10xvolume by adding 4 ml of extract in 36 ml of DI water. This dilutionwas necessary in order to be able to measure both the turbidityand the average particle size in solution. However, reported valuesof lignin and sugar concentrations take this dilution into accountand refer therefore to the neat (i.e. undiluted) extract solution.

Lignin content was determined via UV/Vis spectroscopy bydiluting the solution in 0.01 N NaHCO3, (pH of 9.1) to ensure ligninsolubility after which the acid soluble lignin method from TAPPIStandard T222 was followed. The dilution was done in order forthe maximum absorbance be with the range 0.22 and 0.70. Thepolysaccharides in the extracts were analyzed using proton 1HNMR spectroscopy according to earlier protocols (Kiemle and Sti-panovic, 2001, 2004). Briefly, aliquots of the wood extracts (5 g)were mixed with 96% sulfuric acid to obtain 4% by weight of sulfu-ric acid solution which was autoclaved at 120 �C for 45 min to con-vert the residual oligomeric hemicelluloses into their monomers.This acid-digested samples were filtered through Whatman No. 1filter paper and 0.1 ml of 72% sulfuric acid was added to 1 g ofthe sample. The sample was then analyzed using the 1H NMR spec-troscopic technique (Kiemle et al., 2004). More detailed descriptionof this analysis can be found elsewhere (Mittal, 2006). All experi-ments and analyses were performed in duplicate.

Fig. 1c. Extract pH as a function of mass fraction removed.

3. Results and discussion

The chemical composition of sugar maple wood is given in Ta-ble 1. A more detailed analysis can be found elsewhere (Mittal,2006). A major component of the hemicelluloses in this wood isacetylated glucuronoxylan. The deacetylation of this sugar byhydrolysis decreases the liquor pH (to near 3.2) with the resultingacetic acid auto-catalyzing further deacetylation reactions. Xyloo-ligomers are dissolved in the extract and so are smaller fractionsof lignin and extractives in the wood. The continued hydrolysis

Table 1Composition of sugar maple wood (chips) (results adapted from Mittal (2006)).

Component Wood’s composition (%)

Glucose 45.5Hemicellulosea 25.9Ligninb 26.4Othersc 2.2

a All fermentable sugars (glucose, xylose, mannose, arabinose, rhamnose andgalactose).

b Acid soluble and acid insoluble lignin.c Extractives and ash. Calculated by difference.

of sugar maple at 160 �C increases the amount of solids dissolvedin the extract. The mass removal represents the fraction of themass of wood found in the extract (on an oven dry basis) and in-cludes the particulate (including colloidal) and dissolved compo-nents. The fractional mass removal increases with the time oftreatment in this range as shown in Fig. 1a. Thus, further resultsin this paper are given as functions of mass removal fraction. Abroad categorization of the composition of the extract is shownin Table 2, indicating that the primary chemical constituents arehemicelluloses (denoted as sugars or polysaccharides), lignin andothers.

3.1. Particle size and charge characteristics of extracts

The particle sizes of the extracts obtained at different mass re-moval fractions (i.e. for different treatment times) are shown inFig. 1b. The average particle size of the first particles removed (very

Table 2Composition of the 2 h hot water extracts from sugar maple.

Component Extract’s composition

Concentration (g/l) Fraction (%)

Sugarsa 30–32 57–61Lignin 12.3–13.3 23–25Othersb 7.1–10.2 14–20

a All fermentable sugars (glucose, xylose, manose, arabinose, ramnose andgalactose).

b Un-hydrolyzed hemicelluloses, furfural, acetic acid and extractives. Calculatedby difference.

Fig. 2b. Hemicellulose and lignin fractions in the extract as a function of massremoval.

Fig. 2c. Solids fraction greater than 1.2 lm, between 1.2 lm and 100 nm andsmaller than 100 nm versus mass removal.

Fig. 2d. Effect of electrolyte concentration (KCl) in zeta potential of different massremoval extracts.

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small mass removals) is �225 nm. When the extraction is carriedout further, the particle size initially seems to drop slightly butfor mass removals over 12% the particle size increases significantlyreaching 370 nm. The initial decrease in particle size is not statis-tically different, but it could reflect some degradation of particlesas the extract acidity increases and the pH drops. There is a distinctincrease in particle size with extraction levels above 10%. As foundearlier (Mittal, 2006; Bolton, 2008) wood chip porosity increaseswith continued hydrolysis i.e. with larger mass removal fractions.Such a porosity increase could be accompanied by an increase inpore size, resulting in the removal of larger particles into the ex-tract. An interesting observation is that as the mass removal in-creases, the particle distribution becomes more polydisperse asshown by the increasing standard deviation in Fig. 1b. Anothermechanism which can increase both size and polydispersity couldbe the aggregation/flocculation of smaller particles in the extractwith time. We measured the particle size of a commercial Xylanfor reference and found it to be approximately 330 nm, withinthe range of the particle sizes in the extracts at higher mass re-moval (>18%). Fig. 1c presents the pH of the extracts (measuredat 20 �C) as a function of mass removal. The pH decreases as extrac-tion proceeds as a consequence of the deacetylation of the xylansin the wood. The slow pH decrease below 3.5 beyond mass remo-vals of 7.5% occurs due to the buffering action of the acetic acid.

The zeta potential of the extracts obtained for different massremovals are shown in Fig. 2a. The particles are all anionic andthe zeta potential decreases in magnitude with pH and the point(pH) of zero charge estimated by extrapolation of the curves tothe low pH range is less than 2.0. Furthermore extracts obtainedfrom treatment to different mass removal fractions have differentzeta potential curves. Extracts at the lower mass removal (<7%)have more negative values than the higher mass removal ones(7.7% and above). The zeta potential variation of the commercialxylan sample is also shown in this figure for comparison. As themass removal increases, the extract’s zeta potential comes closer

Fig. 2a. Zeta potential of wood extracts as a function of pH of solution. (curves fordifferent mass removals are shown along with those for a sample of commercialxylan particles).

to that of the commercial xylan. Indeed, at 21% mass removal,the difference between xylan’s zeta potential and that of the ex-tract is practically insignificant. In order to get a better understand-ing of this, we analyzed the hemicelluloses and lignin compositionsof the extracts and the results are shown in Fig. 2b. The lignin frac-tion comprises the bulk of the solids at low mass removals butquickly decreases to 20%. The remainder is the hemicellulose frac-tion which increases steadily with mass removal. Since the hemi-cellulose fraction increases from 23% to 65% the zeta potential athigher mass removals is more affected by the hemicellulose andapproaches that of the xylan. Since lignin contributes more tothe surface charge than the hemicellulose (xylan), the strongernegative charged particles are low mass removals can beexplained.

The general curves of the zeta potential with pH show someadditional interesting features. For higher mass removals, the ex-tract seems to have two dissociation points, one at lower pH(around 3) corresponding to the deprotonation of acidic groupssuch as acetic acid (pKa 4.76), formic acid (pKa 3.75) and glucu-ronic acid (pKa 2.93) and a second one at higher pH (around 12)

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which could correspond to the dissociation of some alcohol func-tional groups in sugars such as xylose, mannose, arabinose and gal-actose among others (all with pKa between 12 and 12.5) (Lide,2009; Robinson et al., 1953). The dilute extracts on the other handshow no such behavior probably because their hemicelluloses frac-tion is small. It also appears that the magnitude of the zeta poten-tial decreases slightly between pH of 8 and 10. This reduction isfound only for the extracts and not for commercial xylan, whichcould occur due to solubilization or desorption of some of thecharge bearing moeities under these pH conditions.

Note also that as the mass removal increases, the fraction ofsuspended particles greater than 1.2 lm increases, mostly at thecost of the fraction smaller than 100 nm, which decreases from�80% to 60% (Fig. 2c). From the particle size results, we know thatnot only the suspended fraction increases with mass removal, butso does the particle size of the solids in solution (smaller than1.2 lm). This will mean that solids found in the higher mass re-moval extracts should be easier to separate via filtration.

3.2. Colloidal stability with electrolytes

Electrolytes can suppress the repulsive electrical double layerpotentials between colloidal particles and hence reduce the energybarrier to coagulation. As can be seen from Fig. 2d, when the indif-ferent electrolyte KCl was added, the zeta potential of the extractsbecomes very close to 0 mV, and the solution was observed to floc-culate. For a concentration of 200 mM, the solids precipitated outonto the electrodes’ surface and the resulting zeta potential mea-

1 2 3 4 4 5 6

Fig. 3a. Duplicate results of dilute extracts with and without flocculant. From left toright – Extract; Extract + 47.3 ppm DADMAC; Extract + 47.3 ppm DADMAC + 5%Kaolin; Water.

Fig. 3b. Wood extracts after flocculation with polymers and/or Kaolin addition. Neat exDADMAC 5% Kaolin; Extract + 47.3 ppm DADMAC; Extract (A) after 10 min; (B) after 3 h

surements fluctuated significantly. Another interesting fact is thatthe particles obtained at the lowest mass removal (1.6%) have thehighest net negative potentials. These require higher KCl concen-trations to reach neutrality, almost 500 mM whereas the extractsat higher mass removals decrease their surface potentials withlower electrolyte concentrations. This effect can of course be ex-pected since as shown in Fig 2a, particles at low mass removal ex-tracts have higher negative potentials over the entire pH range ascompared to those at intermediate or high mass removals. We con-clude that the predominance of lignin and extractives in the earlystages of the extraction imparts a higher measure of colloidal sta-bility to the extracts. On the other hand, extracts under more se-vere conditions (indicated by higher mass removals) are not asstrongly electrostatically stabilized and are more susceptible toflocculation by electrolytes.

3.3. Flocculation with a cationic polyelectrolyte (pDADMAC)

The separation of the extracts upon adding a flocculant poly-mer, pDADMAC, is demonstrated in a set of photographs of sampletubes in Figs. 3a and b. Duplicate tubes are shown in Fig. 3a. Sam-ples 1 and 2 (from the left of the picture) contain extracts dilutedto 10x, 3 and 4 contain the same extract with the polymeric floccu-lant, pDADMAC added at 47.3 ppm dosage and 5 and 6 show theresultant extract with the same flocculant and Kaolin dosed at 5%(based on the mass of solid in the extracts). These pictures were ta-ken after all settling has occurred. Samples 1 and 2 do not showany separation and the initial extract is a turbid solution with a dis-tinct brown coloring. Adding the flocculant results in the settling ofa brown precipitate at the bottom (samples 3 and 4) and a clear li-quid solution at the top. Addition of Kaolin along with the floccu-lant results in a similar separation with a whiter sediment layerat the bottom and clear solutions above them. The supernatantsolutions in samples 5 and 6 appear slightly more turbid thanthose in samples 3 and 4 probably due to some residual fine clayparticles. It was observed that the settling rate for samples 5 and6 was much higher than for samples 3 and 4. The dynamics ofthe separation is illustrated in Fig. 3b with three sub-panels a, b,and c each showing three sample tubes. Samples labeled 3, 6 and9 represent the neat extract allowed to stand for 10 min, 3 h and24 h at room temperature. Sample 9 shows a slight increase inthe color tone from the top to the bottom, as an indication of somemarginal separation of particles by sedimentation after the 24 hperiod. Sample 8 on the other hand shows a clear liquid and a smalldark brown sediment layer at the bottom. The polymer has causedflocculation of the particles and their subsequent sedimentationhas yielded a clear separation. When Kaolin was added to the

tract with and without flocculant addition. From left to right – Extract + 47.3 ppm; (C) after 24 h.

Fig. 4a. Particle size versus time for different amounts of pDADMAC added.

Fig. 4b. Turbidity versus time for different amounts of pDADMAC added.

Fig. 4c. Turbidity versus time for different additions of pDADMAC and salt.

Fig. 4d. Particle size versus time for different additions of pDADMAC and salt.

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extracts along with the flocculant, clarification and separation be-gin almost immediately. Sample 1 shows the separation within a10 min period and sample 4 shows that the separation of the par-ticles is almost complete after 3 h. The supernatant layer in sample4 is more turbid than that in sample 7 showing that waiting till24 h have elapsed results in the clearest degree of separation.These results establish the efficiency of flocculating the extractprior to the separation of the larger particulates.

The average particle size in the extracts after treatment withpoly-DADMAC is shown in Fig. 4a as a function of time after treat-ment. Particle size continues to increase showing flocculation withdifferent dosages of the polymer. Up to an addition of 15.8 ppm ofpDADMAC the particle size increases, reaching a maximum ofapproximately 3 lm after 30 min. The net increase in particle sizeis more than 8 times the initial value. For higher dosages of pDAD-MAC, particle size attained in the same period of time is smaller(approximately 2.5 lm for 47.3 ppm). The rate of flocculation repre-sented by the rate of increase of particle size (floc) is dependent onthe polymer dosage. Higher polymer concentrations show fasterflocculation rates until 15.8 ppm beyond which increased dosageseems to inhibit flocculation rates. Fig. 4b shows the turbidity ofthe extract as a function of time. The suspension turbidity decreasesfrom an initial value above 900 NTU and very significant reductionsin turbidity were observed after 24 h. As the dosage of pDADMAC in-creases up to 47.3 ppm, the turbidity continues to decrease. The flocsize seems to reach a maximum value with dosage of 15.8 ppmwhereas the suspension turbidity continues to decrease significantlywith higher polymer dosages, up to 47.3 ppm. This leads us to con-clude that when the polymer dosage is between 15.8 ppm and47.3 ppm, the flocs obtained are denser thereby promoting faster

Fig. 5a. Extract turbidity after adding pDADMAC followed by Kaolin (0, 3.75% and5%).

Table 3Extract turbidity (NTU) 24 h after adding pDADMAC.

pDADMAC (ppm) Turbidity (NTU) Normalized turbidity (%)

47.3 4.75 0.8223.6 12.5 2.1715.8 20.1 3.497.9 67.2 12.515.7 143 26.630 576 100.00

Fig. 5b. Extract turbidity after adding pDADMAC followed by Kaolin (1.5%, 2.5%,3.75% and 5%).

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and better phase separation. In fact, after a 24 h period, there is aclear phase separation and a drastic reduction of the turbidity asshown in Table 3. When the pDADMAC dosage was increased to94.6 ppm the turbidity did not decrease appreciably, clearly indicat-ing a reversal of charge i.e. over cationization due to excess polymeradsorption on the particles.

We found that the suspensions flocculate when the magnitudeof the zeta potential is relatively small. Reduction in the zeta po-tential could be achieved by increased amounts of pDADMAC upto about 47.3 ppm. Beyond this level, the supernatant turbiditydoes not decrease as much and flocculation seems to be hindered.Reduction in zeta potentials were also observed with the monova-lent electrolyte indicating that charge neutralization is primarilyresponsible for the flocculation process in both these cases. SincepDADMAC has a relatively low MW and high charge density, it ispossible that it adsorbs onto the particle surfaces causing cationicpatches which enable electrostatic binding with the bare anionicsurfaces on other particles. Higher dosage of the polymer resultsin cationization of the particle surfaces and when sufficiently largeamount of polymer is added we found that the suspension was sta-ble and did not flocculate. This is indicative of the patching mech-anism of flocculation.

The supernatants resulting from these settled samples wereanalyzed for sugar and lignin content. The sugar content was deter-mined via NMR and the lignin via UV–Vis spectroscopy. The sugarconcentration in the supernatant was 12.11% based on OD wood(which is equivalent to approximately 30 g/l) whereas the initialsugar content was 12.52% (which is equivalent to approximately31 g/l). The lignin content before and after sedimentation was12.8 g/l and 8.2 g/l, respectively. This corresponds to a removal of36% of the lignin in the extract. As can be seen from the data in Ta-ble 4, a flocculating agent can reduce the concentrations of lignin-derived fermentation inhibitors, while increasing the fermentablefraction of sugars.

Wood extracts produced from large scale industrial hydrolysistreatments using natural water are likely to contain significantquantities of electrolytes in addition to the components presentedabove. Electrolytes can have significant effects on dispersion stabil-ity, particularly with polymeric flocculants. Counter-ions liberatedfrom the electrolytes can shield the charges on the polymer chainas well as the particle surfaces. This electrostatic shielding reducesthe attraction between the polymer and the particles inhibiting the

Table 4Composition of extracts after flocculation and sedimentation.

Component Mixture Supernatant Reduction(%)

Concentration(g/l)

Fraction(%)

Concentration(g/l)

Fraction(%)

Sugarsa 30–32 57–61 29–31 74–77 3Lignin 12.3–13.3 23–25 7.7–8.7 20–22 36Othersb 7.1–10.2 14–20 0.3–2.3 1–6 85

a All fermentable sugars (glucose, xylose, manose, arabinose, ramnose andgalactose).

b Un-hydrolyzed hemicelluloses, furfural, acetic acid and extractives. Calculatedby difference.

polymer adsorption. Furthermore, the polymer size reduces insolution affecting its ability to cause flocculation by patching orbridging mechanisms. In order to investigate the effect of electro-lytes, we added a typical monovalent electrolyte yielding indiffer-ent ions (non-adsorbing), KCl in different dosages to achieveextract concentrations of 0.015, 0.1 and 0.2 M. The effect of KCladdition on the floc size and turbidity is shown in Figs. 4c and d.The addition of electrolyte seems to restrict the drop in suspensionturbidity. When the KCl concentration in the extracts (with15.8 ppm of the pDADMAC) was increased from 0.015 M to0.2 M, flocculation was reduced substantially as indicated by thelarge turbidity values in the latter case. When small amounts ofKCl (0.015 M) was added, flocculation did occur as shown by theincreased particle size and the decrease in turbidity. For largeramounts of salt (0.1 and 0.2 M) the particle size remains unaffectedand the turbidity decrease is very small (less than 8%).

3.4. Acceleration of separation using Kaolin

Flocculation and separation of the extracts using the polymerappear to occur over longer periods of time, of the order of a few

Fig. 5c. Sedimentation velocity of extracts and Kaolin suspensions treated withpDADMAC.

Fig. 6a. Turbidity versus time for different flocculation trials. Solid lines are extracts with pH adjusted to 4.5 and broken lines are for extracts with pH 3.2. (A) Non-normalizedresults. (B) Normalized results. E – Extract; D – $7.3 ppm DADMAC; K – 5% Kaolin.

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hours. A possible way to accelerate the separation is to attach hea-vier particles to cause faster settling. Kaolin clay is anionic, inertand is widely available as filler and coating mineral in papermak-ing. We added Kaolin to the extracts to investigate if it can acceler-ate the separation process. Fig. 5a shows the turbidity of theextract as a function of time after adding pDADMAC and Kaolinin different amounts. The turbidity of the blank control decreasedfrom an initial value of 1200 to 600 and then remained at that levelover a 24 h period. When pDADMAC was added, the reduction inturbidity occurred over an 8 h period and very little turbiditywas found in the supernatant liquid after the 24 h period. Fig. 5bshows the turbidity reductions for the extracts with 47.3 ppmpDADMAC and Kaolin added at 1.5%, 2.5%, 3.75% and 5%, basedon weight of solids in the extracts. A rapid reduction in turbiditywas observed within the first hour itself. Almost complete clarifica-tion was achieved within 3 h pointing out the capability to accel-erate the separation. The overall efficiency (after 24 h) is alsoimproved, as the use of Kaolin allows for a decrease in turbidityof 99.4%, against 96.7% when it is not used. Fig. 5c shows the sed-imentation velocity of each of the suspensions. Native extracts donot settle by themselves within the timescale of the experiments(�48 h). When the extracts were treated with the flocculant(47.3 ppm), the settling velocity was observed to be nearly0.3 mm/min (labeled E + D). For comparison purpose, we showthe settling velocity of a 2.5% suspension of Kaolin in water, whichwas 0.22 mm/min (K). When Kaolin was added to the extract withthe flocculant (47.3 ppm), the sedimentation rate increased drasti-cally to about 13 mm/min (labeled K + D + E). When pDADMAC isadded to the Kaolin suspension alone (same amount as in the sam-ples containing extract), it also causes flocculation and sedimenta-tion of the Kaolin particles at an observed rate of 40 mm/min(K + D).

3.5. Effect of pH

As shown earlier (c.f. Fig 1c), the zeta potential of the particlesin the extracts increases (in magnitude) as pH is increased from thelow acidic range but plateaus out when the pH is in the neutral andalkaline ranges. The affinity between the extract and the flocculant

Fig. 6b. Evolution of particle sizes of extracts flocculated by pDADMAC (dosage at47.3 ppm). Extracts of different initial pH (3.2 and 4.5).

should be proportional to the charge difference between them. Wefirst determined the pKb of pDADMAC to be 11.8, via a titrationusing o-toluidine blue as a charge indicator. This showed thatpDADMAC remains ionized and therefore cationic over much ofthe pH range. Since the extract particles begin to lose charge atthe lowest pH, the charge difference between the pDADMAC andthe extracts is the highest at pH 4.5 and remains at about that levelfor most of the pH range.

We added pDADMAC (47.3 ppm) to two extracts with pH 3.2and 4.5. The resultant turbidities are shown in Fig. 6a as functionsof time. The upper curve represents the turbidity of the neat extractwithout any additions and it shows the relative stability of the ex-tracts over the experimental time scale. From Fig. 2a, increasing thepH from 3.2 to 4.5 results in a larger magnitude of the zeta poten-tial. The extract at pH 4.5 has higher turbidity (�1200) than the ex-tract at pH 3.2 (�900). However, upon the addition of 47.3 ppm ofpDADMAC to both extracts, the 4.5 pH extract flocculated signifi-cantly faster as measured by the relative change in turbidity (seecurves labeled E + D in panel b). This is a consequence of the largeraffinity (potential difference between the anionic particles and thecationic polymer). Turbidities of extracts with both pDADMAC andKaolin added are also shown in this figure (47.3 ppm and 5%,respectively). When Kaolin is added the higher pH extract seemsto flocculate a little slower than the one at lower pH. Since the ini-tial turbidity of the higher pH extract is significantly higher, wescaled the turbidities to the initial values of the neat extracts anddisplayed the results in the panel b. There seems to be no differencein the flocculation and separation performance of both extractswhen Kaolin is added as an enhancer with respect to the relativechange in turbidity. Fig. 6b shows the evolution of particle size dur-ing flocculation of both extracts using pDADMAC alone. The higherpH yields larger floc size, and also increases the flocculation ratereflecting the observations on suspension turbidity. This reinforcesour conclusion that the increased charge difference contributes tofaster flocculation and seems to increase the size of the resultingflocs (zeta potentials at 3.2 are between�7 and�15 mV dependingon the solid concentration whereas at pH 4.5, they are between�20 mV and�30 mV). When 5% Kaolin is added we did not find sig-nificant differences in performance between pH 3.2 and 4.5 with re-spect to particle size evolution. Perhaps the increased floc densitydue to Kaolin outweighs the pH effect.

4. Conclusions

Wood extracts contain particulates which could contribute tofouling of filtration membranes unless they are separated prior toseparations. The particulates’ composition, size and charges are alldependent on the extraction conditions. Short pre-extraction timeslead to relatively dilute extracts with the particulates containingpredominantly lignin compounds. The particles are strongly anio-nic. Longer pre-extraction times yield extracts of higher solid con-

8534 G.V. Duarte et al. / Bioresource Technology 101 (2010) 8526–8534

centrations and also larger particle sizes. The particles in this caseare not as strongly charged though. When a monovalent electrolyte(KCl) was added, at concentrations higher than �150 mM the solu-tions became unstable and flocculation occurred. Another interest-ing observation is that as the mass removal increases, the fractionof suspended solids increases and the fraction of colloidal and dis-solved solids decreases. These results are in agreement with theparticle size results. We also found that particles in extracts pro-duced by less severe treatment result in lower wood mass hydro-lyzed but are more electrostatically stable. Extracts producedunder more severe conditions are relatively less stable. Both ofthese extracts can be clarified by adding electrolytes.

The presence of negatively charged particulates indicates thatseparation processes should be suitably staged incorporating floc-culation or coagulation followed by sedimentation or filtration toseparate larger particulates from the extracts. Flocculation ofhydrolyzates using polymers is a viable option for removing sus-pended and colloidal material before other separation techniquesare employed. These techniques can be applied to reduce down-stream membrane fouling and prevent the reduction in membranefluxes thus improving separation and filtration performance. Sincethe colloidal particles are negatively charged, cationic poly-electro-lytes are the most effective flocculants. In particular, poly-DAD-MAC which is a relatively short chain, low to medium MWcationic polyelectrolyte with high charge density, proved to bean effective flocculant for the extracts. An added advantage of thispolymer is its ability to retain its cationic charge at higher pH lev-els. After 24 h the phases separate clearly with pDADMAC and theremaining liquid is no longer turbid, with turbidity values drop-ping to 4–5 NTU. The lignin concentration of the supernatant is sig-nificantly reduced from approximately 12.8 g/l to approximately8.2 g/l, which translates into a 36% reduction of lignin. The sugarcontent of the supernatant however remains almost unaffected,30 g/l, when compared to 31 g/l in the original extract. This trans-lates to a loss of less than 4%. This seems to be an important resultindicating the possible effectiveness of flocculation processes indetoxifying biomass extracts.

The addition of salt (KCl) was found to have a negative effect onthe purification, shielding the charges from both the extract parti-cles and the flocculant, thus hindering the formation of aggregates.The use of Kaolin as an anchoring agent not only improves theoverall efficiency of the flocculating system (from 96.7% to99.4%), but also drastically reduces the time for the flocs to sepa-rate. After only three hours, the turbidity dropped 91.1% relativeto the blank, when Kaolin is used, opposed to a drop of only20.7% achieved when pDADMAC was used alone. Poly-DADMACalone would require 8–9 h in order to achieve the 3 h results ofKaolin/pDADMAC system when at pH 3.2. If pH 4.5 is used, thenthe use of pDADMAC alone is comparable to the use of pDADMACand Kaolin together after 2 h. Another possible way of increasingthe lignin deposition would be to acidify the solution to a pH lowerthan 2 before adding the flocculating system (pDADMAC/Kaolin).However, the decrease of the pH below 2 would turn off thecharges in the extracts particles, seriously hindering the affinitybetween the flocculant and the particles, thus decreasing the effec-tiveness of the flocculant.

Acknowledgements

We gratefully acknowledge the member companies of the Em-pire State Paper Research Associates Inc., the US Department of En-

ergy (US-DoE) via Grant No. DEFG3607GO87004 and the New YorkState Energy Research and Development Authority (NYSERDA) viaGrant No. 9701 for partial support of this work.

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