UBC Faculty Research and Publications

Development of a green binder system for paper products Flory, Ashley R; Vicuna Requesens, Deborah; Devaiah, Shivakumar P; Teoh, Keat T; Mansfield, Shawn D; Hood, Elizabeth E Mar 26, 2013

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RESEARCH ARTICLE Open AccessDevelopment of a green binder system for paperproductsAshley R Flory1,2, Deborah Vicuna Requesens1, Shivakumar P Devaiah1, Keat Thomas Teoh1,3,Shawn D Mansfield4 and Elizabeth E Hood1,3*AbstractBackground: It is important for industries to find green chemistries for manufacturing their products that haveutility, are cost-effective and that protect the environment. The paper industry is no exception. Renewable resourcesderived from plant components could be an excellent substitute for the chemicals that are currently used as paperbinders. Air laid pressed paper products that are typically used in wet wipes must be bound together so they canresist mechanical tearing during storage and use. The binders must be strong but cost-effective. Although chemicalbinders are approved by the Environmental Protection Agency, the public is demanding products with lowercarbon footprints and that are derived from renewable sources.Results: In this project, carbohydrates, proteins and phenolic compounds were applied to air laid, pressed paperproducts in order to identify potential renewable green binders that are as strong as the current commercialbinders, while being organic and renewable. Each potential green binder was applied to several filter paper stripsand tested for strength in the direction perpendicular to the cellulose fibril orientation. Out of the twenty binderssurveyed, soy protein, gelatin, zein protein, pectin and Salix lignin provided comparable strength results to acurrently employed chemical binder.Conclusions: These organic and renewable binders can be purchased in large quantities at low cost, requireminimal reaction time and do not form viscous solutions that would clog sprayers, characteristics that make themattractive to the non-woven paper industry. As with any new process, a large-scale trial must be conducted alongwith an economic analysis of the procedure. However, because multiple examples of “green” binders were foundthat showed strong cross-linking activity, a candidate for commercial application will likely be found.Keywords: Paper industry, Binders, Enzymes, Plant-produced proteins, Green chemistryBackgroundGlobally, paper companies apply chemical binders duringthe paper-making process to attain target tensile strengthof paper. Some components of these binders are acryl-amide, acetaldehyde, urea-formaldehyde and vinyl acetate.Alternatives to synthetic paper binders have been investi-gated, but a renewable binder that can equal the strengthof the traditional binders has yet to be identified. Plant-based products would be an ideal alternative to chemicalscurrently used in the paper industry.Polymerized plant cell wall components result in a re-markably strong structure in nature. Since paper is madeprimarily of one of those polymers, cellulose, it is possibleto simulate a cell wall assembly by applying and cross-linking other cell wall constituents with enzymes, such aslaccase and peroxidases, which are responsible for thiscross-linking in vivo. The ensuing interwoven network ofsubstrates may increase paper tensile strength and providean alternative to chemical binders. In this work, proteins,carbohydrates, and phenolic compounds were applied topaper with and without enzyme activation to determine ifan increase in tensile strength could be achieved.Phenolic compounds are the first and largest category ofthe three main plant-derived substrates that could poten-tially be utilized to create a strong binder for paper. Theyare made up of aromatic carbon rings with hydroxyl* Correspondence: ehood@astate.edu1Arkansas Biosciences Institute, Arkansas State University, Jonesboro, AR72467, USA3College of Agriculture and Technology, Arkansas State University, Jonesboro,AR 72467, USAFull list of author information is available at the end of the article© 2013 Flory et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the CreativeCommons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, andreproduction in any medium, provided the original work is properly cited.Flory et al. BMC Biotechnology 2013, 13:28http://www.biomedcentral.com/1472-6750/13/28groups attached, and are a major component of the sec-ondary cell wall. Lignin is a complex phenolic polymerwhose composition can change depending on what pre-cursors are involved in its assembly [1]. A few of the pre-cursors to lignin are ferulic acid, and sinapyl and coniferylalcohol. These compounds can cross-link to themselves,to each other, or to a growing lignin polymer with the useof laccase [2].Black liquor is a by-product from the pulp and paper in-dustry generated when wood pulp is chemically digestedand bleached to remove lignin. Bleaching not only resultsin the removal of lignin, but also ferulic acid, cellulose,and hemicelluloses [3]. Because black liquor is a by-product generally used for heat energy, but rich in poten-tially valuable chemicals, it provides an inexpensive sourceof cell wall substrates. Since many of the substrates foundin black liquor are phenolic compounds, this projectsought to employ black liquor as a cross-linking agent inpaper manufacture. However, black liquor obtained fromdifferent sources can have varying properties, dependingon feedstock stream and pulping process employed at thepulp mill, and consequently is compositionally different.Elegir et al. [3] previously described an experimentthat attempted to use phenolic compounds as paperbinders. This group isolated lignin from black liquor andcross-linked it with the use of laccase and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) in anattempt to increase the tensile strength of hot pressedpulp sheets. The authors found that the cross-linking oflignin provided more than a two-fold increase in wettensile strength of the pulp sheet. However, dry tensilestrength was negatively affected. As an alternative ap-proach, Mansfield [4] demonstrated that directly impreg-nating radiate pine with laccase during mechanical pulpproduction could not only reduce the refining energy re-quired to attain a target freeness, but also had enhancedpaper strength properties. This strategy takes advantageof the cross-linking capacity of laccase to enhance theinter-fiber bonding using the native wood fiber ligninremaining on the pulp fibers.Some cross-linking experiments have been performedsimply to understand mechanisms by which cell wall en-zymes and substrates interact. Iiyama et al. [2] describemechanisms for covalent cross-linking of polysaccharides,proteins, phenolic compounds, and hydroxycinnamicacids in cell walls, but any applications of these results wasnot discussed. Given that the mechanisms and compoundsinvolved in these chemical reactions are understood, theseprocesses may be replicated in vitro and then applied inthe paper industry as a renewable, green paper binder.The second type of substrate predicted to be a strongbinder in these experiments was protein. Many types ofprotein have already been utilized for making paperbinders. Fahmy et al. [5] describe the use of plant proteinsfrom soybean and wheat as binders in cellulosic papercomposites. In their experiments, they used the total pro-tein found in soybeans and wheat protein (gluten) to makea slurry with water, urea, sodium hydroxide, and acryl-amide. They found that wheat gluten increased paperstrength as much as 60%, while total soy protein increasedthe paper strength by 46% compared to paper with nobinder added. The authors concluded that although glutenwas more effective at increasing tear strength of paper, soyprotein would be better suited as a bulk binder because itis less expensive than gluten. While these proteins did notprovide enough increase in tensile strength to replace thecurrent chemical binders, it is possible to decrease theamount of synthetic binder used by supplementing the in-dustrial binder with this plant-derived protein binder. Inthe current project, we isolated soy proteins from defattedsoybean meal, which were applied to paper, with and with-out enzyme, to determine if a significant increase in tensilestrength would be possible.Another protein of interest was hydroxyproline-rich gly-coproteins (HRGP), which are highly glycosylated proteinsfound in the cell wall that contain repeated sequences ofserine and proline, as well as an abundance of the aminoacid tyrosine [6]. Peroxidase, which requires hydrogen per-oxide to facilitate its reactions, is responsible for movingelectrons so that tyrosine can attach to other tyrosine resi-dues resulting in cross-linked HRGP in the cell wall [7].Since this protein has been shown to cross-link in aperoxide-mediated fashion, it was also a suitable candidateas a paper binder.Zein is an alcohol-soluble storage protein found inmaize seed that is known as an excellent film-maker be-cause of its ability to cross-link. Kim et al. [8] report thatreagents such as glutaraldehyde, epicholorhydrin and citricacid, induce cross-linking between zein molecules. An in-crease in tensile strength was reported in zein films aftercross-linking occurred. These properties of zein proteinsuggested it could be an organic, renewable alternative fora paper binder.Although plant components are excellent choices forpaper binders, some animal proteins may also serve thesame purpose. Gelatin is a denatured form of collagenthat is derived mainly from skin and bone of bovine andporcine sources (http://www.rousselot.com/en/rousselot-gelatine/gelatine-characteristics/definitions/gelatine-bloom/).Gelatin is tested and graded according to its bloomstrength. Bloom strength is defined as the force, expressedin grams, necessary to depress a 6.67% gel (kept 17 hoursat 10°C) by 4 mm with a standard plunger. Bloom strengthgenerally ranges between 50 to 300 g and a higher bloommeans stronger, usually more expensive gelatin. Gelatin issimilar to HRGP in that it is inherently rich in hydroxy-proline residues, thus it has side chains available forcross-linking with peroxidase, and possesses a nativeFlory et al. BMC Biotechnology 2013, 13:28 Page 2 of 14http://www.biomedcentral.com/1472-6750/13/28conformation that will allow it to lay flat against the cellu-lose of paper. While this protein may provide adequatetensile strength, there may be problems implementing itin a commercial setting because of ethical and health con-cerns about using animal products.The last group of potential binders is carbohydrates.Carbohydrates are the most abundant class of organiccompounds found in living organisms, vegetal and animalindistinctively. In this work, keeping within the scope ofdeveloping a green binder, we chose to test carbohydratesderived from plant sources, algae or fruit. Since these po-tential binders are made up of polysaccharides, laccaseand peroxidase will not effectively cross-link these sub-strates. However, the experiment performed by Fahmyet al. [5] with soy protein and wheat gluten suggests thatenzymatic cross-linking of substrates may not be neces-sary to produce a strong binder.We aimed to produce a renewable binder to replacechemical binders currently used by the paper industry inorder to increase tensile strength of non-woven papers.We studied the potential of oxidoreductase enzymes tocross-link substrates and produce an interwoven networkof substrates within the cellulose of paper, thereby increas-ing tensile strength. Alternatively, these molecules maycross-link without added enzymes. In order to replace thechemical binder with an organic binder, the strength ofpaper had to be at least equal to that of the paper with achemical binder. We found several potential binder com-pounds that produced adequate strength.ResultsOur approach was to test a variety of binders and condi-tions on 1 × 0.5 inch strips of Whatman #1 filter paper.Binders were applied by submersion into solution, thepaper was dried 10 minutes at high temperature and thestrength was tested in the direction perpendicular to theorientation of the cellulose fibers, as this is the weakest dir-ection and the standard in the industry. In some cases en-zymes were used. To determine the utility of the enzymes,they were first tested with substrates without the paperstrips to determine optimal reaction conditions. A list of allpotential binders tested is found in Table 1. Tear weightsfor all groups of green binders were statistically tested fordifferences from the commercial binder control (Table 2).We were interested in binders that were as strong or stron-ger than the commercial binder.Enzyme reactionsTwo enzymes were tested in this study, horseradishperoxidase (HRP) and laccase. Protein gels were used todetermine HRP’s optimum conditions in cross-linkingproteins. Figure 1A shows reactions incubated for 10, 30and 60 minutes at 22, 28 and 37°C. HRP is approximately50 kDa is size, whereas the substrate, hydroxyproline-richglycoprotein, or HRGP extracted from maize silk, is ap-proximately 75 kDa. As heat and reaction time were in-creased, the band corresponding to HRGP at 75 kDalessens in intensity and the bands at 160 kDa and thoseabove 220 kDa become more intense. Gel electrophoresisresults with HRP showed that the increase in HRGP’s mo-lecular weight occurred most efficiently at 50°C after 1Table 1 Summary of all substrates tested as binderSubstratecategorySubstrate SourceProtein Soy Protein Defatted soybean meal(Arkansas State University)HRGP Corn silk (Arkansas State University)Gelatin Knox Gelatin(Kraft Foods, Glenview, IL)JELL-O (Kraft Foods, Glenview, IL)Bovine & Porcine(Great Lakes Gelatin, Grayslake, IL)Zein Acros Organics, Geel, BelgiumCarbohydrates Agar Seng Huad Limited Partnership(Bangkok, Thailand)Agarose Genetic Analysis(Fisher Scientific, Pittsburg, PA)Analytical Grade(Promega, Madison, WI)Pectin Sure-Jell (Kraft Foods, Glenview, IL)Ball (Jarden Home Brands,Daleville, IN)Apple (Sigma ChemicalCompany, St. Louis, MO)Grapefruit (Source Naturals,Inc., Scotts Valley, CA)Gum Arabic Sigma Chemical Company,St. Louis, MOXanthan Gum Kountry Kupboard, Jonesboro, ARLocust BeanGumSigma Chemical Company,St. Louis, MOCarrageen Sigma Chemical Company,St. Louis, MOKelp Powder Now Foods, Bloomingdale, ILFlaxseed Now Foods, Bloomingdale, ILLignin/ Black Liquor Buckeye Technologies Inc.,Memphis, TNPhenolicCompoundsLignin LowSulfonateSigma Chemical Company,St. Louis, MOSodium LigninSulfonateMP Biomedicals, LLC, Solon, OHSalix Vertichem, Toronto, CanadaMarasperse Lignotech, Rothschild, WIFerulic Acid Sigma Chemical Company,St. Louis, MOConiferyl Alcohol Sigma Chemical Company,St. Louis, MOFlory et al. BMC Biotechnology 2013, 13:28 Page 3 of 14http://www.biomedcentral.com/1472-6750/13/28minute of reaction using water as a buffer (pH approxi-mately 7; Figure 1B). Figure 1B shows reactions that wereincubated at 37, 42 and 50°C for 1, 5 and 10 minutes. Thesame concentration of substrate and enzyme was loaded onboth gels shown in Figure 1A and B. The band correspond-ing to HRGP at 75 kDa is clearly seen at room temperatureand 28°C in Figure 1A. This band is fainter and eventuallydisappears as the temperature increases to 50°C.In order to determine the efficacy of HRP on phenoliccompounds, samples were analyzed by gel permeationchromatography. Low and high concentrations of HRP(15 μg and 150 μg) were combined separately with blackliquor, lignin and ferulic acid before chromatographicanalysis. Figure 2 shows a dramatic change in molecularweight when HRP is added to ferulic acid compared towhen ferulic acid was analyzed alone. Only slight in-creases in molecular weight were observed in black li-quor and lignin when HRP was added to these samples.In order to test for activity of laccase on phenolic com-pounds, this enzyme was combined separately with blackliquor, low sulfonate lignin and ferulic acid. It was then an-alyzed for molecular weight changes by gel permeationchromatography. Figure 3 shows that when lignin and lac-case were combined, lignin eluted earlier than when ligninwas run by itself, demonstrating an increase in molecularweight of the lignin stemming from laccase cross-linking.These results also indicate there was no change in mo-lecular weight when laccase was combined with black li-quor and only a slight change when this enzyme wascombined with ferulic acid under the conditions tested.The peaks of ferulic acid in Figures 2 and 3 vary becauseconditions for GPC detection were adjusted for the secondcompound in the mix rather than the ferulic acid and arethus are not quantitative.Protein bindersSeveral proteins were tested as binders using small paperstrips (Figure 4). The units on the y-axis show theweight (in grams) necessary to tear the paper strips.Table 2 Binder differences from the commercial binderBinder Equal tocommercial binder1Higher thancommercial binderProteinsHRGP + 300 ug HRP No3% Zein Yes5% Zein YesTSP (Pellet Resuspended) YesTSP/ 250 μg HRP (10 min) No9% Knox Gelatin Yes9% Knox Gelatin + 200 ugHRPNo Yes9% JELL-O YesPorcine 300 Bloom YesBovine 250 Bloom YesCarbohydrates0.8% Agar No3% Agar (Analytical) No1% Agar (Genetic Analysis) No11% Sure-Jell (low sugar) No9% Sure- Jell No1% Gum Arabic No5% GA + 250 ug HRP NoKelp NoFlax Seed NoXanthan Gum NoLocust Bean Gum No1% Carrageen NoPectins5% Ball Pectin Yes7% Ball Pectin Yes9% Ball Pectin Yes5% Apple No5% Apple + 1N HCl No5% Apple+CA (0.50 g) Yes3% Grapefruit No3% Grapefruit + CA (0.23 g) YesLignins and PhenolicsBlack Liquor (Heat) NoBlack Liquor + HCl NoLignin Low Sulfonate(Heat)NoLignin LowSulfonate + HClNo5% Salix (Heat) YesLignin Sulfonate (Heat) NoTable 2 Binder differences from the commercial binder(Continued)Lignin Sulfonate + HCl NoMarasperse (Heat) NoMarasperse + HCl NoFerulic Acid (2 mg/ml) NoConiferyl Alcohol (2 mg/ml) NoThe goal of these experiments was to find a green binder that was notsignificantly lower than the currently used commercial binder. All binders thatwere not significantly different at the 95% confidence level are marked “Yes”.Only the Knox gelatin binders were significantly higher than thecommercial binder.1Samples that are not significantly different are those that perform as well asthe commercial binder. All samples that are significantly different have lowervalues except for the gelatin, which was significantly higher. See Methods forstatistical treatments.Flory et al. BMC Biotechnology 2013, 13:28 Page 4 of 14http://www.biomedcentral.com/1472-6750/13/28Control strips of paper were received from a papermanufacturer with their vinyl acetate binder already ap-plied. The column labeled “Commercial Binder” showsthe weight needed to tear paper with the industrialbinder applied. The column labeled “No Binder” refersto the average weight needed to tear three pieces of wetpaper in the cross direction with no binder applied. Foreach binder several concentrations of enzyme and sub-strate were tested, as well as different incubation timeswith the various enzyme preparations. Strength valuesfor selected experiments are shown.Knox gelatin with HRP was able to withstand 925grams of weight before the paper tore, a significantlyhigher tear weight (95% confidence level) compared tothe commercial binder which tore at 720 grams weight.Even without enzyme, the Knox gelatin binder was ableto withstand 819 g before tearing which was not differ-ent from the commercial binder. Textured soy proteinheld slightly less weight than gelatin (790 g), but stilloutperformed the commercial binder. Zein proteinserved as an average binder withstanding 574 grams ofweight. Zein and TSP were not significantly differentfrom the commercial binder. The weakest protein binderwas HRGP, which was only able to hold about 77 g be-fore the paper tore.Carbohydrate bindersSeveral carbohydrates were also used as substrates in thisstudy (Figure 5). Results show the concentration of sub-strate that produced the strongest binding strength. Eachvertical bar is an average of the weight it took to tear threeindividual pieces of wet paper in the cross direction. Allcarbohydrate binders were dissolved in boiling water priorto application to the paper strips. Enzymes were only usedwhen working with gum arabic since this polysaccharidealso contains some gylcoprotein [9].Strength results from agar and two different grades ofagarose (analytical grade and genetic analysis grade – seeTable 1) are shown in Figure 5. These binders were able towithstand between 337 and 458 g of weight. Ball pectin’saverage tear weight (745 g) was slightly higher than that ofthe commercial binder but not significantly different.Regular Sure-JellW pectin was the weakest of the threepectins evaluated holding only 495 g, while low sugarSure-Jell was intermediate, withstanding 584 g of weight.Xanthan gum and locust bean gum binders resulted in noextra strength added to the paper. Carrageen also servedas a very weak binder only holding 243 g of weight com-pared to the commercial binder. Brown flax seed wasground with liquid nitrogen, then mixed with water andapplied to paper, and was able to hold 548 g of weight.These carbohydrate binders were all significantly lowerthan the commercial binder.As shown in Figure 6, pure pectin (apple and grapefruit)applied to paper resulted in weak binding, only holdingbetween 230 and 360 g of weight. However, when anacidic component such as citric acid was added, the binderwas capable of withstanding between 680 and 740 g, re-spectively, allowing pectin to equal that of the commercialbinder. Pectin concentration was also shown to affect thebinder strength, e.g., 5% pectin holds about 100 g lessweight than 7 or 9% pectin, although this amount is stillnot significantly lower than the commercial binder.Phenolic bindersFinally, lignin and phenolic compounds were tested as po-tential binders. For each type of lignin applied to the paperFigure 1 SDS-PAGE of HRGP and HRP reactions, A. RT to 37°C; B. 37°C to 50°C.Flory et al. BMC Biotechnology 2013, 13:28 Page 5 of 14http://www.biomedcentral.com/1472-6750/13/28strips, two types of experiments were carried out: lignincombined with hydrochloric acid or lignin alone, dried athigh temperatures, between 100°C and 200°C. Figure 7shows that lignin sulfonate and marasperse were only ableto hold 103 g weight when high heat was applied. How-ever, black liquor and lignin low sulfonate were able towithstand 337 and 456 g, respectively, when they weredried at 200°C. When 1N HCl was sprayed onto lignincoated paper strips, lignin sulfonate and marasperse wereable to hold 123 and 198 g, respectively. The black liquorbinder held 290 g and lignin low sulfonate 282 g after be-ing sprayed with HCl. Neither coniferyl alcohol nor ferulicacid was capable of holding much weight, tearing at 65 g.Salix lignin when dried at 200°C was the strongest lignin/phenolic compound binder, holding 714 g, the only phen-olic binder that was equal to the commercial binder. TheSalix lignin comprises an alcohol soluble fraction fromground wood that contains complex polymerized forms oflignin (USP# 8,053,566).Baby wipe solution testIn order to determine the efficacy of our experiments forcommercial applications, we tested our bound paper soakedin baby wipe solution over a 3 month period. Baby wipe so-lution tests were performed on paper treated with the ballpectin binder exclusively since this was the strongest greenbinder from a non-animal source tested in previous experi-ments. These values were compared to strength values pro-duced by a vinyl acetate binder currently in use. Weobserved no significant differences between the chemicalbinder and the Ball pectin binder used in this experiment,before application of the baby wipe solution or after 3months soaking in the solution (data not shown).DiscussionSeveral proteins, carbohydrates, and lignins were testedas paper binders to explore the possibility of replacingchemical binders currently used by manufacturers ofnon-woven paper products with an alternative green, re-newable compound. Horseradish peroxidase and laccasewere combined with green binders in order to initiatecross-linking between these molecules. We tested envir-onmentally friendly binders that could be implementedinto a commercial setting while still providing the samestrength as the commonly used vinyl acetate binder.In order to determine optimum conditions for the en-zymes, cross-linking tests were performed by using molecu-lar weight determination of cross-linked proteins withSDS-PAGE, or gel permeation chromatography for cross-linked lignins. Protein gels showed that HRP cross-linkedHRGP, increasing its molecular weight. Otte and Barz [10]conducted a similar experiment to investigate if cross-linking was occurring between an extensin-like protein anda proline-rich protein extracted from chickpea cell walls.Figure 2 Gel permeation chromatography of various substrateswith HRP.Flory et al. BMC Biotechnology 2013, 13:28 Page 6 of 14http://www.biomedcentral.com/1472-6750/13/28The authors performed SDS-PAGE analysis to demonstratethe formation of one large band above 205 kDa from twoprotein bands of 60 and 190 kDa when treated with HRPand H2O2, similar to the results shown in the present work.Therefore, it can be concluded that cross-linking of HRGPis occurring, as described by Deepak et al. [11].Gel permeation chromatography (GPC) was used to es-tablish conditions for reactions using phenolic compounds.GPC is commonly used to distinguish differences in mo-lecular weight when trying to determine if cross-linking isoccurring using oxidoreductase enzymes and phenoliccompounds as substrates. Felby et al. [12] described an ex-periment that used laccase to cross-link lignin in order tocreate stronger fiberboards. The authors used GPC tocompare molecular weight of lignin extracted from pulpand fiberboards to lignin from each source treated withlaccase. In a similar experiment, Felby et al. [13] showedan increase in strength properties of beech wood fiber-boards when laccase was used compared to boards that didnot have the enzyme treatment.In the present work, GPC results showed little to nochange in molecular weight when enzymes were added tosubstrates, with the exception of ferulic acid and HRPwhich showed a dramatic increase in molecular weight.Because laccase was shown to have such a minimal effecton its substrates, its use was minimized in binder strengthtests on paper. Also, because of the GPC results, HRP wasprimarily used with proteins and not with lignin or otherphenolic compounds.Lignin samples were analyzed with GPC and prelimin-ary results led us to predict changes could be made toimprove efficiency of laccase reaction conditions. Eventhough pH 5 sodium acetate buffer was used in all reac-tions as suggested by Bailey et al. [14], this buffer wasnot able to establish an ideal pH for the reaction tooccur, as the lignin is extremely basic in nature. Black li-quor has an especially high pH of 10–12 and the sodiumacetate was only able to maintain the reaction conditionsto approximately pH 8. Madzak et al. [15] showed thatlaccase has an activity range of pH 2 to 6.Also, we predict supplementing the reaction with oxy-gen would improve the efficacy of the laccase reaction.Laccase works by removing electrons from oxygen,transferring them to its substrates, thereby creating freeradicals or reactive species that will cross-link with eachother [16]. All reactions analyzed by GPC were carriedout in closed 1.5 ml Eppendorf tubes. Although thesetubes were shaken during the reaction, fresh oxygen ex-change could not occur. Mattinen et al. [17] showed theimportance of oxygen in the reaction by mixing laccaseand lignin together and measuring the amount of oxygenconsumed over a 3 hour period with an oxygen elec-trode. After these solutions were shaken for 3 hours in asealed glass flask, the oxygen concentration remaining inFigure 3 Gel permeation chromatography of various substrateswith laccase.Flory et al. BMC Biotechnology 2013, 13:28 Page 7 of 14http://www.biomedcentral.com/1472-6750/13/28the flask was very low for those reactions that performedefficiently. To resolve this problem of depleted oxygen,Elegir et al. [3] supplemented more oxygen than was re-quired by the reaction, ensuring oxygen would not bethe limiting factor.Mediators such as 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) have also been employed toinduce a conformational change in laccase that results ingreater access to the enzyme’s active site. If the activesite is more accessible, the enzyme should work more ef-ficiently to cross-link substrates [2]. However, enzymesand mediators are expensive and not likely to be used inmanufacturing unless substantial benefit can be gainedin product quality or process efficiency.Gelatin is a substance that is commonly used in papersizing [18]. Paper sizing is defined as a substance appliedto paper in order to provide a glossy finish that will de-crease liquid absorption and improve printability andsmoothness. A Sheffield test is commonly used to test thequality of paper sizing, where the longer it takes for paperto absorb a liquid, the better. Gelatin applied to paper notonly improves the surface properties, but also minimizesthe effect of aging and photooxidation [19]. However, it isnot clear if the use of gelatin in this way has ever beenshown to increase the tear strength of paper.Gelatin could be easily implemented into the paper in-dustry process. It is organic, renewable, readily available atlow price, needs little reaction time to produce high tearstrength, and produces a non-viscous solution that will notclog sprayers. Textured soy protein solution has all of theseadvantages as well. However, if using the resuspended pelletof soy protein, the solution may be too viscous and couldclog sprayers.In the current investigation, gelatin and textured soyprotein showed the best tear strength properties of all theprotein binders assessed when applied to paper, providingFigure 4 Summary of average tensile strength of protein binders. Tear weight is the average of the weight required to tear three strips ofpaper individually. Error bars represent standard deviation. HRP: Horseradish peroxidase. HRGP: Hydroxyproline-rich glycoprotein. TSP: Texturedsoy protein.Figure 5 Summary of average tensile strength of carbohydrate binders. Tear weight is the average of the weight required to tear threestrips of paper individually. Error bars represent standard deviation. GA: Gum arabic. HRP: Horseradish peroxidase.Flory et al. BMC Biotechnology 2013, 13:28 Page 8 of 14http://www.biomedcentral.com/1472-6750/13/28between a 12 and 14-fold increase in wet paper tearstrength when compared to paper with no binder applied.Furthermore, the strength provided by the gelatin bindergradually increases as the percentage of gelatin increases,and is the only green binder tested that was superior tothe commercial binder strength. Also, the strength pro-vided by gelatin increases slightly when HRP is added tothe reaction mixture. Gelatin from both porcine and bo-vine sources was tested along with a high and low bloomfor each source. Neither the source of the gelatin nor thebloom strength seemed to affect the tear strength pro-duced by these binders.The supernatant of the textured soy protein providedapproximately 50% less strength than gelatin and the useof HRP with soy protein did not seem to change thisstrength value. However, when the remaining pellet wasresuspended in water, the wet tear strength of paper in-creased more than 50%, offering a product that wasslightly stronger than the commercial binder product.In theory, HRGP is an excellent substrate to be used asa paper binder. It has side chains available for cross-linking with peroxidase, which will form strong covalentbonds, and its conformation will allow it to lay flat againstthe cellulose of paper. There have been no previous sug-gestions in the literature for industrial applications ofHRGP unlike the plethora of other substrates that havebeen evaluated as binders. However, it is known that thecross-linking of HRGP, as well as lignin and cellulose arecrucial in order to provide structure and strength to theplant cell wall [2]. Therefore, if larger quantities of thisprotein were employed, it is reasonable to assume that anHRGP binder could provide acceptable tear strength toFigure 6 Average tensile strength of pectin binders. Tear weight is the average of the weight required to tear three strips of paperindividually. Error bars represent standard deviation. CA: Citric Acid.Figure 7 Average tensile strength of lignin/phenolic compound binders. Error bars represent standard deviation. HCl: Hydrochloric acidHRP: Horseradish peroxidase.Flory et al. BMC Biotechnology 2013, 13:28 Page 9 of 14http://www.biomedcentral.com/1472-6750/13/28paper products. Nonetheless, in the present study, HRGPdid not provide any extra strength to wet paper tearstrength. This could very likely be due to the very lowquantities of protein applied.Zein protein from corn kernels is also a good candi-date, as when zein is dissolved in ethanol and heated, itis not viscous and would not clog sprayers. Also, zeinprotein provided a 9-fold increase in tear strength whencompared to paper with no binder applied. Because thedevelopment of low-cost manufacturing methods isneeded, this protein could fit this need as it is availablein large amounts. It is 40 to 50% of the protein in cornas reported by Shukla & Cheryan [20].Carbohydrates have already been used as wood andpaper/cardboard adhesives [21,22]. For example, pectinfrom orange peels serves as an excellent binder for drugtablets [23]. These authors also suggest that this binderwould be more appealing than synthetic binders becauseof its availability and low cost. Coffin & Fishman [24] in-vestigated the tensile strength and other physical andmechanical properties produced by citrus pectin filmsand compared those properties to films produced bysugar beet and almond pectin. Determining the differ-ences in the properties of films made by different pectinswill help to understand what type of pectin would makethe best binder.Pectin provided the highest tear strength out of allcarbohydrate binders, equaling the strength produced bythe commercial binder. However, there are several differ-ent types of pectin and all of them are slightly different interms of solubility and strength provided as a binder.While all food grade pectins already contained an acidiccomponent to assist in gelling, usually citric acid or so-dium citrate, these components must be added to purepectin. Without this added acidic component, pure pectinproduced low strength values. We clearly demonstratehow adding citric acid to the pure pectin increasedstrength values, by up to 1.5-fold. Unfortunately, in thiswork, all carbohydrate binders, other than pectin eitherproduced average to low strength results and/or their so-lutions were too viscous to be used in manufacturing.Since lignin is the second most abundant polymer innature, it can be obtained in large quantities and there-fore would be suitable for a commercial setting. It wasobserved that when HCl was added to lignin, a sticky,gel-like mixture formed. This observation implies someform of cross-linking is occurring between the ligninmolecules. Thus, we hypothesized that applying lignin topaper and then spraying that paper with HCl may have apositive effect on the strength of paper. Also, after ob-serving an increase in strength after heat was applied togelatin and pectin samples, it suggested that heat mayhave the same effect on lignin samples, as was previouslyreported by Mansfield [4] with mechanical pulp.Therefore, three treatments were performed on all ligninsamples: addition of laccase, spraying with HCl, and ap-plication of high heat when drying. No increase instrength was apparent in any of the lignin samples whenlaccase was added. Slight increases in strength were ob-served in marasperse, black liquor, and low sulfonate lig-nin binders when HCl was sprayed on lignin coatedpaper. HCl spraying had the most effect on black liquorand low sulfonate lignin binders. High heat obviouslyhad the greatest effect (Figure 7).Results from zein, gelatin, pectin, soy and lignin bindersshow that application of heat dramatically increased thestrength of paper. The possible reasons for this are as fol-lows. First, the application of heat may simply be dehydrat-ing the binder mix and forming a strong film around thecellulose of paper that would not be soluble in water. Theliterature shows that films are formed from several of thebinders used in this work such as zein, pectin and gelatin[20,25,26]. These authors reported remarkable strengthproduced by each of these films and proposed their use inindustrial applications to replace the use of petroleumproducts as adhesives. Another possibility is that chemicalor physical changes may be occurring as a consequence ofthe high heat treatment, changing the flow properties ofthe molecules. Therefore, the strength provided by heatcould be occurring because of dehydration, melting of com-pounds or a combination thereof.The phenolic compounds used did not produce compar-able strength values to the lignin binders. After applicationof ferulic acid and coniferyl alcohol, the paper tore with thesame weight as did paper with no binder applied. The GPCresults showed that ferulic acid had a dramatic increase inmolecular weight when HRP was added, suggesting thatferulic acid could cross-link with cellulose fibers and serveas a strong binder. Unfortunately, only small amounts offerulic acid and coniferyl alcohol could be used when apply-ing solutions to paper because of their low solubility inwater. Using ethanol as a solvent would resolve solubility,but is incompatible with enzymes.The Salix binder provided the same amount of strengthas the commercial vinyl acetate binder. After beingdissolved in ethanol and heated, this binder forms a non-viscous solution that would not likely clog sprayers. Con-sequently, this binder is the most practical out of all otherlignin/phenolic compound binders tested.If black liquor provided adequate strength, it would havebeen the most ideal binder. Paper manufacturers producebetween 250 and 400 gallons of black liquor per ton of pulp(AF&PA, http://www.afandpa.org/). Most of this is used asan energy source to avoid the use of fossil fuels, but muchis excess. If paper manufacturers could use their by-product not only to fuel their mill, but also as the binder intheir manufactured products, this could drastically lowerproduction costs. As shown in the strength results forFlory et al. BMC Biotechnology 2013, 13:28 Page 10 of 14http://www.biomedcentral.com/1472-6750/13/28different lignin binders, slight changes in lignin compos-ition or source of lignin can dramatically affect the strengthof the binder. Therefore, in future experiments, it would beworthwhile to survey black liquor from different paper millsto determine if any samples could be implemented as astrong binder for paper products, and evaluate other reac-tion conditions.Depending on the type of trees used and differences inthe pulping process between facilities, the constituentsof black liquor may be altered which may in turn havean effect on its properties as a binder [1]. In this work,lignin from hardwood trees showed strengths 2-foldhigher than the black liquor from softwood trees. Thelignins were also extracted by very different processes,perhaps contributing to the differences in binding activ-ity. Many studies have been done to determine the struc-tural and chemical differences between hardwood andsoftwood lignin. For example, Mohan et al. [27] foundthat the molecular weight of softwood pyrolysis lignin islarger than that of hardwood pyrolysis lignin. They alsoreported that softwood lignin which consists solely ofguaiacyl-derived monomers results from high amountsof polymerized phenylpropane units, while hardwoodlignin is made up of guaiacyl-syringyl lignin and resultsin mixed polymers of lower molecular mass.The binders developed in this project could be appliedto different commercial products, such as wet wipe appli-cators. Since binders produced adequate strength withoutthe use of enzymes, covalent bonds were not formed, so itwas important to determine if binders could retain theirstrength in commercial solutions. Therefore, paper withbinder applied was soaked in baby wipe solution for up tothree months before tensile strength was tested. The plantbinder retained its strength just as well as the vinyl acetatebinder after soaking in baby wipe solution for these pe-riods of time.ConclusionsThis project aimed to identify potential renewable andgreen replacements for the currently used synthetic paperbinders. A selection of twenty binders was surveyed, andamong them, soy protein, gelatin, zein protein, pectin, andSalix lignin provided wet tear strength equivalent to that ofthe vinyl acetate binder currently used in the manufactur-ing process. All of these binders are organic, renewable,available in large quantities at reasonable prices, requirevery short reaction time and do not form viscous solutionsthat would clog sprayers. For these reasons, all binderschosen in this work could easily be implemented in an in-dustrial process. Oxidoreductase enzymes were combinedwith binder substrates to determine if enzymatic cross-linking could provide an increase in tear strength. Gelatinshowed a slight increase in strength when horseradish per-oxidase was added, but more research in this area isnecessary to determine the true potential of these enzymesin binder systems. This effort clearly shows that there is po-tential for renewable resources, such as plant and animalby-products, to substitute the current level of petroleum-derived chemicals used as binders in the paper industry.Gelatin at 9% concentration combined with HRP providedsignificantly higher tear strength than the current commer-cial binder.MethodsTesting methodA small-scale version of a column-testing instrument(Instron, Norwood, MA) was made in order to test thestrength of paper in the laboratory setting. A die cutterwas used to ensure all pieces of paper were cut exactly thesame in the cross-direction. To test paper strength, thetop portion of a small piece of Whatman #1 filter paper(1.5 in × 0.5 in) was attached to a holder and weight wasadded to the opposite end of the strip. We calculated theexact amount of weight required to tear that individualpiece of paper. This process was repeated three times foreach binder applied.To test all the potential green binders, approximately2 ml of each enzyme/substrate mixture described belowwas dispensed into incubator trays and the strips ofpaper were allowed to incubate in this solution. All reac-tions were incubated at 50°C and the pH used dependedon the results of the enzyme tests previously performed.Reactions containing enzymes were allowed to proceedfor 1 to 15 minutes.After incubating in binder solution, the strips weredried at approximately 200°C for 10 min, submerged inwater, and the strength of each wet paper strip wastested in the cross-direction. Three paper strips (i.e., 3replicates) were used for each treatment. The meanweight needed to tear each set of three strips of paperfor each treatment was recorded. These measurementswere compared to two control treatments: paper withno binder applied and paper with the vinyl acetatebinder applied.Substrate cross-linkingHorseradish peroxidase (HRP) was obtained from SigmaChemical Co. (P6782 St. Louis, MO). Gel electrophoresisof reaction products was used to determine the extent thesubstrate gained molecular weight, or was cross-linked,after reacting with the enzyme under certain conditions.Thermo Scientific gradient gels (4-20% acrylamide) wereused with HEPES buffer.Approximately 6 μg of HRGP, 5 μg of HRP, and 6 μg of8 mM hydrogen peroxide were used, as described byDevaiah & Shetty [28], and incubated at room temperaturefor one hour, then analyzed by SDS-PAGE. The sameFlory et al. BMC Biotechnology 2013, 13:28 Page 11 of 14http://www.biomedcentral.com/1472-6750/13/28concentrations were used in reaction mixtures that were in-cubated at 22, 28, 37, 42 and 50°C, for 1, 5, 10, 30 and 60minutes before being stopped with 0.1% SDS andmercaptoethanol and separated on a gel. The same experi-ment was per-formed at different pH (2, 3, 4, 5 and 6).HRP was also combined with lignin, black liquor andferulic acid, individually. These samples were tested withgel permeation chromatography, as described by Mansfieldet al. [29], to determine if this enzyme caused any molecu-lar weight changes in these phenolic compounds. Gel per-meation chromatography columns, series 60 and 300, wereutilized (YMC Co., Ltd. Kyoto, Japan). The column packingmaterial was silica derivatized with 1,2-dihydroxypropane.Lignin, black liquor and ferulic acid were each combinedwith laccase and gel permeation chromatography wasperformed. Reactions were incubated in sodium acetatebuffer, pH 5, at 50°C as reported by Bailey et al. [14]. Sam-ples were allowed to incubate for 15 and 60 minute timeperiods to ensure that cross-linking occurred. For eachtime period, a high and low concentration of 150 μg and15 μg were chosen for the reaction mixture.Protein bindersGelatin (Great Lakes Gelatin Co., Grayslake, IL), hydroxyproline-rich glycoprotein (HRGP, isolated from maizesilk), textured soy protein (TSP, residue post oil extrac-tion) and zein (Acros Organics, New Jersey, USA) weretested as protein binders. JELL-OW (Kraft Foods, Glenview,IL) and KnoxW unflavored gelatin (Kraft Foods, Glenview,IL) were tested.KnoxW gelatin concentrations of 5, 7 and 9% weredissolved in boiling water then applied to Whatman #1filter paper. For each percentage of KnoxW gelatin used,0, 150, 200, and 250 μg of HRP was added. All reactionswere incubated for 1 minute at 50°C. Gelatin from por-cine sources (blooms 100 and 300) and bovine sources(blooms 100 and 250) were obtained from Great LakesGelatin Company (Grayslake, IL). The highest and low-est bloom strength available from each source was pur-chased. For each source and for each bloom strength, 5,7 and 9% gelatin solutions were made as above and ap-plied to paper. The 9% JELL-OW was applied to paperwith and without HRP.HRGP was extracted from the cell walls of maize silkwith calcium chloride and sodium metabisulfite as de-scribed by Hood et al. [6]. Concentration of HRGP wasdetermined by absorbance at 280 nm using a microplatereader (Synergy HT, Bio-Tek). The concentration ofHRGP applied to paper was approximately 1mg/ml andthe reaction with HRP and H2O2 added was allowed toproceed at 50°C for 15 minutes before the paper wasdried and the strength was tested.Defatted soybean meal was ground in a coffee grinderand then mixed with water at 1:10 (w/v). The mixture ofwater, ground meal, HRP, and H2O2 was analyzed byprotein electrophoresis to determine if cross-linking oc-curred with this substrate. The ground soy slurry wascentrifuged for 5 minutes at 1,860 × g. The supernatantwas then applied to paper strips with 250 μg of HRP and8 mM H2O2 and was carried out for 1 and 10 minutesat 50°C. The supernatant was also applied to paper withno enzyme present. Finally, the pellet was resuspendedin water and applied to paper with no enzyme present.Zein protein was obtained from Sigma Chemical Co. (St.Louis, MO). A cross-linking test for this substrate wasdone by gel electrophoresis. A 3% solution of zein in 50%ethanol was applied to paper with and without HRP.Carbohydrate bindersA variety of carbohydrates that are common substitutesfor gelatin were tested. A range of 1 to 15% of each carbo-hydrate was dissolved in boiling water. Since gum Arabiccontains both polysaccharides and glycoproteins, a 5% so-lution was made with boiling water and applied to paperwith 250 μg of HRP and 8mM H2O2 and without enzyme.Flax seeds were ground with liquid nitrogen and 10 ml ofwater was added to every gram of ground flaxseed beforeapplication to paper.Several different types of pectin were utilized through-out this project. Two types of Sure-JellW pectin and BallWpectin were obtained from local grocers. The first pectinhad less sugar added than the second, regular pectin.These two pectins also contained different acidic compo-nents that assist in gelling. Low sugar pectin contained fu-maric acid and sodium citrate whereas the regular pectinonly contained citric acid. BallW pectin’s ingredients in-cluded citric acid, sodium citrate and potassium sorbate.Boiling water was not necessary to dissolve BallW pectin.Sure-JellW pectins were dissolved in boiling water.To determine the effectiveness of pure pectins asbinders, apple pectin was purchased from Sigma Chem-ical Co. (St. Louis, MO) and grapefruit pectin from alocal health food store. For pure apple and grapefruitpectin, solutions of 1-5% were dissolved in boiling waterand were applied to paper with and without citric acid.The amount of citric acid added was half the amount byweight of pectin added in each solution.Lignin/Phenolic compound bindersBlack liquor, lignin low sulfonate, sodium lignin sulfon-ate, marasperse, ferulic acid, coniferyl alcohol and Salixlignin were the potential substrates used in these setsof experiments.Samples of black liquor were obtained from BuckeyeTechnologies (Perry, FL). Ultra filtration of black liquor,using nitrogen gas and a regenerated cellulose membranewith a 1,000 Dalton molecular weight cutoff, was used toremove unwanted sulfur salts and to concentrate theFlory et al. BMC Biotechnology 2013, 13:28 Page 12 of 14http://www.biomedcentral.com/1472-6750/13/28sample. Approximately 5 ml of black liquor was mixedwith 100 ml of water in order to ensure adequate filtrationwas achieved and to lessen the chance of the black liquorclogging the filter.Three types of tests were carried out with this binder.First, black liquor was applied to Whatman #1 filter paperwith and without enzymes. Three solutions were made;black liquor with 250 μg of laccase, black liquor with 250μg of HRP and 8mM H2O2, and black liquor with no en-zyme. After applying these solutions to paper, they weredried at 100°C for 10 minutes.Second, paper strips were dipped in black liquor, hungup with binder clips in the fume hood and 1N HCl wassprayed on both sides of the strips with a thin-layer chro-matography sprayer. The strips were allowed to air dry for5 minutes then dried completely at 100°C for 10 minutes.Third, high heat was applied to black liquor treatedpaper. After liquor application on to paper, the paperstrips were dried at 200°C for 10 minutes before beingrewet and tested for strength in the cross-direction.Lignin low sulfonate (Sigma Chemical Co. St. Louis,MO), sodium lignin sulfonate, (MP Biomedicals, LLC.Solon, OH) and marasperse (Lignotech Rothschild, WI)were dissolved in water to make 0.05, 0.1 and 0.15 g/mlsolutions for application to paper. Each concentration ofeach type of lignin was applied to paper and sprayedwith HCl as previously described for black liquor. HClwas added to lignin samples in order to reduce the pHof the solution [14]. These paper strips were dried at100°C for 10 minutes. Lastly, all concentrations of eachlignin were applied to paper and dried at temperaturesranging from 100°C to 200°C for 10 minutes before ten-sile strength was tested.Ferulic acid and coniferyl alcohol were obtained fromSigma Chemical Co. (St. Louis, MO). Two mg of ferulicacid were first dissolved in 100 μl of dimethyl sulfoxide(DMSO), then water was added to equal 1 ml. Two mgof coniferyl alcohol were dissolved in 1ml of water. Bothsubstrates were mixed with 250 μg of HRP and 8mMH2O2 for application on to Whatman #1 filter paper.Finally, 0.05 g/ml and 0.10 g/ml of Salix lignin (Vertichem,Toronto, Canada) were dissolved in 60% ethanol. This solu-tion was heated to approximately 55°C in the microwave todissolve any insoluble fractions. The solution was then ap-plied to paper, allowed to dry at room temperature for 5 mi-nutes then dried at 200°C for 10 minutes before rewettingand testing strength in the cross-direction.Baby wipe solution testsSmall strips of Whatman filter paper with binder of inter-est applied were soaked in baby wipe solution, at roomtemperature for up to 3 months. The strength was testedand compared to the factory’s binder (referred to ascommercial binder) that had also been soaked in solutionfor the same amount of time.AbbreviationsABTS: 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid); DMSO: Dimethylsulfoxide; FA: Ferulic acid; GPC: Gel permeation chromatography;HCl: Hydrochloric acid; HRGP: Hydroxyproline-rich glycoprotein;HRP: Horseradish peroxidase; kDa: KiloDaltons; PEO: Poly (ethylene) oxide;SDS-PAGE: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis;TSP: Textured soy protein.Competing interestsThe authors declare that they have no competing interests.Authors’ contributionsAF carried out the experiments with binders and enzyme testing. DVRcoordinated, drafted and edited the manuscript. SD participated in the designof the study and performed some enzyme reactivity assays. SM performed thegel permeation chromatography. KT served as advisor throughout the projectand performed the initial lignin-acid precipitation experiments. EH conceivedthe study, participated in its coordination and helped to draft the manuscript.All authors read and approved the final manuscript.Authors’ informationEEH has worked in the area of cell wall structure and function for 25 years.This project represents an application of that knowledge. SPD is an enzymebiochemist and advised the enzyme reactions. SDM is a lignin chemist andadvised the lignin trials and performed the GPC.AcknowledgementsWe would like to acknowledge Buckeye Technologies Inc. (Perry, FL) forproviding us with samples of black liquor and Vertichem (Toronto, Canada)for supplying Salix lignin.Author details1Arkansas Biosciences Institute, Arkansas State University, Jonesboro, AR72467, USA. 2Department of Biology, Arkansas State University, Jonesboro, AR72467, USA. 3College of Agriculture and Technology, Arkansas StateUniversity, Jonesboro, AR 72467, USA. 4Department of Wood Science,University of British Columbia, 4030-2424 Main Mall, Vancouver, BC V6T 1Z4,Canada.Received: 23 August 2012 Accepted: 18 March 2013Published: 26 March 2013References1. Lora JH, Glasser WG: Recent industrial applications of Lignin: asustainable alternative to nonrenewable materials. J Polym Environ 2002,10(1):39–48.2. Iiyama K, Lam TT, Stone BA: Covalent cross-links in the cell wall. PlantPhysiol 1994, 104:315–320.3. 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Coffin DR, Fishman ML: Physical and mechanical properties of highlyplasticized pectin/starch films. J Appl Polymer Sci 1994, 54(9):1311–1320.25. Hoagland P, Parris N: Chitosan/pectin laminated films. J Agric Food Chem1996, 44:1915–1919.26. Matsuda S, Iwata H, Se N, Ikada Y: Bioadhesion of gelatin films crosslinkedwith glutaraldehyde. J Biomed Mater Res 1999, 45(1):20–27.27. Mohan D, Pittman CU, Steele PH: Pyrolysis of Wood/Biomass for Bio-oil: ACritical Review. Energy Fuel 2006, 20(3):848–889.28. Devaiah SP, Shetty HS: Purification of an infection-related acidicperoxidase from pearl millet seedlings. Pestic Biochem Phys 2009, 94(2–3):119–126.29. Mansfield SD, De Jong E, Saddler JN: Appl Environ Microbiol 1997,63(10):3804–3809.doi:10.1186/1472-6750-13-28Cite this article as: Flory et al.: Development of a green binder systemfor paper products. BMC Biotechnology 2013 13:28.Submit your next manuscript to BioMed Centraland take full advantage of: • Convenient online submission• Thorough peer review• No space constraints or color figure charges• Immediate publication on acceptance• Inclusion in PubMed, CAS, Scopus and Google Scholar• Research which is freely available for redistributionSubmit your manuscript at www.biomedcentral.com/submitFlory et al. BMC Biotechnology 2013, 13:28 Page 14 of 14http://www.biomedcentral.com/1472-6750/13/28

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