RAZISKAVE IN RAZVOJ Papir za notranjost revije PAPIR je prispevala papirnica Goričane, tovarna papirja Medvode, d.d. Sora Matt + 80 g/m 2 . Papir za notranjost revije PAPIR je prispevala papirnica Goričane, tovarna papirja Medvode, d.d. Sora Matt + 80 g/m 2 . RAZISKAVE IN RAZVOJ Raziskujemo in razvijamo Raziskujemo in razvijamo | junij 2018 | 19 | XLVI | junij 2018 | 19 | XLVI 1 INTRODUCTION Pigment coatings are applied to optimize the surface and printing properties of coat- ed papers (Huang and Lepoutre, 1998). A coating color consists of several compo- nents having different functions. To bind the pigments and to fix the coating layer to the base paper, today mainly petrole- um-based latex binders are used. There are efforts to increasingly substitute these petroleum-based products with renewa- ble binders (Flory et al., 2013; Gosselink et al., 2010). Biopolymers like starch, proteins and lignocellulosic materials are gaining in importance in paper coating applications (Imam et al, 2013). Technical lignins, such as lignosulfonates or kraft lignins, are an abundant source of biopolymers which to- day are mainly used for energy production. Material applications of technical lignins for special products range only between 1 and 2% (Gosselink et al., 2004), which is mainly due to the heterogeneity of techni- cal lignins. Still more and more applications of technical lignins as building blocks for polymers, resins or adhesives have been de- veloped recently (Stewart, 2008). The aim of this work was to test the feasi- bility of the development of a renewable binder based on the lignosulfonates con- tained in the spent liquor from an indus- trial source (Mg-sulfite pulp mill) in order to substitute partially petroleum-based SB-latices in pigment coating applications. The potential of enzymatic polymerization of lignosulfonates to enhance the binding properties via the modification of the mo- lecular structure was also studied. 2 MATERIALS AND METHODS All used chemicals were of analytical grade, purchased from Sigma-Aldrich (Steinheim, Germany), Merck (Darmstadt, Germany) or VWR (Vienna, Austria). Laccase from Myce- liophthora thermophila was obtained from Novozymes (Bagsvard, Denmark). Industrial Mg-sulfite spent liquor contain- ing lignosulfonates from an acid magnesi- um bisulfite process from the intermediate evaporation stage (approx. 30 % solids) and precoated base paper was kindly pro- vided by SAPPI Gratkorn (Austria). Ultrafiltration of lignosulfonate A Memcell unit supplied by Osmo Mem- brane Systems GmbH (Korntal-Muenchin- gen, Germany) was used to ultrafiltrate sulfite spent liquor. The sulfite spent liquor having a solids content of 30 % was first pre-filtered with a 5µm paper filter to re- move solid particles and fibrous material. Another prefiltering step using the Mem- cell device was performed using a 2µm membrane. Salts, sugars and smaller lignin fractions were subsequently removed using a 150kDa membrane by repeatedly adding water until the permeate was colorless. Afterwards, the retentate retained on the 150 kDa ultrafiltration membrane (R150), which had a solids content of around 10- 12%, was brought to 30% solids using a rotary evaporator. Enzymatic lignin polymerization A recently described laccase mediated lignosulfonate modification process based on oxygen supplementation was used, which avoids the use of expensive me- diators (Ortner et al., 2015; Huber et al., 2016). Enzymatic polymerization of 30% TDS lignosulfonate with the Myceliophtho- ra thermophila laccase (MTL) (introducing 233 nkatal ml -1 laccase) was carried out at pH 7 in the presence of external oxygen supply (monitored using a FireSting-O 2 device from PyroScience GmbH (Aachen, Germany)). K. HOFER 3 , A. ORTNER 2 , G. S. NYANHONGO 2 , H. WINTER 3 , K.MAHLER 3 , G. GÜBITZ 2 , W. BAUER 1 ABSTRACT The exploitation of renewable and cheaper paper coating formulations especially for graphic paper products is gaining impor- tance due to concerns regarding the use of fossil-based raw materials. For this reason, a new process for enzymatic modification of lignosulfonates to substitute fossil-based styrene-butadiene (SB) latex as a binder in conventional paper coating formulations was developed. Laccase polymerization of lignosulfonates resulted in the increase of molecular weight. When used in coating for- mulations, laccase polymerized lignosulfonate resulted in coated paper with good printing properties (reduced picking) provided that the lignosulfonate was ultrafiltrated before polymerization. Similarly, cross-sectional fluorescence microscopy images showed that ultrafiltration and laccase polymerization reduced the penetration of the polymerized lignosulfonates into the base paper by 33 % in view of coating formulation. These results demonstrate the possibility of substituting fossil-based styrene-butadiene (SB) latex binders with on-site produced lignosulfonates which have traditionally been considered mainly as a by-product used for en- ergy production in the pulp and paper industry. Key words: laccase, paper coating, lignosulfonate, polymerization, binders IZVLEČEK Izkoriščanje obnovljivih in cenejših vrst papirnih premazov predvsem za grafične papirje ima vse večji pomen predvsem zaradi vedno večjih pomislekov pri uporabi fosilnih surovin. Iz tega razloga je bil razvit novejši postopek za encimatsko modifikacijo lignosulfonatov, kot zamenjava za veziva na osnovi stiren butadienskega lateksa (SB), ki se običajno uporabljajo kot vezivo v premazih za premazovanje papirja. Polimerizacija lignosulfonatov z lakazo vpliva na povečanje molekulske mase. Pri uporabi v sestavi premaza za premazovanje papirja vpliva na premazan papir z izboljšanimi tiskovnimi lastnostmi (zmanjša se cepljenje papirja) pod pogojem, da je bil lignosulfonat pred polimerizacijo ultrafiltriran. Tudi prečni posnetki fluorescenčne mikroskopije kažejo, da ultrafiltracija in polimerizacija lakaze za 33 % zmanjša penetracijo polimeriziranih lignosulfonatov v osnovni papir glede na sestavo premaza. Rezultati prikazujejo možnosti zamenjave klasičnega lateks veziva na osnovi stiren-butadiena (SB) z lokalno proizvedenimi lignosulfonati, ki se tradicionalno pridobivajo kot stranski produkt v proizvodnji celuloze in se običajno uporabljajo za proizvodnjo energije v industriji celuloze in papirja. Ključne besede: lakaza, premazovanje papirja, lignosulfonati, polimerizacija, veziva ENZYMATICALLY MODIFIED LIGNOSULFONATES AS COATING BINDERS UPORABNOST ENCIMATSKO MODIFICIRANIH LIGNOSULFONATOV KOT VEZIVA V PREMAZIH Size exclusion chromatography (SEC) The molecular weights of treated and untreated lignosulfonate samples were determined using size exclusion chroma- tography (SEC) equipped with a degasser, a binary pump, an auto sampler, a DAD (Diode Array Detector) and a RI (Refractive Index)-detector system (Agilent Technolo- gies 1260 Infinity). A guard column (Waters Ultrahydrogel, 200 Å, 6 x 40 mm, maxi- mum pressure 3.93 MPa) was placed be- fore the two separating columns (Waters Ultrahydrogel 500, 500 Å, 7.8 x 300 mm, 3.93 MPa and Waters Ultrahydrogel 250, 250 Å, 7.8 x 300 mm, 1.96 MPa) connect- ed in a series. 0.1 M NaNO 3 solution was used as the mobile phase and the runtime was 120 minutes. The lignosulfonates were diluted with the mobile phase to a concen- tration of 1 mg*mL-1 before loading 100µl onto the column. Chromatograms were analyzed with the Agilent GPC/SEC Soft- ware (Version 1.2). The standards used for size exclusion chromatography (SEC) were Polystyrene Sulfonate Sodium Salts with molecular weights ranging from 208 g/mol to 1,188,400 g/mol. Coating color preparation and labora- tory coating process Coating formulations (precoat for a dou- ble coated WFC paper) were prepared using calcium carbonate HC60 from Omya (Oftringen, Switzerland), SB-latex from BASF (Ludwigshafen, Germany), PVOH (Mowiol 4-98) from Kuraray Europe GmbH (Hattersheim am Main, Germany) and CMC (Finnfix 30) from CP Kelco (Cumber- land, USA). Latex was partially substituted by untreated and enzymatically modified lignosulfonate in a ratio of 1:2, i.e. latex amount was reduced by 2%, and 4% of the ultrafiltrated lignosulfonate was add- ed, either with or without enzymatic po- lymerization. Coating was performed on a film press using profiled rod with 30µm depth of profile at a lab coater speed of 15 m/min. The base paper was an uncoat- ed wood-free base paper. The target coat weight was 10g/m² per side. Modified* lignosulfonate Untreated** lignosulfonate Reference Calcium carbonate (HC60) 100 100 100 Modified lignosulfon- ate* 4 Untreated lignosulfon- ate** 4 SB-Latex 6 6 8 PVOH 0.5 0.5 0.5 CMC 0.5 0.5 0.5 Target solids content 60 60 60 * enzymatically polymerized, ultrafiltrated ** no enzymatic polymerization, ultrafiltrated Table 1: Precoat formulations [% dry substance] Preglednica 1: Sestava predpremaza [% suhe snovi] Coating and paper testing Water retention of the coating colors was quantified by measuring the Abo Akademi Gravimetric Water Retention Value (AA- GWR). Furthermore, pH and low shear (Brookfield) viscosity were controlled. Grammage was determined according to EN ISO 536. Optical properties (R457 +/+UV) were measured using Technidyne Color Touch 2 (DIN 6174/ TAPPI T452). Printability parameters (passes-to-fail, set- off, droplet test (ink repellence)) were de- termined using a Prüfbau device and pick- ing resistance using an IGT device. 3 RESULTS AND DISCUSSION Preliminary trials with lignosulfon- ates containing spent liquor First coating trials using lignosulfonate containing spent liquor directly from the pulping process at 30% TDS, only filtered through a 5µm paper filter to remove sol- id particles and fibrous material, showed dusting immediately after coating drying of the reel-to-reel coater, indicating a very low binding force of the lignosulfonates. Ex- tremely poor IGT picking tests verified this observation. For lignosulfonate containing samples, no values could be determined, because the first signs of picking were ev- ident below 2 cm under the lowest loading. As the water retention value (AA-GWR) of 975 g/m² (see also Figure 3, left bar) of this coating was very poor, binder penetra- tion into the base paper was measured for this coating applied directly on an uncoat- ed base paper using a method developed by Hofer et al. (2015), which makes use of the autofluorescence of lignosulfonates. In the BF and GFP images on the left side of Figure 1, a cross-section of a coated paper with untreated lignosulfonate containing spent liquor (no ultrafiltration, no enzy- matic polymerization) is illustrated. The BF Figure 1: Penetration of lignosulfonates from spent liquor into the base paper. Brigthfield (BF) and green fluorescent (GFP) images of blank lignosulfonate (untreated lignosulfonate from spent liquor) (left) compared to BF and GFP image of enzymatically polymerized lignosulfonate from spent liquor (right) Slika 1: Penetracija lignosulfonata iz odpadne lužnice v osnovni papir. Primerjava BF (brigthfield) in GPF (green fluorescent) posnetkov neobdelanega (levo) in encimatsko polimeriziranega lignosulfonata iz odpadne lužnice (desno) (visual light) image shows the coating layer appearing darker, whereas the GFP image (UV light) shows the green fluorescing lignosulfonate; the white lines in the GFP image indicate the segmented coating layer from the BF image. The lack of lignosul- fonate binder in the coating layer is obvi- ous; the lignosulfonate is penetrating deep- er into the base paper and the depletion of the lignosulfonate binder in the coating layer is clearly visible. Almost all of the bind- er penetrated into the porous system of the base paper and the binding force of the coating layer is too low. To overcome this excessive penetration of lignosulfonates contained in the spent liq- uor into the base paper by an increase in molecular weight, enzymatic polymeriza- RAZISKAVE IN RAZVOJ Papir za notranjost revije PAPIR je prispevala papirnica Goričane, tovarna papirja Medvode, d.d. Sora Matt + 80 g/m 2 . Papir za notranjost revije PAPIR je prispevala papirnica Goričane, tovarna papirja Medvode, d.d. Sora Matt + 80 g/m 2 . RAZISKAVE IN RAZVOJ Raziskujemo in razvijamo Raziskujemo in razvijamo | junij 2018 | 19 | XLVI | junij 2018 | 19 | XLVI tion of the lignosulfonates with a laccase mediated modification process based on oxygen supplementation (Ortner et al., 2015; Huber et al., 2016) was tried. First trials with industrial spent liquor contain- ing lignosulfonates originating directly from the intermediate evaporation stage, only filtered through a 5 µm paper filter, showed a significant increase in the average mo- lecular weight after six hours of enzymatic polymerization (Figure 2). The penetration of lignosulfonates into the base paper could be reduced dramatically by enzymatic polymerization (see GFP im- age on the right side of Figure 1). Three-di- mensional evaluation of 100 cross-sections similar to/like the ones depicted in Figure 1 Figure 2: Molecular weight distribution (SEC) of enzymatically polymerized lignosulfonate from spent liquor with 30% TDS (right) compared to blank lignosulfonate (untreated lignosulfonate) at 30 % TDS (left, no enzyme treatment) and their average molecular weights (table below figure) Slika 2: Porazdelitev molske mase (SEC) encimatsko polimeriziranega lignosulfonata iz odpadne lužnice s 30 % suhe snovi (TDS) (desno) v primerjavi z neobdelanim lignosulfonatom s 30 % suhe snovi (levo, brez encimatske obdelave) in njihova povprečna molska masa (preglednica pod sliko) Figure 3: Penetration depth of lignosulfonates into the base paper (measured according to Hofer et al. (2015)) and AA-GWR water retention of coatings containing lignosulfonates from spent liquor compared to the reference Slika 3: Globina penetracije lignosulfonata v osnovni papir (meritve izvedene v Hofer et al., 2015) in retencija vode (AA-GWR) v premazu, ki vsebuje lignosulfonate, v primerjavi z referenčnimi vrednostmi (image length > 5mm) resulted in the pene- tration depth illustrated in Figure 3. Due to enzymatic polymerization, the penetration depth was decreased by around 10µm. The penetration depth for the SB-latex refer- ence was not determined because SB-latex needs to be stained by a fluorescent dye and the results are not directly comparable to the lignosulfonate coatings. The observations regarding penetration depth were confirmed by measuring a more than 50% higher water retention (low AA-GWR) for the enzymatically polym- erized lignosulfonates compared to untreat- ed samples. As can be seen in Figure 3, the modified lignosulfonates were in the same AA-GWR range as the reference. Although binder penetration was signifi- cantly reduced using enzymatically polym- erized lignosulfonates from the spent liq- uor, the binding force was still insufficient, which we attributed to a large amount of impurities contained in the industrial ligno- sulfonates, as e.g. salts, sugars or extrac- tive. Therefore, ultrafiltration was applied in all further trials to eliminate the impurities, which were assumed not to contribute to the binding function on the one hand and to hinder the lignosulfonate binder to form a continuous film on the other. Trials using ultrafiltrated lignosulfon- ate Using such ultrafiltrated lignosulfonate samples, the incubation time in enzyme polymerization could be reduced from six to two hours to reach a similar molecular weight increase compared to non-ultrafil- trated samples. Thus a sufficiently reproducible high degree of polymerization was achieved after mere- ly two hours of incubation at an oxygen supply rate of 15cm 3 , leading to an increase of the average molecular weight from 14092 to 97574 Da (see Figure 4). It should be noted that ultrafiltration alone leads to a higher molecular weight of the lignosul- fonates (compare blank in Figure 4 to blank in Figure 2). Lignosulfonates as binders in precoat application Ultrafiltrated and enzymatically polymerized lignosulfonates were tested in the applica- tion in a precoat (for recipes see Table 2) to hide the brown color of lignosulfonates un- der a white top-coating layer. Precoats were applied with a film press in single sheet mode, which also allows better adjustment of the coat weight. The results of the IGT pick test of the precoated samples are listed in Table 2. Untreated (ultrafiltrated, no en- zyme treatment) lignosulfonate showed a slightly lower picking resistance than mod- ified (ultrafiltrated, enzymatically polym- erized) lignosulfonates, and both showed clearly lower values than the reference. The three precoated papers were topcoat- ed using a standard topcoat. In Table 3, the Prüfbau printing results for the topcoat- ed papers are shown. The offset suitability of all three samples is comparable, with a slight advantage for the reference in pass- es-to-fail wet. Ink setting of the modified lignosulfonate containing precoat is com- parable to the reference, while untreated lignosulfonate containing precoat shows slower ink setting. The droplet test as a measure for water repellence shows signif- icantly higher values for the lignosulfonate containing samples compared to the reference. The brightness level of the double coat- ed samples is listed in Table 3. Brightness measured with and without UV is signifi- cantly lower compared to the reference. Figure 4: Molecular weight distribution (SEC) of enzymatically polymerized lignosulfonate (ultrafiltrated 2µm > x > 150kDa) with 30% TDS (right) compared to blank lignosulfonate (untreated ultrafiltrated lignosulfonate) at 30% TDS (left, no enzyme treatment,) and their average molecular weights (table below figures). Slika 4: Porazdelitev molekularne mase (SEC) encimatsko polimeriziranih lignosulfonatov suhe snovi (levo, brez encimatske obdelave) in povprečna molska masa (preglednica pod sliko). Modified Lignosul- fonate in PC Untreated Lignosul- fonate in PC Reference Precoat weight [g/m²] 10.4 9.9 9.6 IGT pick test [cm/s] 118 109 141 Modified lignosul- fonate in PC Untreated lignosul- fonate in PC Reference Offset suitability dry [passes to fail] 3 3 3 Offset suitability wet [passes to fail] 2 2 2,5 Ink setting after 30 s 0.38 0.48 0.36 Droplet test [%] 81.3 84.3 62.9 R457 +UV 83.23 81.7 92.38 R457 -UV 79.34 78.02 85.56 Table 2: Coat weight and IGT picking of precoatings Preglednica 2: Nanos premaza in IGT suho cepljenje predpremaza Table 2: Coat weight and IGT picking of precoatings Preglednica 2: Nanos premaza in IGT suho cepljenje predpremaza 4 SUMMARY AND OUTLOOK Ultrafiltrated and enzymatically polym- erized lignosulfonates were applied as partial replacement of SB-latex in wood- free coated paper coating formulations. Lower penetration depth of lignosulfon- ate-based binder into the base paper was achieved by ultrafiltration and enzymatic polymerization of the spent liquor con- taining lignosulfonates. This also led to a significant improvement in water reten- tion of the coating formulations contain- ing lignosulfonates. The coated papers containing a lignosulfonate-based binder showed promising results regarding off- set printability, which justifies further research. As expected, brightness values are signifi- cantly below the reference and need to be improved. Therefore, trials to bleach the lignosulfonates are currently in prog- ress with promising first results. Enzymatically polymerized lignosulfonates might also be interesting in size press applications for packaging papers, where brightness is not an issue. First results in such applications have shown promising results. ACKNOWLEDGEMENTS The authors acknowledge the indus- trial partners Sappi Gratkorn, Heinzel Pulp Pöls, Norske Skog Bruck and Mondi Frantschach, the Austrian Research Pro- motion Agency (FFG), COMET, BMVIT, BMWFJ, and the Provinces of Styria and Carinthia for their financial support of the K-project Flippr. 5 REFERENCES Flory, A.R., Vicuna Requesens, D., Devaiah, S.P ., Teoh, K.T., Mansfield, S.D., Hood, E.E. (2013). Development of a green binder system for paper products. BMC Biotechnology, 13 (28) Gosselink, R.J.A., de Jong, E., Guran, B., Abächerli, A., (2004). Co-ordination network for lignin—standardisation, production and applica- tions adapted to market requirements (EUROLIG- NIN). Ind. Crops Prod, 20, 121–129. Gosselink, R.J.A., Van Dam, J.E.G., De Jong, E., Scott, E.L., Sanders, J.P .M., Gellerstedt, G. (2010). Fractionation, analysis, and PCA modelling of properties of four technical lignins for prediction of their application potential in binders. Holzfor- schung, 64(2), 193-200. Hofer, K., Seidl, B., Ortner, A., Nyanhongo, G.S., Winter, H., Kozich, M., Gübitz, G., Bauer, W. (2016). Serial Sectioning of Coated Paper as a Novel Method to analyze Binder Penetration, Pa- per Technology, 57 (1), 39 – 41. Huber, D., Ortner, A., Daxbacher, A., Nyanhongo, G.S., Bauer, W. Guebitz, G. (2016). Influence of Oxygen and Mediators on Laccase-Catalyzed Po- lymerization of Lignosulfonate. ACS Sustainable Chemistry & Engineering 4 (10), 5303-5310 Imam, S.H., Bilbao-Sainz, C., Chiou, B-S., Glenn, G.M., Orts, W.J. (2013). Biobased adhesives, gums, emulsions, and binders: currrent trends and future prospects. Journal of Adhesion Sci- ence and Technology, 27(18-19), 1972 – 1997. Nugroho Prasetyo, E., Kudanga, T., Steiner, W., Murkovic, M., Nyanhongo, G., Guebitz, G. (2009). Antioxidant activity assay based on lac- case-generated radicals. Analytical and Bioanalyt- ical Chemistry, 393(2), 679-687. Ortner, A., Huber, D., Haske-Cornelius, O., We- ber, H., Hofer, K., Bauer, W., Nyanhongo, G., Guebitz, G. (2015). Laccase mediated oxidation of industrial lignins: Is oxygen limiting? Process Biochem., 50(8). Stewart, D. (2008). Lignin as a base material for materials applications: Chemistry, application and economics. Industrial Crops and Products, 27(2), 202 – 207. 1 Institute of Paper, Pulp and Fiber Technology, Graz University of Technology, Inffeldgasse 23, 8010 Graz, Austria 2 University of Natural Resources and Life Sciences Vienna, Institute of Environmental Biotechnology, Konrad-Lorenz Str. 20, 3430 Tulln, Austria 3 Sappi Paper Holding GmbH, Brucker Strasse 21, 8101 Gratkorn, Austria Corresponding Authors: wolfgang.bauer@tugraz.at