The modification of the lignin structure in an eucalypt feedstock after alkaline deconstruction by the kraft, soda-AQ and soda-O2 processes was assessed. For this purpose, the residual lignins isolated from the pulps at kappa levels 50, 35, 20 and 15 were analyzed by different analytical techniques, including 2D-NMR, 31P-NMR and SEC (size exclusion chromatography). In addition, dissolved lignins precipitated from the black liquors of the kraft and soda-AQ processes at kappa 20 and 15 were also analyzed. The distribution of the different lignin substructures in the residual lignins was similar to that in the native lignin, although with a drastic reduction in their content. This reduction was more evident in pulps with lower kappa number due to the more drastic pulping conditions.The research leading to these results has received funding from the European Community's Seventh Framework Programme FP7/2007-2013 under grant agreement no KBBE-2009-3-244362 LignoDeco (EU/Brazil co-operation). We thank Suzano Papel e Celulose (Brasil) for providing the pulps and the black liquors.Peer reviewe

Berbís, Manuel Álvaro

Gutiérrez Suárez, Ana

Jiménez-Barbero, Jesús

Liitiä, Tiina

Martínez, Ángel T.

Prinsen, Pepijn

Río Andrade, José Carlos del

Tamminen, Tarja

Digital.CSIC

MODIFICATION OF THE LIGNIN STRUCTURE OF EUCALYPT FEEDSTOCKS DURING CHEMICAL DECONSTRUCTION BY KRAFT, SODA-AQ AND SODA-O2 PROCESSING  Pepijn Prinsen1*, Ana Gutiérrez1, José C. del Río1, Manuel A. Berbis2, Jesús-Jiménez Barbero2, Angel T. Martínez2, Tiina Liitiä3 and Tarja Tamminen3. 1 Instituto de Recursos Naturales y Agrobiología de Sevilla, CSIC, PO Box 1052, E-41080 Seville, Spain; 2 Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, E-28040 Madrid, Spain; 3 VTT Technical Research Centre of Finland, Biologinkuja 7,  Espoo, P.O. Box 1000, FI-02044 VTT, Finland  *Email: prinsen@irnase.csic.es ABSTRACT The modification of the lignin structure in an eucalypt feedstock after alkaline deconstruction by the kraft, soda-AQ and soda-O2 processes was assessed. For this purpose, the residual lignins isolated from the pulps at kappa levels 50, 35, 20 and 15 were analyzed by different analytical techniques, including 2D-NMR, 31P-NMR and SEC (size exclusion chromatography). In addition, dissolved lignins precipitated from the black liquors of the kraft and soda-AQ processes at kappa 20 and 15 were also analyzed. The distribution of the different lignin substructures in the residual lignins was similar to that in the native lignin, although with a drastic reduction in their content. This reduction was more evident in pulps with lower kappa number due to the more drastic pulping conditions. I. INTRODUCTION During the deconstruction of the lignin polymer in pulping processes, degradation of preferentially non-condensed moieties is the main reaction, although the accumulation of condensed moieties may also result from condensation reactions. This lowers the delignification rate drastically. Soda processes can induce recalcitrant structures to posterior oxygen bleaching stages. In the production of bioethanol and other fermentation products, the mechanical characteristics of the cellulose pulp fibers are not essential as deconstruction of the whole lignocellulosic matrix is desired for the posterior hydrolysis steps. Therefore the oxygen treatment in alkaline conditions (soda-O2), which has more drastic consequences for the mechanical fiber properties, is a promising alternative to kraft and soda-AQ as pretreatment for biorefinery processes. In this work, we have studied the modifications of the lignin structure during alkaline processes (kraft, soda-AQ and soda-O2) at different kappa numbers. For this purpose, the residual lignins were isolated from these pulps and subsequently analyzed by an array of analytical techniques (2D-NMR, 31P-NMR and SEC). II. EXPERIMENTAL Samples. The pulps and black liquors selected for this study arise from the alkaline pulping of a Eucalypt hybrid E. grandis × [E. urophylla × E. globulus] (G1×UGL) feedstock, from the Brazilian Network of Eucalyptus Genome Research (Grattaplagia, 2004). Lignin isolation. The residual lignins were isolated from the pulps by acidolysis according to (Evtuguin et al., 2001). A two stage extraction of both 40 minutes under argon atmosphere using a dioxane:water (82:18 v/v) mixture with 0.1 M HCl was carried out, first at 13.3 and then at 10.0 ml/g dry pulp. After freeze-drying, the lignins were extracted with n-pentane during 8 hours in Soxhlet devices. The lignins of the black liquors were precipitated and air dried at 40 ºC.   2D-NMR. Around 40 mg of lignin was dissolved in 0.750 mL of DMSO-d6. NMR spectra were recorded at 25 °C on a Bruker AVANCE 600 MHz instrument equipped with a cryogenically cooled z-gradient triple-resonance probe. The acquisition and processing parameters are described in (del Río et al., 2012). 31P-NMR. The phosphitylation of dry lignin samples was performed according to (Granata and Argyropoulos, 1995). The 31P-NMR spectra were recorded on a Bruker Avance III 500 MHz spectrometer using a 90º pulse width and an inversegated decoupling sequence. CDCl3 was  the  locking solvent and 512 scans were accumulated with a delay time of 5 s between successive pulses. For processing data, the chemical shifts were calibrated to the sharp signal (132.2 ppm) of the reaction product between water and phosphitylation reagent and the line broadening was set to 2 Hz, followed by exponential window multiplication, Fourier transformation, automatic/manual phase correction and automatic baseline correction. For all samples the gain of 203 scans was attained and the integration of the internal standard (IS) signal was set to 1. Additionally the peak width at half height of the IS signal was checked to guarantee a satisfactory resolution and an exact quantification. After establishing optimal deconvolution parameters used for all samples, the deconvolutions of the syringylic - and condensed hydroxyl group signals were processed with a line broadening factor of 8 Hz. Between 12 and 20 peaks were used in the 144.2 – 141.ppm region to obtain an exact deconvolution result (Bruker topspin software). SEC. The molar mass measurements are relative measurements. They were performed by Waters HPLC in 0.1M NaOH eluent using PSS’s MCX 1000 and 100 000 Å columns with UV detection at 280 nm. The average molar masses (Mn, Mw) and the molar mass distributions were calculated relative to the sulphonated Na-polystyrene (Na-PSS, 3 420-148 500 g/mol) standards using Waters Empower  2  software.  For  SEC  analysis,  about  4  mg  lignin  was  dissolved  overnight  in  4  ml  analytical NaOH (0.1 M) and filtered with 0.45 µm PTFE membrane syringe filters (VWR).    III. RESULTS AND DISCUSSION Lignin isolation Table 1 shows that the extraction yield was higher at higher kappa levels, and higher for the soda-AQ than the Soda-O2 pulp. The yield at kappa 15 is similar for the three different pulps.   Table 1: Extraction yield (%) of lignin isolation by acidolysis of different pulps of G1×UGL eucalyptus based on theoretical content of:  % lignin in pulp  =  0.15 × K ( K = kappa number). Process Kraft Soda-AQ Soda-O2 Kappa number K20 K15 K50 K35 K20 K15  K50 K35 K15  35 26 82 63 28 28 60 41 28  13C - 1H HSQC 2D-NMR  The detailed structural characteristics of the MWL of G1×UGL eucalyptus from HSQC data has been reported in (Prinsen et al., 2012). From Table 2 it  can  be  seen  that  the  distributions  of  the  inter-unit linkages in the residual lignins are similar to that in the native lignin, although with a drastic reduction in their content. This reduction was more evident in the pulps with lower kappa number due to the more drastic pulping conditions.   Table 2: Content of lignin inter-unit linkages (expressed as linkages per 100 unmodified aromatic units) and S/G ratio in MWL of G1?UGL eucalypt wood, and in residual - and black liquor lignin from kraft, soda-AQ and soda-O2 processes at different kappa levels. MWL Residual lignin Black liquor lignin     Kraft Soda-AQ Soda-O2 Kraft Soda-AQ Inter-unit linkage   K20 K15 K50 K35 K20 K15 K50 K35 K15 K20 K15 K20 K15 ?-O-4´ ether 84 35 24 77 68 35 23 48 44 19 2 2 1 0 ???´ resinol 11.3 10.6 13.9 14.9 12.6 12.2 14.5 17.0 15.7 13.3 14.0 14.0 25.3 25.3 ?-5´ phenylcoumaran 4.3 0.8 0.2 1.4 1.2 0.9 0.8 1.0 1.6 1.7 0.0 0.0 0.0 0.0 S/G ratio 2.8 4.4 3.1 4.5 4.4 4.5 3.6 4.2 3.6 3.0 13.5 21.0 18.2 21.9  At kappa number 20 and 15, the content of ?-O-4´ ether linkages in the residual lignins was almost identical for the soda-AQ - as for the kraft process. The lignins precipitated from the respective black liquors were enriched in S-units, which are predominantly forming ?-O-4´ ether structures. This indicates that they are preferentially removed during pulping. Interestingly, the black liquor lignins from soda-AQ were more enriched in S-lignin units than those from the kraft process, suggesting that the soda-AQ is more efficient to depolymerize eucalypt lignin, although differences were not significant. From higher to lower kappa numbers the estimated S/G ratios of the residual lignins decreased as their respective ?-O-4´ linkages content decreased. At kappa 15 the degradation of ?-O-4´ ether - and ???´ resinol linkages was similar in the kraft - and soda-AQ pulps. The soda-O2 process seems to be more efficient in the degradation of ?-O-4´ ether linkages.  31P-NMR  The hydroxyl group (OH) content of lignins can be determined with quantitative 31P-NMR (Granata and Argyropoulos, 1995). When using 2-chloro-4,4,5,5-tetramethyl-l,3,2-dioxaphospholane as phosphitylating reagent, the aliphatic -, phenolic - and carboxylic OH signals are well resolved. In the case of hardwoods, the syringylic OH signals overlap with the condensed guayacyl OH signals. The same is observed in grasses, which show an additional p-hydroxyphenol OH signal.  The  aim of  this  work  was  the  accurate  separation  of  syringylic  -  and total  condensed OH signals, although some condensed structures like 4-O-5’ may overlap completely with the syringylic OH (high resolution needed for deconvolution). The deconvolution was carried out for all samples to quantifiy syringylic - and condensed OH content. The results are shown in Table 3.   Table  3: Aliphatic -, phenolic - (syringylic + guayacylic + condensed + catechol) and carboxylic hydroxyl content determined by quantitative 31P-NMR (mmol OH /g pure lignin) in G1?UGL eucalypt wood, residual - and black liquor lignin from kraft, soda-AQ and soda-O2 processes at different kappa levels. The standard desviation was calculated from 3 independent samples of one bulk sample + phosphitylation + 31P-NMR recording + integration + deconvolution for syringylic OH, total condensed OH and 5-5’ biphenylic content. MWL Residual lignin Black liquor lignin     Kraft Soda-AQ Soda-O2 Kraft Soda-AQ  OH group   K20 K15 K50 K35 K20 K15 K50 K35 K15 K20 K15 K20 K15 Aliphatic  (± 0.023) 4.69 2.20 1.69 3.33 2.80 2.07 1.73 2.81 2.55 1.75 1.07 0.60 1.06 0.83 Phenolic  (± 0.010) 0.67 1.72 2.05 1.47 1.56 2.13 2.25 1.64 1.71 1.82 2.50 2.83 2.57 2.42 Syringylic         (± 0.034) 0.21 0.75 0.85 0.86 0.69 1.01 1.13 0.87 0.79 0.79 1.52 1.74 1.51 1.37 Total condensed (± 0.041) 0.07 0.64 0.78 0.34 0.62 0.72 0.69 0.43 0.57 0.64 0.52 0.51 0.60 0.62 5-5‘ biphenylic (± 0.003 ) 0 0.096 0.098 0.052 0.090 0.072 0.066 0.048 0.036 0.033 0 0 0 0 Guayacylic        (± 0.002) 0.38 0.27 0.36 0.25 0.21 0.35 0.39 0.32 0.33 0.37 0.46 0.58 0.46 0.43 Catechol           (± 0.001) 0 0.057 0.065 0.019 0.033 0.044 0.037 0.016 0.023 0.022 0 0 0 0 Carboxylic        (± 0.002) 0.005 0.25 0.27 0.09 0.09 0.20 0.26 0.14 0.18 0.24 0.66 0.54 0.66 0.53 Total OH          (± 0.034) 5.37 4.17 4.01 4.89 4.44 4.40 4.24 4.59 4.43 3.81 4.24 3.97 4.29 3.77 S/G ratio           (± 0.11) 0.6 2.7 2.4 3.5 3.2 2.9 2.9 2.7 2.4 2.1 3.3 3.0 3.3 3.3  A negative linear correlation (r2 =  0.99)  was  observed  between  the  aliphatic  -  and  phenolic  OH  content for both MWL/kraft and MWL/soda-AQ data. In the case of MWL/soda-O2 the phenolic OH content remained more constant (r2 = 0.94 for linear correlation). The higher phenolic content in the residual lignin at K15 of the soda-AQ process was not a consequence of a higher condensed OH content, but mainly resulted from a higher syringylic OH content. Identical condensed OH contents were calculated from samples of two independent but similar soda-AQ pulping processes and were in contrast with the kraft residual lignin (higher condensed OH content at K15). The carboxylic OH content in the black liquor lignins was 2-3 times higher than in the corresponding residual lignins, althought it remained unclear if the signals at 134.61 (black liquors) and 134.69 (residual lignins) ppm represented carboxylic groups present in lignin or in fatty acids (Dyer et al., 2002). Interestingly, a linear correlation (r2 = 0.91) was observed between the content of 5-5‘ biphenyl structures and catechol structures. This indicates that both signals might come from one structure. Further investigation is needed to confirm this.   SEC  From the results in Table 2 and 4 an exponential correlation (r2 > 0.99) was found between the ?-O-4´ linkages content and the molar mass Mw of the residual lignins of the soda-AQ process. For the soda-O2 process the exponential fit was less appropriate (r2 = 0.88), because the ?-O-4´ linkages content was significant lower at kappa 50 and 35. At kappa 20 and 15 the molar masses of the residual lignins were similar for the different processes. The lowest molar mass at kappa 15 was found in the kraft residual lignin. The black liquor lignins had a molar mass of 55 and 65 % of the corresponding residual lignins at respectively kappa 20 and 15. Similar results were observed for the soda-AQ black liquor lignins (53 and 61 %).     Table  4:  Molar masses Mn and Mw (Daltons) and polydispersity of MWL of G1?UGL eucalypt wood, and residual - and black liquor lignin from kraft, soda-AQ and soda-O2 processes at different kappa numbers. MWL Residual lignin Black liquor lignin     Kraft Soda-AQ Soda-O2 Kraft Soda-AQ     K20 K15 K50 K35 K20 K15 K50 K35 K15 K20 K15 K20 K15 Mn 1891 2440 2370 2480 2510 2710 2530 2380 2360 2300 1680 1640 1660 1580 Mw 4430 4340 3490 10550 8300 4770 3870 9380 6080 3640 2390 2280 2540 2350 Polydispersity 2.34 1.78 1.47 4.25 3.30 1.76 1.53 3.93 2.58 1.58 1.42 1.39 1.53 1.48 IV. CONCLUSIONS The residual lignins of G1*UGL eucalypt isolated by acidolysis and the black liquor lignins from the kraft, soda-AQ and soda-O2 processes at different kappa levels showed rather similar results for the interunit linkages distribution, hydroxyl group content and molar mass. The residual lignin of the soda-O2 process showed more significant differences: lower ?-O-4´ linkages at similar molar mass (or lower molar mass at the same kappa number), more constant phenolic hydroxyl group content at different kappa levels and lower total hydroxyl group content.  V. ACKNOWLEDGEMENT The research leading to these results has received funding from the European Community's Seventh Framework Programme FP7/2007-2013 under grant agreement no KBBE-2009-3-244362 LignoDeco (EU/Brazil co-operation). We thank Suzano Papel e Celulose (Brasil) for providing the pulps and the black liquors. VI. REFERENCES del Rio J.C., Prinsen P., Rencoret J., Nieto L., Jiménez-Barbero J., Ralph J., Martínez A.T. and Gutiérrez A. Structural characterization of the lignin in the cortex and pith of elephant grass   (Pennisetum purpureum) stems. J. Agric. Food Chem. 2012, 60, 3619-3634.  Dyer T., Pu Y., Ragauskas A. J. and Nilvebrant N.-O. Application of lignin NMR techniques for wood extractives.  223rd ACS National Meeting, 2002, Orlando, U.S.A.  Evtuguin D.V., Neto C.P., Silva A.M.S.,  Domingues P.M., Amado F.M.L., Robert D. and Faix O. Comprehensive study on the chemical structure of dioxane lignin from plantation Eucalyptus globulus wood, J. Agric. Food Chem. 2001, 49, 4252-4261.  Granata A. and Argyropoulos D.S. 2-Chloro-4,4,5,5-tetramethyl-1,3,2-dioxaphospholane, a reagent for the accurate determination of the uncondensed and condensed phenolic moieties in lignins, J. Agric. Food Chem. 1995, 43, 1538-1 544.  Grattaplagia D. Integrating genomics into Eucalyptus breeding, Genet. Mol. Res. 2004, 3, 369–379.  Prinsen P.,  Gutiérrez A., Rencoret J.,  Nieto L., Barbero J.-J,  Burnet A., Petit-Conil M., Colodette J.L., Martínez A.T. and del Río J.C. Morphological characteristics and composition of lipophilic extractives and lignin in Brazilian woods from different eucalypt hybrids, Ind. Crop. Prod. 2012, 36, 572-583.  

Modification of the lignin structure of eucalypt feedstocks during chemical deconstruction by kraft, soda-AQ and soda-O2 processing

Modification of the lignin structure of eucalypt feedstocks during chemical deconstruction by kraft, soda-AQ and soda-O2 processing

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