Characterization of Residual Lignins in Pulps Delignified by Laccase

Chapter 22

Characterization of Residual Lignins in Pulps Delignified by Laccase/N-Hydroxyacetanilide Downloaded by STANFORD UNIV GREEN LIBR on September 17, 2012 | http://pubs.acs.org Publication Date: March 26, 2001 | doi: 10.1021/bk-2001-0785.ch022

Kristiina Poppius-Levlin, Tarja Tamminen, Anna Kalliola, and Taina Ohra-aho K C L , Science and Consulting, P.O. Box 70, FIN-02151 Espoo, Finland

A pine kraft pulp and a two-stage oxygen-delignified pine kraft pulp were delignified with laccase in the presence of N-hydroxyacetanilide(NHA). Laccase/NHA was selective in its reactions with lignin: 30% delignification was obtained under the moderate reaction conditions employed, while the carbohydrates remained unattacked. Laccase/NHA did not react with neutral pulp carbohydrates or with uronic acids. Beside delignification, laccase/NHA caused chemical modification of fiber lignin, lowering the content of free phenolic hydroxyl groups and thus verifying their importance in delignification. Oxidation of the residual lignins was clearly observed as a decrease in methoxyl groups and a simultaneous increase in conjugated carbonyl groups, carboxyl and/or ester groups. Only 50-60% of the pulp residual lignin retained its aromatic structure. Pyrolysis-GC/MS gave important information, not only of the chemical structure of residual lignins, but also of the behavior of the mediator during delignification. Residuals of aniline and acetanilide, i.e. degradation products of NHA, were found among the pyrolysis products of residual lignins. However, only trace amounts of NHA-derived nitrogen-containing products were present in the lignins.

358

© 2001 American Chemical Society In Oxidative Delignification Chemistry; Argyropoulos, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 2001.

359

Downloaded by STANFORD UNIV GREEN LIBR on September 17, 2012 | http://pubs.acs.org Publication Date: March 26, 2001 | doi: 10.1021/bk-2001-0785.ch022

Introduction Laccases are lignin oxidizing enzymes, whose potential for pulp bleaching has been widely investigated since the invention of the laccase/mediator system, which enables the large enzyme molecule to react with fiber lignin via the small molecular mediator molecule (1). Investigations in this area are now aimed primarily at finding new efficient mediators with low production costs. Suitable mediators have been found to be compounds containing the N-OH group. One of the most widely studied mediators is 1-hydroxybenzotriazole (HBT) (1). More recently developed mediators include violuric acid and N hydroxyacetanilide, NHA (2), which was chosen for the present study. It has been suggested that NHA is superior to HBT as a mediator, because, unlike HBT, it does not inhibit laccase and is a more specific laccase substrate. It is also biodegradable and contains less nitrogen per molecule than HBT. NHA is also cheaper to produce than HBT (2). Pulps delignified with laccase in the presence of mediator are known to be more reactive towards final bleaching than the reference pulps (1,2,3,4,5). In order to explain this increased reactivity, as well as the reactions occurring during delignification, the chemical structures of the pulps themselves and of the residual lignins from a pine kraft pulp and from an oxygen-delignified pine kraft pulp before and after laccase/NHA delignification were studied. NHA was also compared to HBT as a mediator in laccase delignification.

Materials and Methods Pulps and laccase. Pine (Pinus sylvestris) kraft pulp was prepared in the laboratory. The sulphidity was 35% and the effective alkalinity was 3.9 mol/kg. The pulp was subjected to a two-stage oxygen delignification (00). The oxygen stages were carried out at 95°C for 60 min and 75 min using 2.0% and 1.4% NaOH, respectively, and at 0.8 MPa oxygen pressure at 10% consistency. The MgS0 x7H 0 charge was 0.5% on pulp in both stages. The properties of the pulps are shown in Table I. Laccase was produced by Trametes versicolor and was kindly donated by Consortium fiir electrochemische Industrie GmbH. 4

2

2

Pulp Treatments Laccase/NHA Treatment (L/NHA). Never-dried pulp (60 g abs. dry) was treated with laccase (15 IU/g pulp = 250 nkat/g pulp) in the presence of NHA (obtained from Consortium fiir electrochemische Industrie GmbH) at pH 4.5 and 45°C for 2 hours under 0.3 MPa oxygen pressure at 10% consistency in a

In Oxidative Delignification Chemistry; Argyropoulos, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 2001.


360 rotating autoclave. The NHA charge was 1.83% (121 mmol/kg pulp) on pine kraft pulp and 0.73% (48 mmol/kg pulp) on oxygen-delignified pulp. Thus the ratio of NHA to lignin was the same in both pulps. Oxygen-delignified pulp was also treated with laccase in the presence of 1.12% NHA (74 mmol/kg pulp). Laccase/HBT treatments were performed under the same conditions with 1.00% HBT (74 mmol/kg pulp) on oxygen-delignified pulp. Laccase treatments (L) without NHA and reference treatments without laccase or NHA were carried out under the same conditions as described for the laccase/NHA system. Alkaline extraction (E) was carried out at 10% pulp consistency, with 1% sodium hydroxide on pulp at 60°C for 1 h with occasional stirring. Residual lignins Residual lignins were isolated from the pine kraft pulp (NHA: 1.83%) and from the OO-delignified pine kraft pulp (NHA: 0.73%) before and after laccase and laccase/NHA treatments using an enzymatic method (6). The lignins were further purified as described earlier (7). Analyses Kappa number and viscosity were determined using the standard methods SCAN-C 1:77 and SCAN-CM 15:88, respectively. Pulp brightness was measured with an Elrepho instrument. Polysaccharides were hydrolysed to monosaccharides, which were quantitatively determined by anion exchange chromatography (8). The pulps were subjected to total enzymatic hydrolysis and the 4-O-methylglucuronic acid (MeGlcA) and hexenuronic acid (HexA) contents were determined from the hydrolysates (9). Lignin kappa number was calculated using the reported finding that 10 mmol HexA/kg pulp is equal to 0.86 kappa units (10). Carbonyl group and carboxyl group contents of the pulps were determined according to the published methods (11,12). Pyrolysis-GC/MS of the samples was based on an earlier work (13). The pulps and residual lignins were pyrolysed at 580°C for 2 s (Pyrolab 2000), and the resulting fragments separated and identified using GC/MS (Varian 3800 and Varian Saturn 2000). Nitrogen analysis and methoxyl group determinations of the lignins were performed at the Analytical Laboratories, Engelskirchen, Germany. The protein content was obtained by multiplying the percentage nitrogen content by 6.25 (14). Phenol groups in the residual lignins were determined using an ionization difference-UV method (14). FT1R spectra were obtained with a Nicolet 740 FTIR spectrometer (KBr technique). FTIR spectra corrected for the purified residual lignins were


361 obtained by subtracting the FTIR spectrum of the protein impurity from the FTIR spectra of residual lignins (14).

Results and Discussion


Delignification by Laccase/NHA A pine kraft pulp and the same pine kraft pulp after a two-stage oxygen delignification were treated with either laccase alone or laccase in the presence of NHA as a mediator. The ratio of mediator to lignin was the same for both pulps. Table I shows the properties of the pulps before and after the treatments. The kappa numbers of the pine kraft pulp and the oxygen-delignified pulp were lowered by 27% and 25%, respectively, by laccase/NHA and subsequent alkaline extraction under the conditions used. Kappa number reduction by laccase was no higher than that in the reference treatment under the same reaction conditions without laccase. Taking the content of hexenuronic acids into account (lignin kappa number), the delignification achieved with laccase/NHA was calculated to be 29% for the pine kraft pulp and 31% for the oxygen-delignified pulp. The hexenuronic acid content of pine kraft pulp and oxygen-delignified pine kraft pulp was 23 mmol/kg; this was unaffected by the laccase and laccase/NHA treatments (Table I). The results thus clearly show that laccase in the presence of NHA did not react with the double bond in hexenuronic acid either under oxygen pressure or at atmospheric pressure (15). The methylglucuronic acid content of the pulps was insignificant. The high pulp yields, and the fact that there were no changes in pulp viscosity and no significant changes in total carbohydrate content and composition suggest a high selectivity of laccase/NHA towards lignin (Table I). These benefits have also been reported for laccase/HBT treatments (3). Laccase/NHA treatment increased the content of carboxyl groups and carbonyl groups in pine kraft pulp. As with laccase/HBT treatment (3), both are probably introduced into lignin and not into carbohydrates. The low lignin content of oxygen-delignified pulp after laccase/NHA treatment prevents any increase in carbonyl group content from being seen. Unlike with laccase/HBT delignification (3), the carboxyl content decreased. Dissolution of the highly oxidized lignin in the OO-pulp during laccase/NHA treatment can explain the overall drop in the carboxyl content of the pulp in spite of the oxidative conditions during the treatment.


362 Table I. Properties of Pine Kraft Pulp and Two-Stage Oxygen-Delignified Pine Kraft Pulp (OO), Before and After Laccase Treatment and Laccase/NHA Treatment and Subsequent Alkaline Extraction (E).


Κ^φ

LE

(UNHA)E 00

OOLE

ÔÔ~

Kappa no. Lignin kappa no. Brightness, % Viscosity, ml/g Yield, % on wood

25.4 23.4 30.4 1110 45.5

22.4 20.3 30.1 1140 45.1

18.6 16.7 23.2 1170 45.3

10.1 8.1 38.9 890 43.7

9.1 7.1 44.3 890 43.3

(UNHA)E 7.6 5.6 42.5 890 43.3

HexA, mmol/kg MeGlcA, mol/kg COOH, mmol/kg CO, mmol/kg

23 + 74.3 15

24 + 75.3 17

22 + 92.8 28

23 + 81.3 17

23 + 79.1 18

23 + 71.8 18

95.5

93.3

94.3

95.8

95.7

94.8

0.8 + 84.1 8.2 6.9

0.8 0.4 84.4 7.7 6.7

0.7 0.4 84.4 7.7 6.8

0.7 + 84.8 7.7 6.8

0.7 + 85.0 7.5 6.8

0.7 + 84.8 7.8 6.7

Tot. monosacch., mg/100mg Ara, % Gal, % Glc, % Xyl, % Man, % b

a

3

+ = below determination limit of 15 mmol/kg. +=below determination limit of 0.2 mg/100 mg.

b

Comparison of Laccase/NHA and Laccase/HBT Delignification There was no difference in kappa reduction between laccase/HBT and laccase/NHA-treated and alkali-extracted oxygen-delignified pine kraft pulps, when equal amounts (74 mmol/kg pulp) of HBT and NHA on molar basis were used (Figure 1). However, the brightness improvement was a little greater for laccase/HBT-treated pulp than for laccase/NHA-treated pulp. Earlier studies on comparison of mediator indicated that the kappa reduction gained with laccase/NHA was slightly greater (2,16,17) for softwood kraft pulps of low kappa numbers and slightly lower (18) for softwood kraft pulps of higher kappa numbers compared to laccase/HBT. However, a much higher degree of delignification was obtained with the same HBT charge using a higher charge of a different laccase in the case of an oxygen-delignified pine kraft pulp with slightly lower kappa number (3).


363 Comparing the effects of NHA charges showed that increasing the NHA dosage is unnecessary, because the 25% kappa reduction was achieved with NHA charges of 48 and 74 mmol/kg pulp.


| w e e Kappa number '

00(17HBT)E 74

" Brightness, %

00(L/NHA)E 74

00(L/NHA)E 48

Figure!. Comparison of the effects of HBT and NHA on oxygen-delignified pine kraft pulp, when equal amounts of mediator (74 mmol/kg pulp) were used. Residual Lignins The pine kraft pulp and the two-stage oxygen-delignified pine kraft pulp, before and after laccase and laccase/NHA treatment followed by alkaline extraction, were subjected to total enzymatic hydrolysis in order to obtain the residual lignins. The lignin yields obtained ranged from 77% to slightly over 100% calculated from the total lignins in the pulps (Table II). This method (6) is known to give the lowest yield for the residual lignin from the pulp with the lowest kappa number, which probably indicates that some hydrophilic part of the lignins was not precipitated. However, despite the use of protease purification, the isolated lignin still contained some protein impurities originating from the cellulases used in the isolation procedure. The protein contents of the residual lignins were determined from their nitrogen contents (Table II). All nitrogen was assumed to originate from the protein residues of the enzymes. The protein contents of the lignins were also determined by pyrolysis-GC/MS. The protein impurity gives rise to three degradation products, which can be identified and quantified, although the accuracy of this method is not as good as that based on nitrogen content. However, the two methods gave comparable results, indicating that only trace



364 amounts of NHA-derived nitrogen-containing products were present in the samples. Detected as monosaccharides, the total carbohydrate contents of all isolated residual lignins were 6.3-8.2 mg/100mg (Table II), which are also typical values for lignins from other alkaline pulp types, as well as for laecase/HBTdelignified pulps. Some carbohydrates originate from the cellulase enzyme, which contained 10.2% by weight of carbohydrates, the monosaccharide composition being 85% mannose, 8.5% glucose and 6.5% galactose (14). Most, however, originated from the carbohydrates in the pulp, representing lignincarbohydrate complexes. The monosaccharide content and composition of carbohydrates originating from the pulps show that the carbohydrate contents in the residual lignins from the laccase/NHA-treated pulps were slightly lower than in the residual lignin of the initial pulps or of the laccase-treated pulps. The decrease was in the mannose and galactose contents in the case of laccase/NHAtreated pine kraft pulp lignin and in the mannose content in the case of laccase/NHA-treated oxygen-delignified pine kraft pulp lignin. On the other hand, oxygen delignification had a smaller effect on carbohydrates, the main difference being the decrease in galactose content, which agrees with earlier results (19). The results may thus indicate that some of the linkages between lignin and carbohydrates were cleaved during laccase/NHA treatments, which may partly explain the improvement in the bleachability of laccase/mediatortreated pulps (3,4,5). Table IL Yields and Nitrogen, Protein and Total Carbohydrate Contents and Monosaccharides (After Acid Hydrolysis) of Residual Lignins of the Pulps Shown in Table I. Res.lignins a

Yield, % N,% Protein, % Carbohydr., mg/100mg Man, mg/100mg Xyl, mg/100mg Glc, mg/100mg Gal, mg/100mg Ara, mg/100mg

Kraft

LE

96 0.49 3.1 7.51

109 0.61 3.8 8.22

110 1.89 11.8 6.98

92 2.30 14.4 7.46

91 2.20 13.8 7.33

OO (UNHA)E 77 2.47 15.4 6.28

2.31 1.68 1.43 1.72 0.37

2.52 2.09 1.56 1.61 0.43

1.78 1.91 1.44 1.47 0.39

2.13 1.97 1.90 0.97 0.49

2.25 1.91 1.88 0.83 0.46

1.60 1.79 1.66 0.80 0.43

(UNHA)E

OO

OOLE

b

a

Yield of isolated lignin as % of pulp lignin content; pulp lignin content calculated by multiplying lignin kappa number by 0.15. b

Corrected for carbohydrates originating from cellulases.


365 Functional Groups of the Residual Lignins The FTIR spectra of residual lignins obtained by subtracting the FTIR spectrum of the protein impurity from the spectra of the original residual lignins are shown in Figure 2. The most useful region in lignin analysis is the carbonyl region at 1750-1650 cm* , which gives information about changes in the oxidation degree in relation to the aromatic bands at 1500 cm" . The FTIR spectra of the residual lignins show that laccase/NHA treatment increased the intensity ratio of the carbonyl band at 1720 cm" to that of the aromatic band at 1510 cm' indicating an increase of carboxylic acid or ester groups in relation to aromaticity. Laccase/NHA treatment of kraft pulp also increased the intensity of the band at 1660 cm" in relation to the aromatic band, indicating formation of conjugated carbonyl structures such as quinone structures. Similar increases in carboxylic and carbonyl groups have been seen to occur on laccase/HBT treatment (20). Unconjugated carbonyl groups in aldehydes and ketones would also stretch in this region. The phenol content of the residual lignin of the pine kraft pulp and the oxygen-delignified pine kraft pulp decreased by about 30% during laccase/NHA treatment, indicating that free phenolics are reactive sites in delignification (Figure 3). The results thus agree with those obtained for other types of pulps (18,21) and for HBT-mediated delignification (5,20). Model compound studies, however, have shown that laccase in the presence of mediator also degrades non-phenolic structures (22,23). The increase in the proportion of conjugated phenols due to the formation of α-carbonyl groups was similar to that brought about by laccase in the presence of HBT. As with other laccases (20), Trametes versicolor left the fiber phenols intact, probably because of the fiber matrix. Increasing the NHA charge from 48 to 74 mmol/kg pulp did not affect the concentration of free phenols in the residual lignins of oxygen-delignified pine kraft pulp. Comparing the effects of NHA and HBT as a mediator showed that the contents of phenolic hydroxyl groups in residual lignins were equal when equal amounts of mediator (on a molar basis) were used. These results agree well with the delignification efficiencies obtained (Figure 1). In another investigation it was found that laccase in the presence of NHA lowered the concentration of free phenols in the residual lignin of softwood kraft pulp slightly more than laccase in the presence of HBT (18). The decrease in the methoxyl group content of pulp lignins with increasing delignification degree (Figure 4) was much smaller than the decrease in aromatic structures (Figure 5). The results thus show that cleavage of aromatic rings occurred without simultaneous demethylation. 1

1


1

1

1



366

2000

1600

1200

Wavenumber (cm-1)

800

2000

1600

1200

800

Wavenumber (cm-1)

Figure 2. Difference FTIR spectra of residual lignins (protein impurities have been subtracted) from pine kraft pulp, laccase-treated and laccase/NHA-treated pine kraft pulps after alkaline extraction, and from oxygen-delignified pine kraft pulp before and after laccase or laccase/NHA treatment followed by alkaline extraction.



367

(UNHA)E

Figure 3. Total phenols and conjugated phenols (% of total) in residual lignins ofpine kraft pulp, laccase-treated and laccase/NHA-treated pine kraft pulps after alkaline extraction, and from oxygen-delignified pine kraft pulp before and after laccase or laccase/NHA treatment followed by alkaline extraction. (Values corrected for protein impurities and carbohydrates).

Kraft

LE

(L/NHA)E

OO

OOLE

OO (L/NHA)E

Figure 4. Methoxyl groups in residual lignins ofpine kraft pulp, laccase-treated and laccase/NHA-treated pine kraft pulps after alkaline extraction, and from oxygen-delignified pine kraft pulp before and after laccase or laccase/NHA treatment followed by alkaline extraction. (Values corrected for protein impurities and carbohydrates).


368


Lignin Structure Analysed by Analytical Pyrolysis The isolated residual lignins were weighed (80 μg) and subjected to pyrolysis. A total of 19 monomeric compounds representing guaiacyl (G) and phydroxyphenyl (H) lignin degradation products were separated and identified by GC/MS (15). The peak areas were quantified and normalized to the weight of sample. Repeatability was 10% (RSD) calculated from the peak areas of dublicate samples. A black liquor lignin sample was analysed similarly in the same series and used as an external standard representing totally aromatic lignin. The peak areas for the residual lignin of pine kraft pulp and the external standard were equal, showing that these lignins have the same aromaticity. Laccase treatment did not change the content of aromatic residual lignin, as shown in Figure 5. However, the residual lignins of both oxygen-delignified pulp and laccase/NHA-treated pine kraft pulp contained significantly lower concentrations of aromatic subunits. After laccase/NHA treatment, the aromaticity of the residual lignin from oxygen-delignified pulp was further diminished. These results clearly show that laccase/NHA treatment oxidized aromatic residual lignin in the pulps to non-aromatic lignin structures such as muconic acids. The change caused by oxygen was very similar to that brought about by laccase/NHA. Only 60% of the residual lignin in the oxygendelignified pulp and the laccase/NHA-treated kraft pulp retained its aromatic structure. Less than 50% of the residual lignin in the laccase/NHA-treated oxygen-delignified pulp was aromatic (Figure 5). The less aromatic nature of the lignins was not, however, seen as a decrease in U V absorptivity. On the contrary, the absorptivities of the laccase/NHAtreated samples were even higher than those of the untreated, as seen in Figure 5. The formation of highly UV-active structures like quinones could explain this phenomenon. On the other hand, the composition of the aromatic pyrolysis products showed that there are only insignificant differences in the chemical structures of the aromatic parts of the different isolated residual lignins (Figure 6). Laccase and laccase/NHA treatment, however, caused a small increase in the content of α-carbonyl structures, which agrees with the results shown in Figures 2 and 3. The increase in α-oxidized structures was greater with oxygen treatment than with laccase/mediator treatment. Pyrolysis gives information about residual lignin structures without the need to isolate the residual lignin from the pulp. Direct pyrolysis analysis of the pulps showed a significant enrichment of p-hydroxyphenyl structures caused by laccase/NHA treatment, indicating their stability against mediator-aided delignification (Figure 7). The enrichment of p-hydroxyphenyl structures seen in the isolated residual lignins was insignificant (Figure 6). The probable reason for the difference in the concentration of /?-hydroxyphenyl units obtained by pyrolysis of pulp and isolated lignins is that p-hydroxyphenyl lignin was


369 Β Sum of the pyrolysis products •UV-absorptivity, l/g*cm 100OO0 g

90000

g

80000

τ 25

• 23.6 •

A20.4

CO 70000

20.0

• 20.0

• 21.3

H

60000

2L 50000 I 40000

20

i

15

&

30000


ε w

20000

5

>

10000

id Kraft

LE

(L/NHA)E

OO

OOLE

0 0 (L/NHA)E

Ref. lignin

Figure 5. Swm of the pyrolysis products showing the content of aromatic lignin in residual lignins ofpine kraft pulp, laccase-treated and laccase/NHA-treated pine kraft pulps after alkaline extraction, and from oxygen-delignified pine kraft pulp before and after laccase or laccase/NHA treatment followed by alkaline extraction. The absorptivities of the residual lignins are included in the Figure. A black liquor lignin sample (reference) was used as an external standard. Ε Coniferaldehyde Btrans-Coniferyl alcohol ®cis-Coniferyl alcohol •Dihydroconiferyl alcohol •4-(1-hydroxy-prop-2-enyt)guaiacol •4-(oxy-aliyl)guaiaeol •Acetoguaiacone ^Home-vanillin ^Vanillin Btrans-lsoeugenol •cis-lsoeugenol ÛEugenol •4-Vinylguaiacol 04-Ethylguaiacol •4-Methylguaiacol 04-Methyiphenol D2-Methylphenol •Guaiaco! (L/NHA)E

® Phenol

Figure 6. Composition of the pyrolysis products in the residual lignins ofpine kraft pulp, laccase-treated and laccase/NHA-treated pine kraft pulps after alkaline extraction, and from oxygen-delignified pine kraft pulp before and after laccase or laccase/NHA treatment followed by alkaline extraction. A black liquor lignin sample (reference) was used as an external standard.


370 precipitated in the isolation procedure to a lower extent than guaiacyl lignin (24). The increase in p-hydroxyphenyl content agrees with that obtained for birch pulp, which showed that the syringyl structures were more reactive than guaiacyl structures and that p-hydroxyphenyl structures were stable against laccase/HBT treatment (25). 100% Downloaded by STANFORD UNIV GREEN LIBR on September 17, 2012 | http://pubs.acs.org Publication Date: March 26, 2001 | doi: 10.1021/bk-2001-0785.ch022

y80%

;

60% '\ '

Characterization of Residual Lignins in Pulps Delignified by Laccase

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