The effects of various nutrients on extracellular peroxidases and acid-precipitable polymeric lignin production byStreptomyces chromofuscus A2 andS. viridosporus T7A

Summary The relationships between growth, medium pH, assimilation of glucose and amino acids, presence or absence of lignocellulose in the medium, lignin solubilization, and the appearance of extracellular peroxidase activity were compared for two lignin-solubilizing actinomycetes, Streptomyces chromofuscus A2 and S. viridosporus T7A. In a mineral salt medium containing yeast extract and three amino acids S. chromofuscus A2 grew faster than S. viridosporus T7A. When d-glucose was added to this medium, it was used in preference to the amino acids, the assimilation of which was delayed. Extracellular peroxidase activity peaked during the stationary phase, and glucose supplementation delayed peroxidase production. The eventual peak in peroxidase activity was higher in glucose-containing medium than in medium without glucose. Supplementation of the medium with lignocellulose did not affect either the level or time of appearance of extracellular peroxidase. However, lignin solubilization in lignocellulose-supplemented medium correlated positively with peroxidase activity: both increased after the cells entered the stationary phase. Supplementation of lignocellulose-containing medium with glucose delayed peroxidase production and lignin solubilization until the glucose had been assimilated. With S. viridosporus T7A, addition of d-glucose to the standard medium affected amino acid assimilation differently from S. chromofuscus A2. Glucose was consumed concomitantly with the amino acids. In the medium supplemented with lignocellulose, peroxidase activity and lignin solubilization correlated as they did for S. chromofuscus A2. A correlation of unknown significance was observed between the peroxidase activities of both strains and increasing medium pH. S. chromofuscus A2 produced more peroxidase and solubilized more lignin from lignocellulose than did S. viridosporus T7A. Overall, these findings show that extracellular peroxidases of both Streptomyces ssp. appear extracellularly primarily after cells cease growing and nutrients have been depleted from the medium. Also, increasing extracellular peroxidase activity and rates of lignin solubilization in both organisms are correlated and subject to glucose repression. These results point to the involvement of stationary-phase active peroxidases in the Streptomyces-catalyzed solubilization of lignin.Paper No. 90518 of the Idaho Agricultural Experiment Station Offprint requests to: D. L. Crawford     

Production and Characterization of Polymeric Lignin Degradation Intermediates from Two Different Streptomyces spp.

Previous investigations have identified a quantitatively major intermediate of lignin degradation by Streptomyces viridosporus. The intermediate, a modified lignin polymer, acid-precipitable polymeric lignin (APPL), is released as a water-soluble catabolite and has been recovered in amounts equivalent to 30% of the lignin originally present in a corn stover lignocellulose substrate after degradation by this actinomycete. In the present work, APPLs were collected at various time intervals from cultures of two highly ligninolytic Streptomyces sp. strains, S. viridosporus T7A and S. badius 252, growing on corn stover lignocellulose. APPL production was measured over time, and the chemistry of APPLs produced by each organism after different time intervals was compared. Chemical characterizations included assays for lignin, carbohydrate, and ash contents, molecular weight distributions by gel permeation chromatography, and chemical degradation analyses by permanganate oxidation, acidolysis, and alkaline ester hydrolysis. Differences between the organisms were observed in the cultural conditions required for APPL production and in the time courses of APPL accumulation. S. viridosporus produced APPL in solid-state fermentation over a 6- to 8-week incubation period, whereas S. badius produced as much or more APPL, but only in liquid culture and over a 7- to 8-day incubation period. The chemistry of the APPLs produced also differed. S. viridosporus APPL was more lignin-like than that of S. badius and was slowly modified further over time, although no change in molecular weight distribution over time was observed. In contrast, S. badius APPL was less lignin-like and increased substantially in average molecular weight over time. Results indicated that differing mechanisms of lignin metabolism may exist in these two Streptomyces sp. strains. S. viridosporus APPL probably originates from the heart of the lignin and is released largely as the result of β-ether cleavage and other oxidative reactions. S. badius APPL probably originates in the same manner; however, after release as a water-soluble catabolite, lower-molecular-weight intermediates of lignin degradation are repolymerized with APPL in a reaction catalyzed by an extracellular phenol oxidase. The chemical analyses and the presence of extracellular phenol oxidase in S. badius, but not in S. viridosporus, support this conclusion.     

Purification of lignin peroxidase isoforms from <Emphasis Type="Italic">Streptomyces viridosporus</Emphasis> T7A by hydrophobic based chromatographies

Five lignin peroxidases ALiP-P3 isoenzymes from Streptomyces viridosporus T7A were separated by hydrophobic interaction chromatography. The most abundant isoenzyme was purified by reversed phase chromatography and it was analyzed by mass spectrometry.     

Chemistry of softwood lignin degradation byStreptomyces viridosporus

Polymeric lignin isolated from ground spruce phloem/bark tissue following decay by the actinomyceteStreptomyces viridosporus (T7A) was characterized chemically and compared to undergraded lignin from the same source. The chemical transformations resulting from degradation were compared to those that result from fungal degradation of softwood lignins by brown- and white-rot fungi. Degradative chemical analyses showed thatS. viridosporus-degraded lignin was significantly altered in structure. Much of the integrity of the basic 4-hydroxy-3-methoxyphenylpropane subunit structure was lost. Actinomycete-decayed lignin was decreased in carbon and enriched in oxygen and hydrogen contents. It also had been extensively demethylated. Chemical analysed indicated that phenylpropanoid side-chains had been oxidized by introduction of -carbonyls and by side-chain shortening reactions. Although the degraded lignin remained polymeric, it was significantly dearomatized. These changes are similar to those previously reported for white-rotted lignins, except for the increased hydrogen content. The evidence indicated that lignin degradation byS. viridosporus is oxidative and involves demethylations, ring cleavage reactions, and oxidative attack on phenylpropanoid side-chains. Also, some reduced structures accumulate in the polymer and some low molecular weight intermediates are released into the growth medium.Abbreviations MWL milled wood lignin - TMS trimethylsyily - PCA protocatechuic acid Paper nunfber 81512 of the Idaho Agricultural Experiment Station     

Biodegradability of lignosulphonate by Streptomyces viridosporus strain T7A and a mixed natural microbial population antagonistic effects

The ability of a mixed natural microbial population, collected in an aerated lagoon treating Fluff pulp effluent and Streptomyces viridosporus strain T7A, to degrade lignosulphonate was evaluated. S. viridosporus growing in a mineral medium containing glycerol (7 g/l) and lignosulphonate (1 g/l) allowed 20% of lignosulphonate to be degraded after 18 days of incubation. A culture of the mixed population on culture medium after S. viridosporus growth was unable to degrade lignosulphonate products. Moreover, antagonism between S. viridosporus and the mixed population or between S. viridosporus and the isolated strains from this population was observed. The enhancement of lignosulphonate biodegradation by naturally occurring microorganisms in association with S. viridosporus (bioaugmentation strategy) seems to be difficult.     

Catabolic Fate of Streptomyces viridosporus T7A-Produced, Acid-Precipitable Polymeric Lignin upon Incubation with Ligninolytic Streptomyces Species and Phanerochaete chrysosporium

Degradation of ground and hot-water-extracted corn stover (Zea mays) lignocellulose by Streptomyces viridosporus T7A generates a water-soluble lignin degradation intermediate termed acid-precipitable polymeric lignin (APPL). The further catabolism of T7A-APPL by S. viridosporus T7A, S. badius 252, and S. setonii 75Vi2 was followed for 3 weeks in aerated shake flask cultures at 37°C in a yeast extract-glucose medium containing 0.05% (wt/vol) T7A-APPL. APPL catabolism by Phanerochaete chrysosporium was followed in stationary cultures in a low-nitrogen medium containing 1% (wt/vol) glucose and 0.05% (wt/vol) T7A-APPL. Metabolism of the APPL was followed by turbidometric assay (600 nm) and by direct measurement of APPL recoverable from the medium. Accumulation and disappearance of soluble low-molecular-weight products of APPL catabolism were followed by gas-liquid chromatography and by high-pressure liquid chromatography, utilizing a diode array detector. Identified and quantified compounds present in culture media included p-coumaric acid, ferulic acid, p-hydroxybenzoic acid, p-hydroxybenzaldehyde, protocatechuic acid, vanillic acid, and vanillin. The further catabolism of these APPL-derived aromatic compounds varied with the culture examined, and only S. setonii and P. chrysosporium completely degraded all of them. Some new intermediates of APPL metabolism also appeared in culture media, but the patterns were culture specific. Additional evidence from high-pressure liquid chromatography analyses indicated that one strain, S. badius, converted a water-soluble fraction evident by high-pressure liquid chromatography (7 to 10 min retention time range) into new products appearing at shorter retention times. Mineralization of a [¹⁴C-lignin]APPL was also followed. The percent ¹⁴C recovered as ¹⁴CO₂, ¹⁴C-APPL, ¹⁴C-labeled water-soluble products, and cell mass-associated radioactivity, were determined for each microorganism after 1 and 3 weeks of incubation in bubbler tube cultures at 37°C. P. chrysosporium evolved the most ¹⁴CO₂ (10%), and S. viridosporus gave the greatest decrease in recoverable ¹⁴C-APPL (23%). The results show that S. badius was not able to significantly degrade the APPL, while the other microorganisms demonstrated various APPL-degrading abilities. The significance of these findings relative to the fate of APPLs in nature was discussed.     

Isolation from haddock tissue of psychrophilic bacteria with maximum growth temperature below 20 degrees C.

Protoplast fusion was investigated as a technique for genetically manipulating two lignin-degrading Streptomyces strains, Streptomyces viridosporus T7A and Streptomyces setonii 75Vi2. Four of 19 recombinants tested showed enhanced production of acid-precipitable polymeric lignin (APPL), producing 155 to 264% more APPL from corn stover lignocellulose than was produced by the wild-type S. viridosporus T7A. APPLs are lignin degradation intermediates known to be potentially valuable chemical products produced by bioconversion of lignin with Streptomyces spp. The prospects of utilizing protoplast fusion to construct APPL-overproducing Streptomyces strains was considered especially promising.     

Oxidation of phenolic and non-phenolic substrates by the lignin peroxidase of Streptomyces viridosporus T7A

Summary Numerous single-ring, aromatic, phenolic and non-phenolic compounds were tested as substrates of Streptomyces viridosporus T7A extracellular lignin peroxidase. Oxidations were monitored by spectroscopy, with and without 4-aminoantipyrine (4-AAP) as a color-forming reagent. The oxidation of phenols containing one or no carbon groups in the para position resulted in coupling with 4-AAP to form a red color. Thin layer chromatography and mass spectroscopy showed that the oxidation of vanillic acid (4-hydroxy-3-methoxybenzoic acid) and syringic acid (4-hydroxy-3,5-dimethoxybenzoic acid) resulted in a direct coupling between 4-AAP and the phenol ring to form a quinone structure. In the reaction with vanillyl acetone (4-(4-hydroxy-3-methoxyphenyl)-3-buten-2-one) and 4-AAP, 4-AAP coupled to Á-carbon of vanillyl acetone. As shown by UV-visible spectroscopy, S. viridosporus T7A peroxidase oxidized phenolic compounds, but was unable to oxidize non-phenolic ones.Paper no. 91 517 of the Idaho Agricultural Experiment Station Correspondence to: D. L. Crawford     

Biodegradation of kraft lignin by a newly isolated bacterial strain, <Emphasis Type="Italic">Aneurinibacillus aneurinilyticus</Emphasis> from the sludge of a pulp paper mill

A kraft lignin-degrading bacterium (ITRC S ₇ ) was isolated from sludge of pulp and paper mill and characterized as Aneurinibacillus aneurinilyticus by biochemical tests and 16SrRNA gene sequencing. The bacterium did not utilize kraft lignin (KL) as the sole source of carbon and energy. However, this strain reduced the color (58%) and lignin content (43%) from kraft lignin-mineral salt medium when supplemented with glucose at pH 7.6 and 30°C after 6 days. The degradation on addition of glucose in culture medium is clear evidence of co-metabolism of KL by A. aneurinilyticus. The analysis of lignin degradation products by GC-MS in ethyl acetate extract from an A. aneurinilyticus-inoculated sample revealed the formation of low molecular weight aromatic compounds such as guaiacol, acetoguaiacone, gallic acid and ferulic acid, indicating that the bacterium can oxidize of the sinapylic (G units) and coniferylic (S units) alcohol units which are the basic moieties that build the hardwood lignin structure. The low molecular weight aromatic compounds identified in extracts of the inoculated sample favors the idea of biochemical modification of the KL to a single aromatic unit.     

Lignin Degradation by Streptomyces viridosporus: Isolation and Characterization of a New Polymeric Lignin Degradation Intermediate

A new, quantitatively significant intermediate formed during lignin degradation by Streptomyces viridosporus T7A was isolated and characterized. In Streptomyces-inoculated cultures, the intermediate, an acid-precipitable, polyphenolic, polymeric lignin (APPL), accumulated in the growth medium. The APPL was a water-soluble polymer probably consisting of a heterogeneous mixture of molecular weight components of ≥20,000. APPLs were precipitable from culture filtrates after they had been acidified to pH <3 to 5. Noninoculated controls yielded little APPL, but supernatant solutions from inoculated cultures produced quantities of APPL that correlated with the biodegradability of the lignocellulose type. Maximal recovery of APPL was obtained from corn lignocellulose, reaching 30% of the initial lignin present in the substrate. APPLs contained small amounts of carbohydrate, organic nitrogen, and inorganic materials. The lignin origin of APPLs was confirmed by chemical analyses, which included acidolysis, permanganate oxidation, elemental analyses, functional group analyses, nuclear magnetic resonance spectroscopy, and ¹⁴C isotopic techniques. Analyses of APPLs from corn lignocelluloses showed that S. viridosporus-degraded APPLs were lignin derived but significantly different in structure from APPLs derived from uninoculated controls or from a standard corn milled-wood lignin. Degraded APPLs were enriched in phenolic hydroxyl groups and, to a small extent, in carboxyl groups. Degradative changes appeared to be largely oxidative and were thought to involve substantial cleavage of p-hydroxy ether linkages and methoxyl groups in the lignin.     

Activities of cellulase and other extracellular enzymes during lignin solubilization by Streptomyces viridosporus

Summary Two mutant strains of the lignin degrading bacterium Streptomyces viridosporus strain T7A with enhanced abilities to produce a soluble lignin degradation intermediate, acid-precipitable polymeric lignin (APPL) and several mutants derepressed for cellulase production were compared with the wild type to examine the roles of cellulase and selected other extracellular enzymes in lignin solubilization by S. viridosporus. The two APPL-overproducing mutants, T-81 and T-138, had higher cellulase activities than the wild type. Mutants specifically derepressed for cellulase were also isolated and were found to produce more APPL than the wild type. The results are indicative of some involvement of cellulase in the lignin solubilization process. The lignin solubilized from corn (Zea mays) lignocellulose by the mutants was slightly different chemically as compared to wild type solubilized lignin in that it had a higher coumaric acid ester content. The production of extracellular coumarate ester esterase, aromatic aldehyde oxidase, and xylanase was also examined in the mutants. Xylanase and aromatic aldehyde oxidase production did not differ significantly between the mutants and the wild type. Mutant T-81 was found to have a slightly lower activity for esterase as compared with the wild type. It was concluded that xylanase, oxidase and esterase are not the enzymes directly responsible for enhanced lignin solubilization. The results, however, do implicate cellulase in the process.Paper number 86 511 of the Idaho Agricultural Experiment Station     

Degradation of Softwood, Hardwood, and Grass Lignocelluloses by Two Streptomyces Strains

Two Streptomyces strains, S. viridosporus T7A and S. setonii 75Vi2, were grown on softwood, hardwood, and grass lignocelluloses, and lignocellulose decomposition was followed by monitoring substrate weight loss, lignin loss, and carbohydrate loss over time. Results showed that both Streptomyces strains substantially degraded both the lignin and the carbohydrate components of each lignocellulose; however, these actinomycetes were more efficient decomposers of grass lignocelluloses than of hardwood or softwood lignocelluloses. In particular, these Streptomyces strains were more efficient decomposers of grass lignins than of hardwood or softwood lignins.     

Catabolism of the lignin substructure model compound dehydrodivanillin by a lignin-degrading Streptomyces

The lignin-degrading actinomycete Streptomyces viridosporus T7A readily degrades the lignin model compound dehydrodivanillin. Four mutants of this organism (produced by irradiation of spores with ultraviolet light) were shown to have lost the ability to catabolize dehydrodivanillin. These mutant strains retained an undiminished ability to degrade Douglas-fir lignin (¹⁴C-lignin ¹⁴CO₂) as compared to the wild-type strain. None of the strains accumulated detectable quantities of dehydrodivanillin when grown on lignocellulose. Thus it appears that the enzymes involved in dehydrodivanillin catabolism are not a part of the streptomycete's system for degrading polymeric lignin. It is concluded that dehydrodivanillin is probably not a relevant model compound for study of lignin polymer degradation by Streptomyces viridosporus. Since many stable mutants completely lacking DHDV-degrading ability were readily obtained, it is suggested that the relevant catabolic enzymes may be encoded on a plasmid.Abbreviations DHDV dehydrodivanillin     

Identification of low molecular weight aromatic compounds by gas chromatography–mass spectrometry (GC–MS) from kraft lignin degradation by three Bacillus sp.

Three bacterial strains identified as Paenibacillus sp., Aneurinibacillus aneurinilyticus and Bacillus sp. have been shown to decolourise kraft lignin in 6 days of incubation. The release of low molecular weight aromatic compounds by these bacterial strains during degradation of kraft lignin was analysed by GC–MS analysis. The total ion chromatograph (TIC) of ethyl acetate extract from kraft lignin sample inoculated by Paenibacillus sp. was similar to control except some minor changes in the chromatographic profile indicating incapability of this bacterium to modify kraft lignin. On the other hand the TIC of ethyl acetate extract from kraft lignin inoculated by A. aneurinilyticus and Bacillus sp. caused formation of several aromatic lignin-related compound that were not present in the extract of control. The compounds identified in extract of the sample degraded by A. aneurinilyticus were guaiacol, acetoguiacone, gallic acid and ferulic acid while t-cinnamic acid, 3,4,5 trimethoxy benzaldehyde, and ferulic acid by Bacillus sp. indicating oxidization of coniferylic (G units) and sinapylic (S units) alcohol of lignin polymer. Differences between the identified compounds from different bacterial treatment were strain-specific. Among the identified aromatic compounds, ferulic acid and 3,4,5-trimethoxy benzaldehyde could be useful to the industry of preservatives, aromas and perfumes.     

Production of Major Extracellular Enzymes during Lignocellulose Degradation by Two Streptomycetes in Agitated Submerged Culture

Streptomyces viridosporus T7A and S. badius 252 were grown in 1 to 2% (wt/vol) slurry cultures with mineral salts solution containing 0.6% yeast extract and 100/200 mesh ground and extracted corn lignocellulose at 37°C. Enzyme activities rapidly increased in the first 3 to 4 days and then declined and remained at a relatively constant level. Concentrations of endoglucanase and xylanase produced by S. badius were lower than those produced by S. viridosporus. However, the lignin-peroxidase peak concentration was threefold higher than with S. viridosporus and was obtained at 9 to 10 days of incubation. By polyacrylamide gel analysis, it was determined that peroxidases from both species consisted of four enzymes, with only one, the lignin peroxidase, having high activity. A culture pH of 8.5 was preferable for lignocellulose degradation by S. badius.     

Solubilisation and mineralisation of [14C]lignocellulose from wheat straw by Streptomyces cyaneus CECT 3335 during growth in solid-state fermentation

Nine Streptomyces strains were screened for their ability to solubilise and mineralise ¹⁴C-labelled lignin during growth in solid-state fermentation. Streptomyces viridosporus was confirmed as an active lignin-degrading organism along with a new isolate, Streptomyces sp. UAH 15, further classified as Streptomyces cyaneus CECT 3335. This organism was able to solubilise and mineralise the [¹⁴C]lignin fraction of lignocellulose (44.96 ± 1.77% and 3.41 ± 0.48% respectively) after 21 days of incubation. Cell-free filtrates from Streptomyces sp. grown in solid-state fermentation were capable of solubilising up to 20% of the [¹⁴C]lignin after 2 days incubation, with most of the product detected in the acid-soluble rather than in the water-soluble fraction. Identification of the extracellular enzymes produced during growth of S. cyaneus CECT 3335 revealed that extracellular peroxidase and phenol oxidase activities were present, with the activity of phenol oxidase being 100 times greater than peroxidase activity. The activity of these two enzymes was found to correlate with both solubilisation and mineralisation rates. This is the first report of phenol oxidase activity produced by a Streptomyces strain during growth in solid-state fermentation. A role for the enzyme in the solubilisation and mineralisation of lignocellulose by S. cyaneus is suggested. Received: 12 May 1997 / Accepted: 19 May 1997     

Biodegradation of kraft lignin by a bacterial strain Comamonas sp. B-9 isolated from eroded bamboo slips

Aims: The aim was to obtain evidences for lignin degradation by unicellular bacterium Comamonas sp. B‐9. Methods and Results: Comamonas sp. B‐9 was inoculated into kraft lignin‐mineral salt medium (KL‐MSM) at pH 7·0 and 30°C for 7 days of incubation. The bacterial growth, chemical oxygen demand (COD) reduction, secretion of ligninolytic enzymes and productions of low‐molecular‐weight compounds revealed that Comamonas sp. B‐9 was able to degrade kraft lignin (KL). COD in KL‐MSM reduced by 32% after 7 days of incubation. The maximum activities of manganese peroxidase (MnP) of 2903·2 U l^?1 and laccase (Lac) of 1250 U l^?1 were observed at 4th and 6th day, respectively. The low‐molecular‐weight compounds such as ethanediol, 3, 5‐dimethyl‐benzaldehyde and phenethyl alcohol were formed in the degradation of KL by Comamonas sp. B‐9 based on GC‐MS analysis. Conclusions: This study confirmed that Comamonas sp. B‐9 could utilize KL as a sole carbon source and degrade KL to low‐molecular‐weight compounds. Significance and Impact of the Study: Comamonas sp. B‐9 may be useful in the utilization and bioconversion of lignin and lignin‐derived aromatic compounds in biotechnological applications. Meanwhile, using Comamonas sp. B‐9 in treatment of wastewater in pulp and paper industry is a meaningful work.     

Growth Regulators in Picea abies

The occurrence of growth regulators active in the Avena coleoptile straight-growth test in sprouting buds and seedlings of Norway spruce (Picea abies Karst.) was investigated. The acid ether fraction contained a growth stimulator, the Rf of which in isopropanol: ammonia: water was 0.2–0.4. This substance behaved as indole-3-acetic acid (IAA) in elec-trophoresis, in chromatography in various solvent systems on paper and on a Sephadex column. It gave the colour typical of IAA when sprayed with Ehrlich reagent and its fluorescence characteristics corresponded to IAA. Acid ether-soluble inhibitors showed most activity at Rf 0.4–0.7, but due to tailing they interfered with the determination of the stimulator at the Rf of IAA in the bioassay. They also masked the activity of other stimulators. Colour reactions were obtained with Ehrlich reagent in the inhibiting chromatogram zone. When eluates from this zone were tested in high dilutions or after gel filtration growth stimulation was obtained. The acid fraction of seedling shoots also contained a stimulator with Rf 0.7–0.8. In the neutral-basic ether-soluble fraction growth stimulation was obtained at Rf 0.5–0.7. The extracts also contained stimulatory substances insoluble in ether but soluble in n-butanol and partly in ethyl acetate. When the butanol fraction was hydrolyzed in 1 M NaOH a substance behaving as IAA when chromatographed was released.     

Depolymerization and decolorization of kraft lignin by bacterium Comamonas sp. B-9

There is no commercial or industrial-scale process for the remediation of black liquor using microorganisms to date. One of the most important causes is that most microorganisms are not able to use lignin as their principal metabolic carbon or energy source. The bacterial strain Comamonas sp. B-9 has shown remarkable ability to degrade kraft lignin and decolorize black liquor using lignin as its principal metabolic carbon and energy source. This report looks at the depolymerization and decolorization of kraft lignin by Comamonas sp. B-9. The degradation, decolorization, and total carbon removal reached 45, 54, and 47.3 %, respectively, after 7 days treatment. Comamonas sp. B-9 was capable of depolymerizing kraft lignin effectively as analyzed by gel permeation chromatography and decolorization via degrading benzene ring structures as shown using Fourier transform infrared spectroscopy analysis.     

Laccase-initiated cross-linking of lignocellulose fibres using a ultra-filtered lignin isolated from kraft black liquor

In this work, the effect of Trametes pubescens laccase (TpL) used in combination with a low-molecular-weight ultra-filtered lignin (UFL) to improve mechanical properties of kraft liner pulp and chemi-thermo-mechanical pulp was studied. UFL was isolated by ultra-filtration from the kraft cooking black liquor obtained from softwood pulping. This by-product from the pulp industry contains an oligomeric lignin with almost twice the amount of free phenolic moieties than residual kraft pulp lignin. The reactivity of TpL on UFL and kraft pulp was studied by nuclear magnetic resonance spectroscopy and size exclusion chromatography. Laccase was shown to polymerise UFL and residual kraft pulp lignin in the fibres, seen by the increase in their average molecular weight and in the case of UFL as a decrease in the amount of phenolic hydroxyls. The laccase initiated cross-linking of lignin, mediated by UFL, which gives rise to more than a twofold increase in wet strength of kraft liner pulp handsheets without loosing other critical mechanical properties. Hence, this could be an interesting path to decrease mechano-sorptive creep that has been reported to lessen in extent as wet strength is given to papers. The laccase/2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) mediator system showed a greater increase in wet tensile strength of the resulting pulp sheets than the laccase/UFL system. However, other mechanical properties such as dry tensile strength, compression strength and Scott Bond internal strength were negatively affected by the laccase/ABTS system.