Study of lignin biotransformation by Aspergillus fumigatus and white-rot fungi using (14)C-labeled and unlabeled kraft lignins

The biodegradation of lignin by fungi was studied in shake flasks using (14)C-labeled kraft lignin and in a deep-tank fermentor using unlabeled kraft lignin. Among the fungi screened, A. fumigatus-isolated in our laboratories-was most potent in lignin biotransformation. Dialysis-type fermentation, designed to study possible accumulation of low MW lignin-derived products, showed no such accumulation. Recalcitrant carbohydrates like mi-crocrystalline cellulose supported higher lignolytic activity than easily metabolized carbohydrates like cellobiose. An assay developed to distinguish between CO(2) evolved from lignin and carbohydrate substrates demonstrated no stoichiometric correlation between the metabolism of the two cosubstrates. The submerged fermentations with unlabeled lignin are difficult to monitor since chemical assays do not give accurate and true results. Lignolytic efficiencies that allowed monitoring of such fermentations were defined. Degraded lignins were analyzed for structural modifications. A. fumigatus was clearly superior to C. versicolor in all aspects of lignin degradation; A. fumigatus brought about substantial demethoxylation and dehydroxylation, whereas C. versicolor degraded lignins closely resembled undegraded kraft lignin. There was a good agreement among the different indices of lignin degradation, namely, (14)CO evolution, OCH(3) loss, OH loss, and monomer and dimer yield after permanganate oxidation.     

Thermophilic anaerobic biodegradation of [C]lignin, [C]cellulose, and [C]lignocellulose preparations

Thermophilic (55 degrees C) anaerobic enrichment cultures were incubated with [C-lignin]lignocellulose, [C-polysaccharide]lignocellulose, and kraft [C]lignin prepared from slash pine, Pinus elliottii, and C-labeled preparations of synthetic lignin and purified cellulose. Significant but low percentages (2 to 4%) of synthetic and natural pine lignin were recovered as labeled methane and carbon dioxide during 60-day incubations, whereas much greater percentages (13 to 23%) of kraft lignin were recovered as gaseous end products. Percentages of label recovered from lignin-labeled substrates as dissolved degradation products were approximately equal to percentages recovered as gaseous end products. High-pressure liquid chromatographic analyses of CuO oxidation products of sound and degraded pine lignin indicated that no substantial chemical modifications of the remaining lignin polymer, such as demethoxylation and dearomatization, occurred during biodegradation. The polysaccharide components of pine lignocellulose and purified cellulose were relatively rapidly mineralized to methane and carbon dioxide; 31 to 37% of the pine polysaccharides and 56 to 63% of the purified cellulose were recovered as labeled gaseous end products. An additional 10 to 20% of the polysaccharide substrates was recovered as dissolved degradation products. Overall, these results indicate that elevated temperatures can greatly enhance rates of anaerobic degradation of lignin and lignified substrates to methane and low-molecular-weight aromatic compounds.     

Biodegradation of radiolabelled synthetic lignin (14C-DHP) and mechanical pulp in a compost environment

Mineralization of radioactive synthetic lignin (14C-DHP) was studied in a compost environment at 35, 50 and 58 degrees C. Compost samples were successively extracted with water, dioxane and alkali, and the molecular weight distribution of some extracts was determined by gel permeation chromatography (GPC). Biodegradation of lignin-containing spruce groundwood (SGW) and pine sawdust was concurrently determined in controlled composting tests by measuring evolved CO2. The temperatures were the same as in the 14C-DHP mineralization experiment and bleached kraft paper, with a lignin content of 0.2%, was used as a reference. The mineralization of 14C-DHP was relatively high (23-24%) at 35 degrees C and 50 degrees C, although the mixed population of compost obviously lacks the most effective lignin degraders. At 58 degrees C the mineralization of 14C-DHP, as well as the biodegradation of SGW and sawdust, was very low, indicating that the lignin-degrading organisms of compost were inactivated at this temperature. SGW was poorly biodegradable (<40%) in controlled composting tests compared with kraft paper (77-86%) at all temperatures, which means that lignin inhibits the degradation of carbohydrates. During the incubation, water-soluble degradation products, mainly monomers and dimers, and the original 14C-DHP were either mineralized or bound to humic substances. A substantial fraction of 14C-DHP was incorporated into humin or other insolubles.     

Biokraft pulping of European black pine with Ceriporiopsis subvermispora

European black pine (Pinus nigra Arn.) chips were treated with the white-rot fungus Ceriporiopsis subvermispora for periods ranging from 20 to 100 days. The effects of pretreatment on the chemical composition of wood and kraft pulping were investigated. The results showed that fungal pretreatment reduced the lignin and extractive content of wood chips. Also, weight losses occurred. Kappa number, viscosity, and reject ratio of biokraft pulps decreased. Biokraft pulps gave better response to beating, which led to significant energy saving during refining. The tear index, burst index, and tensile index of biokraft pulps were found to be lower than those of kraft pulps. However, the tensile index and burst index of 20-day biotreated and unbeaten pulp was higher than those of kraft pulp. Also, the tear index of 20-day biotreated and beaten pulp was higher than that of kraft pulp. The brightness of biokraft pulps decreased irregularly with increasing incubation time.     

Factors affecting the activation of pulps with laccase

Activation of fibres by radical formation is the first step when aiming at oxidative coupling of new functional groups on the fibre bound lignin. In this work, factors affecting the amount of phenoxy radicals created to unbleached TMP, CTMP, softwood kraft and hardwood kraft pulp fibres in the laccase catalysed oxidation were determined by EPR. Laccase was able to catalyse the oxidation of all the pulps studied. The reactivity of the pulp was found to be affected by both the physical accessibility of lignin in the fibres and the chemistry of the surface lignin accessible to laccase. Laccase dosage, use of extra oxygen in the laccase catalysed radicalization reaction, treatment time and also the amount and type of low-molecular weight compounds (LMWC) present in the pulp were all found to contribute to the radical content of pulp fibres measured after the enzymatic reaction. It could not been excluded that two types of reactions take place during the radical formation in fibres. Within the fibre matrix there may be both fibre material bound and soluble lignin fragments differing with respect to accessibility, molecular weight or chemical structure which can be radicalized at various rates, and the formed radicals may also undergo cross-coupling reactions reducing the amount of the total radicals.     

Thermophilic Anaerobic Biodegradation of [14C]Lignin, [14C]Cellulose, and [14C]Lignocellulose Preparations

Thermophilic (55°C) anaerobic enrichment cultures were incubated with [¹⁴C-lignin]lignocellulose, [¹⁴C-polysaccharide]lignocellulose, and kraft [¹⁴C]lignin prepared from slash pine, Pinus elliottii, and ¹⁴C-labeled preparations of synthetic lignin and purified cellulose. Significant but low percentages (2 to 4%) of synthetic and natural pine lignin were recovered as labeled methane and carbon dioxide during 60-day incubations, whereas much greater percentages (13 to 23%) of kraft lignin were recovered as gaseous end products. Percentages of label recovered from lignin-labeled substrates as dissolved degradation products were approximately equal to percentages recovered as gaseous end products. High-pressure liquid chromatographic analyses of CuO oxidation products of sound and degraded pine lignin indicated that no substantial chemical modifications of the remaining lignin polymer, such as demethoxylation and dearomatization, occurred during biodegradation. The polysaccharide components of pine lignocellulose and purified cellulose were relatively rapidly mineralized to methane and carbon dioxide; 31 to 37% of the pine polysaccharides and 56 to 63% of the purified cellulose were recovered as labeled gaseous end products. An additional 10 to 20% of the polysaccharide substrates was recovered as dissolved degradation products. Overall, these results indicate that elevated temperatures can greatly enhance rates of anaerobic degradation of lignin and lignified substrates to methane and low-molecular-weight aromatic compounds.     

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.     

Effect ofStreptomyces viridosporus T7A on kraft lignin

Summary The ability of the lignino-cellulolytic actinomyceteStreptomyces viridosporus T7A to attack purified fractions of kraft lignin was examined. In the presence of 0.3% yeast extract, high-molecular weight kraft lignin (MW>3000, ether-insoluble fraction) does not affect growth of this microorganism significantly, whereas low-molecular weight kraft lignin (MW<3000, ether-soluble fraction) inhibits its development. Accordingly, average molecular weight of the ether-insoluble fraction after bacterial growth remained unaltered, as measured by Sephadex G-50 gel permeation chromatography. Slight modifications were detected by high performance liquid chromatography in the ether-soluble fraction after incubation with the microorganism.S. viridosporus T7A partially decolorized Remazol Brilliant Blue R during growth on wheat lignocellulose. However, decolorization of either fraction of kraft lignin was not observed. These results suggest that the filamentous bacteriumS. viridosporus T7A is not suitable for pulp mill effluent treatment.     

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.     

Arundo donax L. reed: new perspectives for pulping and bleaching. Part 4. Peroxide bleaching of organosolv pulps

A comparative study on TCF (totally chlorine-free) bleachability of organosolv pulps from the annual fibre crop Arundo donax L. (giant reed) was carried out using a simple three-stage peroxide bleaching sequence without oxygen pre-bleaching. ASAM (alkali-sulfite-anthraquinone-methanol), Organocell (alkali-anthraquinone-methanol) and ethanol-soda organosolv pulps were bleached and compared with kraft pulp, as a reference. The final brightness of 76-78% ISO was attained for all tested pulps. The chemical charge required to reach this level of brightness varied for different pulps (despite the equal initial content of the residual lignin) and directly related to starting brightness values. No direct correlation between brightness improvement and lignin removal during bleaching was found, indicating the influence of the specific pulp properties introduced by pulping process on bleaching chemistry. The general higher bleaching response of organosolv pulps from A. donax was noted in comparison with kraft.     

Laccase-mediated synthesis of lignin-core hyperbranched copolymers

Lignin, one of the major chemical constituents of woody biomass, is the second most abundant biopolymer found in nature. The pulp and paper industry has long produced lignin on the scale of millions of tons annually as a by-product of the pulping process, and the dawn of cellulosic ethanol production has further contributed to this amount. Historically, lignin has been perceived as a waste material and burned as a fuel for the pulping process. However, recent research has been geared toward developing cost-effective technologies to convert lignin into valuable commodities. Attributing to the polyphenolic structure of lignin, enzymatic modification of its surface using laccases (benzenediol:oxygen oxidoreductases, EC 1.10.3.2) has demonstrated to be highly successful. The current study aims at developing lignin-core hyperbranched copolymers via the laccase-assisted copolymerization of kraft lignin with methylhydroquinone and a trithiol. Based on the physical properties of the resulting material, it is likely that crosslinking reactions have taken place during the drying process to produce a copolymeric network rather than discrete hyperbranched copolymers, with NMR data providing evidence of covalent bonding between monomers. Preliminary thermal analysis data reveals that the copolymeric material possesses a moderate glass transition temperature and exhibits good thermostability, thus may have potential application as a lignin-based thermoplastic. Scanning electron microscopy images confirm the smooth, glossy surface of the material and that it is densely packed. The presented results are a sustainable, ecofriendly, economic method to create an exciting novel biomaterial from a renewable feedstock while further enhancing lignin valorization.     

Investigation of lignin-carbohydrate complexes in kraft pulps by selective enzymatic treatments

The occurrence of covalent bonds between residual lignin and polysaccharides in birch and pine kraft pulps was investigated by specific enzymatic treatments. Pure enzymes degrading cellulose, xylan and mannan were used both separately and in combination. Comparison of the molar masses of polysaccharides and lignin in the orginal pulps and in the residual pulps after enzymatic treatments showed that residual lignin in birch kraft pulp is linked at least to xylan. A minor portion may also be linked to cellulose. In pine kraft pulp some of the residual lignin appears to be linked to cellulose, glucomannan and xylan. The linkages between lignin and cellulose and hemicelluloses may be either native or formed during pulp processing. The results also provided new information on the synergistic action of cellulose- and hemicellulose-degrading enzymes on pulp fibres. The synergism appears to be mainly due to the structure of the pulp fibres, with different layers of cellulose sheets, hemicelluloses and lignin. On the other hand the results also provided information about fibre structure. The degradation of xylan clearly enhanced the action of enzymes on cellulose, suggesting that xylan partially covers the cellulose. A similar phenomenon was not observed in the simultaneous hydrolysis of glucomannan and cellulose. However, the results suggest that glucomannan does interact with cellulose, possibly by non-covalent linkages. Received: 8 July 1998 / Received revision: 7 October 1998 / Accepted: 11 October 1998     

Biotransformation of kraft lignin fractions by Serratia marcescens

The ability of a Serratia marcescens strain to transform ethyl acetate soluble (EASF) and insoluble (EAIF) fractions of kraft pine lignin, was studied, In media containing EASF or EAIF and 0.1% glucoes, the bacterium degraded 15 and 9% respectively, as shown by ultraviolet spectrophotometry. The greates percentages degradation, 48 and 43% were obtained when S. marcescens grew on neutral (N-EASF) and acid (A-EASFF) compnents of the EASF of kraft lignin     

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.     

Identifying the characteristic secondary ions of lignin polymer using ToF-SIMS

The chemical structure of lignin, a complex, irregular polymer of phenylpropane units that occurs in plant cell walls, was investigated using time-of-flight secondary ion mass spectrometry (ToF-SIMS). The positive ToF-SIMS spectra of lignin isolated from pine and beech wood showed prominent secondary ions possessing guaiacyl (at m/z 137 and 151) or syringyl (at m/z 167 and 181) rings, which are the basic building units of lignin polymer. This shows that ToF-SIMS is a useful tool for lignin structural analysis. The peaks at m/z 137 and 167 were assigned as the C6-C1 ion, and the peaks at m/z 151 and 181 may be double-component, the C6-C1 ion and the C6-C2 ion. We confirmed the characteristic guaiacyl ions using a synthetic lignin model compound, dehydrogenation polymer (DHP), which was formed by polymerizing of unlabeled and deuterium-labeled coniferyl alcohols. The formation mechanism of the main secondary ions was deduced by labeling specific positions of coniferyl alcohols with a stable isotope to study the relationship between chemical structure and secondary ion formation in ToF-SIMS.     

A novel and efficient oxidative functionalization of lignin by layer-by-layer immobilised Horseradish peroxidase

Horseradish peroxidase (HRP) was chemically immobilised onto alumina particles and coated by polyelectrolytes layers, using the layer-by-layer technique. The reactivity of the immobilised enzyme was studied in the oxidative functionalisation of softwood milled wood and residual kraft lignins and found higher than the free enzyme. In order to investigate the chemical modifications in the lignin structure, quantitative (31)P NMR was used. The immobilised HRP showed a higher reactivity with respect to the native enzyme yielding extensive depolymerisation of lignin.     

Quantitative 13C NMR spectroscopy. Chemical structure of kraft and nitrosated lignins

Quantitative ¹³C NMR spectroscopy has been used to study the chemical structure of industrial kraft lignin, obtained from softwood pulping, and its nitrosated derivatives, which demonstrate high inhibition activity in the polymerization of unsaturated hydrocarbons.     

Oligomerization and chaperone activity of a plant 2-Cys peroxiredoxin in response to oxidative stress

Plant 2-Cys peroxiredoxins (2-Cys Prxs) have been reported to localize to chloroplasts and perform antioxidative roles during plant development and photosynthesis. In this study, we identified that, in addition to the well-known function of thioredoxin (Trx)-dependent peroxidase, the plant 2-Cys Prx in Chinese cabbage 2-Cys Prx1, designated C2C-Prx1, also behaves as a molecular chaperone under oxidative stress conditions, like the yeast and mammalian 2-Cys Prxs. By the chaperone function of C2C-Prx1, the protein efficiently prevented the denaturation of citrate synthase and insulin from heat shock and dithiothreitol (DTT)-induced chemical stresses. Also, the protein structure of C2C-Prx1 was shown to have discretely sized multiple structures, whose molecular sizes were in the diverse ranges of low molecular weight (LMW) proteins to high molecular weight (HMW) protein complexes. The dual functions of C2C-Prx1 acting as a peroxidase and as a molecular chaperone are alternatively switched by heat shock and oxidative stresses, accompanying with its structural changes. The peroxidase function predominates in the lower MW forms, but the chaperone function predominates in the higher MW complexes. The precise regulation of C2C-Prx1 structures and functions may play a pivotal role in the protection of plant chloroplasts from photo-oxidative stress.     

Effect of Penicillium chrysogenum on Lignin Transformation

A strain of Penicillium chrysogenum has been isolated from pine forest soils in Tenerife (Canary Islands). This strain was capable of utilizing hydroxylated and nonhydroxylated aromatic compounds, in particular cinnamic acid, as its sole carbon source. In an optimum medium with high levels of nitrogen (25.6 mM) and low levels of glucose (5.5 mM), it was able to decolorize Poly B-411 and to transform kraft, organosolv, and synthetic dehydrogenative polymerisate lignins. After 30 days of incubation, the amount of recovered kraft lignin was reduced to 83.5 and 91.3% of that estimated for uninoculated controls by spectrophotometry and klason lignin, respectively. At the same time, the pattern of molecular mass distribution of the lignin remaining in cultures was changed. The amount of organosolv lignin recovered from cultures was reduced to 90.1 and 94.6% of the initial amount as evaluated by spectrophotometry and klason lignin, respectively. About 6% of total applied radioactivity of O¹⁴CH₃-organosolv lignin was recovered as ¹⁴CO₂ after 30 days of incubation, and 18.5% of radioactivity from insoluble O¹⁴CH₃-organosolv lignin was solubilized. After 26 days of incubation, 2.9% of ¹⁴C-β-dehydrogenative polymerisate and 4.1% of ¹⁴C-ring-dehydrogenative polymerisate evolved as ¹⁴CO₂.     

Biological pretreatment of softwood Pinus densiflora by three white rot fungi

The effects of biological pretreatment on the Japanese red pine Pinus densiflora, was evaluated after exposure to three white rot fungi Ceriporia lacerata, Stereum hirsutum, and Polyporus brumalis. Change in chemical composition, structural modification, and their susceptibility to enzymatic saccharification in the degraded wood were analyzed. Of the three white rot fungi tested, S. hirsutum selectively degraded the lignin of this sortwood rather than the holocellulose component. After eight weeks of pretreatment with S. hirsutum, total weight loss was 10.7%, while lignin loss was the highest at 14.52% among the tested samples. However, holocellulose loss was lower at 7.81% compared to those of C. lacerata and P. brumalis. Extracelluar enzymes from S. hirsutum showed higher activity of ligninase and lower activity of cellulase than those from other white rot fungi. Thus, total weight loss and changes in chemical composition of the Japanese red pine was well correlated with the enzyme activities related with lignin- and cellulose degradation in these fungi. Based on the data obtained from analysis of physical characterization of degraded wood by X-ray Diffractometry (XRD) and pore size distribution, S. hirsutum was considered as an effective potential fungus for biological pretreatment. In particular, the increase of available pore size of over 120 nm in pretreated wood powder with S. hirsutum made enzymes accessible for further enzymatic saccharification. When Japanese red pine chips treated with S. hirsutum were enzymatically saccharified using commercial enzymes (Cellulclast 1.5 L and Novozyme 188), sugar yield was greatly increased (21.01%) compared to non-pretreated control samples, indicating that white rot fungus S. hirsutum provides an effective process in increasing sugar yield from woody biomass.