Delignification of an unbleached hardwood kraft pulp by Phanerochaete chrysosporium

Summary Culture conditions affecting lignin degradation of an unbleached hardwood kraft pulp by Phanerochaete chrysosporium have been examined. Optimum pH and temperature for lignin degradation (about 33%) were 3.5 and 38°C, respectively. Optimum fungal growth was at a pH of 4.5 and a temperature of around 32°C. Addition of exogeneous glucose to the cultures lessened the degradation of pulp carbohydrates. Lignin degradation was stimulated by oxygen atmosphere and non-agitated cultures. Increased surface to volume ratio (decreased culture depth) enhanced lignin degradation (about 56% at a depth of 1.2 cm). Finally, the correlations: pulp yield vs. residual glucose, ligninase activity vs. mycelium, and extent of delignification vs. residual extracellular H₂O₂ were discussed in light of recent findings of ligninases responsible for ligninolysis.     

Effect of Residual Lignin Type and Amount on Bleaching of Kraft Pulp by Trametes versicolor

The white rot fungus Trametes (Coriolus) versicolor can delignify and brighten unbleached hardwood kraft pulp within a few days, but softwood kraft pulps require longer treatment. To determine the contributions of higher residual lignin contents (kappa numbers) and structural differences in lignins to the recalcitrance of softwood kraft pulps to biobleaching, we tested softwood and hardwood pulps cooked to the same kappa numbers, 26 and 12. A low-lignin-content (overcooked) softwood pulp resisted delignification by T. versicolor, but a high-lignin-content (lightly cooked) hardwood pulp was delignified at the same rate as a normal softwood pulp. Thus, the longer time taken by T. versicolor to brighten softwood kraft pulp than hardwood pulp results from the higher residual lignin content of the softwood pulp; possible differences in the structures of the residual lignins are important only when the lignin becomes highly condensed. Under the conditions used in this study, when an improved fungal inoculum was used, six different softwood pulps were all substantially brightened by T. versicolor. Softwood pulps whose lignin contents were decreased by extended modified continuous cooking or oxygen delignification to kappa numbers as low as 15 were delignified by T. versicolor at the same rate as normal softwood pulp. More intensive O₂ delignification, like overcooking, decreased the susceptibility of the residual lignin in the pulps to degradation by T. versicolor.     

Litter decomposition contrasts in second- and old-growth Douglas-fir forests of the Pacific Northwest, USA

Litterfall and its subsequent decomposition are important feedback mechanisms in the intrasystem cycling of nutrients in forest ecosystems. The amount of litterfall and the rate of decomposition are expected to vary with stand age and climate. Over a 2-year period, decomposition of five litter types were measured in two second-growth forest stands and one old-growth stand in the Cascade Mountains of southern Washington state, USA. Both second-growth stands were dominated by Douglas-fir [Pseudotsuga menziesii (Mirb.,) Franco] but one had a significant proportion of red alder (Alnus rubra Bong.), a nitrogen (N) fixer. The old-growth stand was dominated by Douglas-fir and western hemlock [Tsuga heterophylla (Raf.) Sarg.]. All stands had a relatively shallow layer of forest floor mass. The five litter types were placed in each stand to evaluate decomposition patterns. Despite significant differences in stand age, microclimate and mean residence times for carbon (C) and N, the rates of litter mass loss varied only slightly between sites. The relative order of species litter mass loss was: vine maple ≫ salal = western hemlock > Douglas-fir (from the youngest stand) > Douglas-fir (from the N rich stand with red alder). The initial litter lignin concentration, not lignin:N, was the primary determinant of decomposition rates, although the initial N concentration was the predictor for mass loss after 2 years in the N rich Douglas-fir-alder stand. All litter types showed immobilization of N for nearly 2 years. Data for Douglas-fir litter suggest that higher levels of N may retard decomposition of tissues with greater amounts of lignified material. The retention of N by the litter appeared influenced by the nutrient capital of the stands as well as the forest floor C:N ratio. Decomposition was minimal during the cold winter months, but displayed a definitive peak period during early Fall with wet weather, warm soils, and fungal activity. Thus, long-term climatic change effects on forest floor C storage may depend more on changes in seasonality of precipitation changes than just temperature changes.     

Decomposition of roots of black alder and hybrid poplar in short-rotation plantings: Nitrogen and lignin control

The decomposition of the roots (0–2 mm, 2–5 mm and 5–10 mm) of black alder (Alnus glutinosa (L.) Gaertn.) and hybrid poplar (Populus nigra L. X Populus trichocarpa Torr & Gray) was followed over a 462-day period in pure and mixed plantings in southern Quebec. Small roots of alder had the highest initial concentrations of nitrogen and lignin, and lost 9 and 10% less mass than medium and large roots, respectively. Large roots of poplar had the highest lignin-to-nitrogen ratio and showed the smallest loss of mass over the total incubation period. Slow root decomposition of black alder and hybrid poplar was characterized by a greater proportion of initial root nitrogen immobilized per unit of carbon respired. Lignin concentration in roots of alder and poplar increased rapidly at the beginning of the incubation. Our results suggest that high levels of nitrogen in roots of alder could contribute in slowing the rate of decomposition by allowing the formation of nitrogen-lignin derivatives and low levels of nitrogen in roots of poplar may limit the growth of microorganisms and the rate of root decomposition. A multiple regression was developed using initial nitrogen, lignin concentration and the ratio of lignin to nitrogen to produce an index of the rate of root decomposition. The correlation between the index values and the percentage of residual root mass was significant (r=0.98, p<0.01).     

Biodegradation of 2,4,6-trinitrotoluene byPhanerochaete chrysosporium: Identification of initial degradation products and the discovery of a TNT metabolite that inhibits lignin peroxidases

Biodegradation of 2,4,6-trinitrotoluene (TNT) by the wood-rotting BasidiomycetePhanerochaete chrysosporium was studied in a fixed-film silicone membrane bioreactor and in agitated pellected cultures. The initial intermediate products of TNT biodegradation were shown to be 2-amino-4,6-dinitrotoluene (2amDNT) and 4-amino-2,6-dinitrotoluene (4amDNT). These intermediates were also degraded byP. chrysosporium. However, their rates of degradation were slow and appeared to represent rate-limiting steps in TNT degradation. The fact that 2amDNT and 4amDNT were further degraded is of importance. In most other microbial systems these compounds are typically not further degraded or are dimerized to even more persistent azo and azoxydimers. Similar to previous studies performed in stationary cultures, it was shown that substantial amounts of [¹⁴C]-TNT were degrade to [¹⁴C]-carbon dioxide in agitated pelleted cultures. Lignin peroxidase activity (assayed by veratryl alcohol oxidation) virtually disappeared upon addition of TNT to ligninolytic cultures ofP. chrysosporium. However, TNT, 2amDNT, and 4amDNT did not inhibit lignin peroxidase activity, nor were they substrates for this enzyme. Subsequent studies revealed that 4-hydroxylamino-2,6-dinitrotoluene, an intermediate in TNT reduction, was a potent lignin peroxidase inhibitor. Further studies revealed that this compound was also a substrate for lignin peroxidase H8.     

Fate of Residual Lignin during Delignification of Kraft Pulp by Trametes versicolor

The fungus Trametes versicolor can delignify and brighten kraft pulps. To better understand the mechanism of this biological bleaching and the by-products formed, I traced the transformation of pulp lignin during treatment with the fungus. Hardwood and softwood kraft pulps containing ¹⁴C-labelled residual lignin were prepared by laboratory pulping of lignin-labelled aspen and spruce wood and then incubated with T. versicolor. After initially polymerizing the lignin, the fungus depolymerized it to alkali-extractable forms and then to soluble forms. Most of the labelled carbon accumulated in the water-soluble pool. The extractable and soluble products were oligomeric; single-ring aromatic products were not detected. The mineralization of the lignin carbon to CO₂ varied between experiments, up to 22% in the most vigorous cultures. The activities of the known enzymes laccase and manganese peroxidase did not account for all of the lignin degradation that took place in the T. versicolor cultures. This fungus may produce additional enzymes that could be useful in enzyme bleaching systems.     

Evaluation of the white-rot fungi Ganoderma australe and Ceriporiopsis subvermispora in biotechnological applications

Ganoderma australe is a white-rot fungus that causes a selective wood biodelignification in some hardwoods found in the Chilean rainforest. Ceriporiopsis subvermispora is also a lignin-degrading fungus used in several biopulping studies. The enzymatic system responsible for lignin degradation in wood can also be used to degrade recalcitrant organic pollutants in liquid effluents. In this work, two strains of G. australe and one strain of C. subvermipora were comparatively evaluated in the biodegradation of ABTS and the dye Poly R-478 in liquid medium, and in the pretreatment of Eucalyptus globulus wood chips for further kraft biopulping. Laccase was detected in liquid and wood cultures with G. australe. Ceriporiopsis subvermispora produce laccase and manganese peroxidase when grown in liquid medium and only manganese peroxidase was detected during wood decay. ABTS was totally depleted by all strains after 8 days of incubation while Poly R-478 was degraded up to 40% with G. australe strains and up to 62% by C. subvermispora after 22 days of incubation. Eucalyptus globulus wood chips decayed for 15 days presented 1–6% of lignin loss and less than 2% of glucan loss. Kraft pulps with kappa number 15 were produced from biotreated wood chips with 2% less active alkali, with up to 3% increase in pulp yield and up to 20% less hexenuronic acids than pulps from undecayed control. Results showed that G. australe strains evaluated were not as efficient as C. subvermispora for dye and wood biodegradation, but could be used as a feasible alternative in biotechnological processes such as bioremediation and biopulping.     

Production of Phanerochaete chrysosporium lignin peroxidase under various culture conditions

Lignin peroxidase production by the white-rot fungus Phanerochaete chrysosporium is markedly influenced by the buffer system employed. In immobilized P. chrysosporium cultures with carbon-limited glucose medium, the use of acetate buffer resulted in higher lignin peroxidase activities than tartrate. With acetate as the buffer in shake-flask cultures a 20% to over 100% improvement in lignin peroxidase production was obtained as compared to tartrate-buffered systems. Of trace elements, Cu²⁺, Mn²⁺ and Zn²⁺ seemed to have the greatest influence on lignin peroxidase production. Furthermore, an increase in the Cu²⁺ and Zn²⁺ concentrations resulted in considerably higher ligninase activities. Although it has been shown previously that high manganese levels repress ligninase production, for maximum ligninase production the presence of some Mn²⁺ appeared to be necessary. The concentration of phosphorus had surprisingly little effect on ligninase production. Highest lignin peroxidase activities were obtained with lower phosphorus concentrations, but reasonably high activities were obtained within the whole studied phosphorus range of 0.12–4.60 g l^–1. Diammonium tartrate alone was a better nitrogen source than a mixture of diammonium tartrate, proteose peptone and yeast extract. The addition of solid manganese (IV) oxide to 3-day-old immobilized biocatalyst cultures increased the maximum ligninase activity obtained by about one-third. Correspondence to: S. Linko     

Fast degradation of kraft lignin by bacteria

Summary Lignin degrading bacteria were isolated directly by an enrichment culture technique using an industrial kraft lignin (Indulin AT) as the sole carbon source. The lignin degrading ability of these isolates was assayed in pure cultures. One strain (Aeromonas sp.) had degraded 98% of the lignin (1 g/l) after 5 days of incubation. Different genera have been identified including Corynebacterium, Agrobacterium, Pseudomonas, Aeromonas, but also Klebsiella and Enterobacter. These strains were also able to assimilate different phenolic compounds considered as lignin related simple monomers.     

Effect of biodelignification of rice straw on humification and humus quality by Phanerochaete chrysosporium and Streptomyces badius

The effect of biodelignification of rice straw by two different ligninolytic organisms, Phanerochaete chrysosporium (white-rot fungus) and Streptomyces badius (actinomycetes), on humus quality was investigated during a 56-day incubation at 30 °C. Lignin degradation, the release of humic extract (HE), humic acid (HA) and fulvic acid (FA), E4/E6 ratio of HA, and humification index (HI, HA/FA) were measured during the incubation. Lignin was degraded by both organisms, but to different extents. Lignin was degraded to 41% and 31% by P. chrysosporium and S. badius, respectively. HE released by P. chrysosporium and S. badius were, respectively, 2.10 and 2.13 times larger than that in the control at the maximum values. A significant correlation between lignin degradation and humus-related parameters involving HA fraction showed that both organisms are converting lignin to humic substances.     

Biodelignification of rice straw by <Emphasis Type="Italic">Phanerochaete chrysosporium</Emphasis> in the presence of dirhamnolipid

Lignin degradation by white-rot fungi has received considerable attention as a means for reducing accumulation of lignocellulosic wastes in the environment. The stimulatory effect of surfactants on fungal lignocellulose bioconversion also has attracted wide interest. In this study the influence of dirhamnolipid biosurfactant on biodegradation of rice straw by Phanerochaete chrysosporium was investigated. It was shown that the biodelignification process of rice straw can be significantly enhanced by the presence of dirhamnolipid biosurfactant. In particular, the dirhamnolipid at the concentration of 0.007% increased the peak activity of lignin peroxidase (LiP) by 86% without affecting the manganese peroxidase (MnP) activity. The water-soluble organic carbon (WSOC) contents in the straw substrates as well as the microbial growth and activity were effectively improved by dirhamnolipid, while the degradation rate of lignin increased by 54% with dirhamnolipid of 0.007%. Observed chemical structural and morphological changes showed that the straw substrates were delignified in the presence of dirhamnolipid with the formation of terrace-like fragments separated from the inner cellular fibers and the release of simple compounds. Variation partitioning analysis revealed that the dirhamnolipid addition induced a significant straw biodelignification which explained 22.1% (P = 0.013) of the variance.     

Factors Involved in the Regulation of a Ligninase Activity in Phanerochaete chrysosporium

The regulation of an H₂O₂-dependent ligninolytic activity was examined in the wood decay fungus Phanerochaete chrysosporium. The ligninase appears in cultures upon limitation for nitrogen or carbohydrate and is suppressed by excess nutrients, by cycloheximide, or by culture agitation. Activity is increased by idiophasic exposure of cultures to 100% O₂. Elevated levels of ligninase and, in some cases, of extracellular H₂O₂ production are detected after brief incubation of cultures with lignins or lignin substructure models, with the secondary metabolite veratryl alcohol, or with other related compounds. It is concluded that lignin degradation (lignin → CO₂) by this organism is regulated in part at the level of the ligninase, which is apparently inducible by its substrates or their degradation products.     

Lignin decomposition along an Alpine elevation gradient in relation to physicochemical and soil microbial parameters

Lignin is an aromatic plant compound that decomposes more slowly than other organic matter compounds; however, it was recently shown that lignin could decompose as fast as litter bulk carbon in minerals soils. In alpine Histosols, where organic matter dynamics is largely unaffected by mineral constituents, lignin may be an important part of soil organic matter (SOM). These soils are expected to experience alterations in temperature and/or physicochemical parameters as a result of global climate change. The effect of these changes on lignin dynamics remains to be examined and the importance of lignin as SOM compound in these soils evaluated. Here, we investigated the decomposition of individual lignin phenols of maize litter incubated for 2 years in‐situ in Histosols on an Alpine elevation gradient (900, 1300, and 1900 m above sea level); to this end, we used the cupric oxide oxidation method and determined the phenols’ ¹³C signature. Maize lignin decomposed faster than bulk maize carbon in the first year (86 vs. 78% decomposed); however, after the second year, lignin and bulk C decomposition did not differ significantly. Lignin mass loss did not correlate with soil temperature after the first year, and even correlated negatively at the end of the second year. Lignin mass loss also correlated negatively with the remaining maize N at the end of the second year, and we interpreted this result as a possible negative influence of nitrogen on lignin degradation, although other factors (notably the depletion of easily degradable carbon sources) may also have played a role at this stage of decomposition. Microbial community composition did not correlate with lignin mass loss, but it did so with the lignin degradation indicators (Ac/Al)s and S/V after 2 years of decomposition. Progressing substrate decomposition toward the final stages thus appears to be linked with microbial community differentiation.     

Factors affecting lignin degradation in lignocellulose by Phanerochaete chrysosporium

Cultural conditions affecting lignin degradation by Phanerochaete chrysosporium in various lignocellulosic materials were studied in comparison to an isolated lignin preparation. With shallow mycelial cultures, the degradation of lignin in wood proceeded more slowly in a 100% O₂-atmosphere than in an air atmosphere, indicating that pure oxygen was toxic to the fungus. The organism was able to degrade lignin efficiently even under 30% CO₂ and 10% O₂ concentrations. Evolution of ¹⁴CO₂ from labelled lignocellulosic materials was shown not to be representative of total lignin degradation. Addition of glucose to the culture did not affect lignin degradation measured by ¹⁴CO₂ evolution, whereas lignin degradation measured by Klason lignin method stopped completely (poplar) or slowed considerably (straw). Due to partial depolymerization of lignin to soluble products, measuring only the evolution of ¹⁴CO₂ results in an underestimation of the total amount of lignin bioaltered. The soluble products from all of the tested lignocellulosic materials and from the isolated lignin had an average molecular weight of about 1,000 and the products could be further fractionated by ion exchange chromatography. The relative amount of these products could be varied from 15 to 45% from the original lignin.     

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     

Degradation of alkali-lignin residues from solid-state fermentation of wheat straw by streptomycetes

The ability of three Streptomyces strains to degradealkali-lignin, produced from the treatment of wheat straw by the same organisms, was examined. Decolourisation and loss of alkali-lignin was only detected in cultures supplemented with ammonium as an inorganic N source. The pH of cultures supplemented with inorganic N reached lower pH than in those supplemented with yeast extract. From FT-IR spectra corresponding to the alkali-lignin obtained from the same cultures, a degradation of carbohydrate component concomitant with a modification in the aromatic moiety of lignin could be inferred. The results indicate that streptomycetes are suitable for use in the treatment of alkali-lignin effluents from the biological treatment of wheat straw by the same organisms and therefore support the role for these organisms in the development of clean technologies in pulp and paper industry.     

Biotreatment of pulp mill bleachery effluents with the coelomycetous fungusStagonospora gigaspora

Summary The coelomyceteStagonospora gigaspora degrades lignin derivatives within pulp mill bleachery effluents. Besides dechlorination, 90% of the color was removed from CEH bleachery effluents. Lignin derivatives in the effluents of the EOP bleaching stages revealed more persistent against fungal attack. Toxicity of both effluents was diminished significantly byS. gigaspora.     

Isolation of Bacterial Strain from Sediment Core of Pulp and Paper Mill Industries for Production and Purification of Lignin Peroxidase (LiP) Enzyme

Five bacterial strains were isolated and purified (CSA101 to CSA105) from the sediment core of the effluent released from the Century Pulp and Paper Mill Ltd., India. These strains were grown in minimal salt medium (MSM) containing pulp (10% as a carbon source). The production of lignin peroxidase, CMCase, Fpase, and xylanase together with protein and reducing sugar by all bacterial strains was observed. All of the bacterial isolates responded differently with respect to growth and ligninocellulolytic enzyme production. The maximum lignin peroxidase (LiP) was obtained from the cell extract of Bacillus sp. (CSA105) strain, which was used for purification, fractionation and characterization. The culture filtrate from Bacillus sp. (CSA105) was purified with ammonium sulfate precipitation. Crude protein was desalted by dialyzing with Tris buffer. The lignolytic enzyme produced in the liquid medium was fractionated by gel filtration on Sephadex G-100. In the present study, 12.4-fold purification of LiP enzyme was obtained and 35.85% yield of lignin peroxidase was achieved in the cell extract of Bacillus sp. (CSA105). Lignin peroxidase enzyme plays an important role in lignin degradation process. The ligninolytic enzymes were produced by all of the bacterial strains but maximum lignin peroxidase activity was found in cell extract of CSA105. On the basis of the results obtained, the bacterial strain (CSA105) was found most suitable for the purification of the LiP enzyme.     

White-rot fungal growth on sugarcane lignocellulosic residue

Summary Twelve white-rot fungi were grown in solid state culture on sugarcane chips previously fermented by yeast employing the EX-FERM process. The lignocellulosic sugarcane residue had 12.5% permanganate lignin and 81.3% holocellulose. After 5 to 6 weeks at 20° C, all fungi produced a solid residue which had a lower in vitro dry matter enzymatic digestibility than the original bagasse, with the exception of Coriolus versicolor which showed a slight increase of 0.6 units. Four fungi produced a residue with higher soluble solids than the original sample. Lignin losses were rather similar for all fungi tested, an average value of 38.64% of the original value was obtained. About the same amount of hemicellulose was degreaded, 32.22%. Most fungi showed a preference for hemicellulose hydrolysis over cellulose degradation. The two fungi that showed greater cellulolytic activity were Sporotrichum pulverulentum and Dichomitus squalens. No appreciable dry matter losses were detected for Agrocybe aergerita and Flammulina velutipes.     

Biotechnology in the degradation and utilization of lignocellulose

Lignocellulose is the predominant renewable resource. It uses include fuel, as the feedstock for the pulp and paper industry, and for animal nutrition. It also constitutes a large proportion of agricultural and urban waste. Biotechnology has roles in its efficient production and utilisation. The types of lignin substrates available for study of lignin biodegradation are described. The white rot fungus Phanerochaete chrysosporium is the archetypal system for the study of lignocellulose degradation, since it mineralises lignin and degrades both cellulose and hemicellulose. The salient features of the P. chrysosporium system are described. The lignin peroxidases are a family of proteins, and it is shown that expression of their genes is differential. P. chrysosporium is heterokaryotic with two gene equivalents that have abundant RFLPs. A set of basidiospore-derived strains with genetic compositions defined by such RFLPs provided the potential basis for a strain improvement programme for lignin degradation. However, analysis of this system using radiolabelled synthetic lignin (DHP) as the substrate confirmed previous evidence that both the substrate and the fungal cultures displayed much variation, so that it was difficult to quantify performance for this property. The cellobiohydrolase I enzymes are also coded for by a family of genes, and evidence is also presented for allelic variants, for differential expression and for differential splicing. In contrast, the cellobiohydrolase II function is encoded at a unique genetic locus. Approaches to an homologous integrative transformation system are discussed. Some actinomycete bacteria represent an alternative system for lignin solubilisation in which strains differ in their spectra of activities on lignocellulose substrates. The xylanase system of Streptomyces cyaneus is shown to include three enzymes, two of which are inducible by xylan. A novel assay method was developed and used to demonstrate that the third is constitutive and also non-repressible by glucose. It is proposed that this acts as a sensor for xylans in the environment that can yield breakdown products that are taken up and can then act as inducers of the other two enzymes. The studies on microbial lignocellulose degradation from different laboratories have allowed the formulation of specific biotechnological goals, and some of the problems and opportunities in this area are identified.