Comparative ligninolytic and polysaccharolytic potentials of an alkaliphilic basidiomycete on native ligninocellulose

A comparison of the ligninolytic, cellulolytic and hemicellulolytic abilities of an alkaliphilic white-rot fungus. Coprinus fimetarius, on wheat straw under varying conditions of solid-substrate fermentation is presented. The extent of fractional degradation (percentage of the original dry weight of the fraction) of straw under an optimized set of cultural conditions (pH 9·0, moisture 65%, temperature 37°C, period 21 days) was in the following order: lignin (45%), cellulose (42%), hemicellulose (27%). Urea nitrogen favoured the degradation of lignin as well as cellulose and hemicellulose up to a certain level (1·5% sterile urea or 3% unsterile urea on a dry weight basis) beyond which the degradation of lignin was relatively more adversely affected than cellulose. The addition of phosphorus and sulphur was found essential for selective lignin removal. Increasing the C:N ratio by addition of free carbohydrates resulted in an overall decrease in the degradation wherein cellulose utilization was the most affected event. The pre-treatment (physical or chemical) of the substrate caused a general increase in biodegradation of lignin, cellulose and hemicellulose. The degrading activity of the fungus declined with the scaling-up of the fermentation particularly under non-sterile conditions.     

Bioconversion and biotransformation of coir pith for economic production of Pleurotus florida: chemical and biochemical changes in coir pith during the mushroom growth and fructification

Coir pith represents ∼50% of the waste from the coir industries and was tested for its potential in serving as a growth substrate for the production of species of oyster mushroom, Pleurotus florida. Due to its high lignin (∼48%) content and amorphous powdery nature, coir pith supported poor mushroom mycelial growth and yields were considerably low (∼25% bioconversion efficiency). Pre-treating coir pith with hot water did not prove economical to produce the mushroom yields. Acid swelling and alkali delignification of coir pith though served to change the structure of coir pith; the mushroom yields were not improved. Amendment of coir pith with rice (Oryza sativa) straw and horse gram (Dolichos biflorus) plant residue tended to greatly modify the physical characteristics of the inoculated mushroom bed. Such a supplementation of coir pith growth substrate resulted in production of mushroom yields with 110–125% bioconversion efficiency. Implications of supplementing coir pith with rice straw/horse gram plant residue in terms of holocellulose:lignin ratio are discussed. Sensorially, the mushrooms so produced did not differ from that on rice straw, the economic growth substrate recommended for production of the mushroom yields on commercial scale. Changes in cellulose, hemicellulose and lignin contents of coir pith amended with rice straw were studied. Cellulase, hemicellulase and protease enzyme activities in the amended coir pith substrate showed a continuous increase from inoculation till the end of fructification, whereas laccase activity decreased during fructification, in consonance with decreased lignin degradation during fructification.     

The key role of lignin decomposing fungi in the decay of roofs thatched with water reed

We investigated the involvement of microorganisms in the rapid reed decay of roofs thatched with water reed. Numerous bacteria and fungi were isolated by enrichment cultures from reed samples and from fungal fruit bodies on roofs. All strains were characterised in respect to their abilities to degrade cellulose, hemicelluloses and the lignin model substance Poly-R-478. Only 15 of the 92 isolated bacterial strains were capable of degrading cellulose and hemicelluloses. However, nearly all 61 of the identified fungal isolates had these abilities. Nevertheless, only 14 of the isolated fungal strains as well as a reference isolate of Trametes versicolor were capable of degrading Poly-R-478. The ability of the microorganisms to attack complete reed was assessed using a newly developed test system which measures the loss of dry weight during the incubation. A significant loss of dry weight was apparent only in tests using the ligninolytic fungi Pycnoporus cinnabarinus, Trametes versicolor, Phlebia tremellosa and some Mycena species, but not in the case of the majority of cellulolytic bacteria and fungi. From these results, we conclude that ligninolytic fungi are capable of destroying complete reed structure and that they play the key role in the process of the rapid decay of roofs thatched with reed. Directly after the initial lignin attack, cellulose and hemicellulose were degraded to a great extent, evidenced by the large loss of dry weight (up to 72 %), which significantly exceeds the lignin content of reed (ca. 15 %). However, after the initial attack by ligninolytic fungi, bacteria or other fungi capable of degrading cellulose and hemicelluloses may contribute to the progressive decay of reed under natural conditions. Furthermore, we show that the rate of decay depends on the source of the reed and on the reed quality.     

Fungal succession and decomposition of <Emphasis Type="Italic">Camellia japonica</Emphasis> leaf litter

Decomposition processes of Camellia japonica leaf litter were investigated over an 18-month period with reference to the role of fungal succession in the decomposition of lignin and holocellulose. Decomposition and fungal succession were studied in bleached and nonbleached portions of litter, which were precolonized by ligninolytic and cellulolytic fungi, respectively. Coccomyces nipponicum and Lophodermium sp. (Rhytismataceae), which can attack lignin selectively, caused mass loss of lignin and were responsible for bleaching during the first 4 months (stage I), whereas cellulolytic fungi caused mass loss of holocellulose in adjacent nonbleached portions. Soluble carbohydrates and polyphenols also decreased rapidly during this stage. Pestalotiopsis guepini, coelomycete sp.1, and the Nigrospora state of Khuskia oryzae caused mass loss of holocellulose between 4 and 14 months (stage II) and Xylaria sp. caused mass loss of both lignin and holocellulose from 14–18 months (stage III). In stages II and III, decomposition was more rapid in bleached portions than in nonbleached portions probably due to the prior delignification of lignified holocellulose in bleached portions. Frequencies of these fungi showed different responses among species to the pattern of changes in lignin and holocellulose contents during decomposition. Total hyphal length increased in both portions over the study period, but mycelia of basidiomycetes accounted for about 2% of total hyphal length, suggesting that their role in fungal succession and decomposition was low. Lignin and nitrogen contents were consistently lower and holocellulose content was higher in bleached portions than in nonbleached portions during decomposition. The succession of ligninolytic and cellulolytic fungi was a major driving factor that promoted decomposition and precolonization by ligninolytic fungi enhanced decomposition.     

Inheritance of cellulose- and lignin-degrading ability as well as endoglucanase isozyme pattern in Dichomitus squalens

Summary The progeny of Dichomitus squalens CBS-432-34 is heterogeneous with respect to specific growth rate on glucose, cellulolytic ([U¹⁴C]cellulose ¹⁴CO₂) and ligninolytic ([¹⁴C]synthetic lignin ¹⁴CO₂) activities with little correlation between these metric characters. Variations do not show clear-cut phenotypes but rather a continuous range between extreme values pointing to multigenic control of these characters. Most homocaryons showed decreased cellulolytic or ligninolytic activity compared to the parent dicaryon. However a few homocaryons were comparable or even superior to the parent dicaryon for ligninolytic or cellulolytic activity with no correlation between each factor. Strains with reduced cellulolytic activity and altered isozyme patterns of endoglucanases were isolated in the progeny of D. squalens CBS-432-34. While the parent strain produced three main endoglucanase multiple enzymes designated EnI, EnII and EnIII, several strains in the progeny produced a different multiple enzyme pattern. In contrast to the quantitative ability to degrade cellulose, multiple enzyme pattern variation in the progeny did not show continuous variations. characterization of heterocaryon phenotypes derived from Ien⁺ and Ien 1 homocaryons and first filial generation (f1) analysis showed that genetic control of the multiple enzyme pattern (Ien 1 phenotype) in D. squalens is complex. Offprint requests to: E. Odier     

Effect of coculture of anaerobic fungi isolated from ruminants and non-ruminants with methanogenic bacteria on cellulolytic and xylanolytic enzyme activities

Neocallimastix strain N1, an isolate from a ruminant (sheep), was cocultured with three Methanobacterium formicicum strains, Methanosarcina barkeri, and Methanobrevibacter smithii. The coculture with Methanobacterium formicicum strains resulted in the highest production of cellulolytic and xylanolytic enzymes. Subsequently four anaerobic fungi, two Neocallimastix strains (N1 and N2) from a ruminant and two Piromyces species from non-ruminants (E2 and R1), were grown in coculture with Methanobacterium formicicum DSM 3637 on filter paper cellulose and monitored over a 7-day period for substrate utilisation, fermentation products, and secretion of cellulolytic and xylanolytic enzymes. Methanogens caused a shift in fermentation products to more acetate and less ethanol, lactate and succinate. Furthermore the cellulose digestion rate increased by coculture. For cocultures of Neoallimastix strains with Methanobacterium formicicum strains the cellulolytic and xylanolytic enzyme production increased. Avicelase, CMCase and xylanase were almost completely secreted into the medium, while 40–60% of the -glucosidase was found to be cell bound. Coculture had no significant effect on the location of cellulolytic and xylanolytic enzymes.     

Laccase, cellulase and xylanase activities during growth ofPleurotus sajor-caju on sagohampas

Sagohampas, the fibrous pith residue left after starch extraction from sago palm, is abundant at sago-processing factories and can be used as a substrate for the production of laccase by solid substrate fermentation (SSF) withPleurotus sajorcaju, an edible mushroom. The fungus grown onhampas with an adjusted carbon : nitrogen ratio of 35:1, exhibited high laccase activity together with variable cellulase (0.3-2.8 U/g) and xylanase (0.9-10.1 U/g) activity. The maximum amount of laccase produced was approximately 17.7 U/g after 6 days of SSF using 4-week-old inoculum at a density of 10%. With the mature four-week inoculum, laccase activity increased 12-fold compared to that achieved with two-week-old inoculum. The optimum pH and temperature of the crude laccase were 6.0 and 50‡C, respectively. The apparent K_m and V_max values obtained were 0.073 mM and 0.962 U/min, respectively. The maximum laccase activity could be almost doubled after 6 days of fermentation by addition of 0.2 mM vanillin or ferulic acid; the cellulose to lignin ratio increased significantly during the 12 days of SSF, from 2.74 in the control to 3.3, when 0.2 mM of either vanillin or ferulic acid was added to the substrate.     

Effect of substrate nitrogen on lignin degradation by Pleurotus ostreatus

In order to determine the effect of substrate nitrogen (N) on ligninolytic activity, Pleurotus ostreatus was grown in solid media containing either growth-limiting (1mM) or excess (10mM) NH₄Cl. After 25 days, ¹⁴C–CO₂ production from ¹⁴C-cornstover lignin in low-N medium was 3 times that in nitrogen (N)-rich medium. Supplementation of low-N medium with glucose (0.3%) further enhanced ligninolytic activity. Decolorization of an aromatic, polymeric dye, Poly R-481, in solid media was also greatest under N-limiting conditions.     

Relationship of nitrogen to the onset and suppression of ligninolytic activity and secondary metabolism in Phanerochaete chrysosporium

Ligninolytic activity in the white-rot fungus Phanerochaete chrysosporium was previously found not to be induced by lignin, but to develop in cultures in response to nitrogen starvation. Added NH ₄ ⁺ suppressed existing activity. The present study examined amino acid profiles and protein concentrations during onset of ligninolytic activity (synthetic ¹⁴C-lignin¹⁴CO₂) in nitrogen-limited cultures, and defined some characteristics of subsequent suppression by added nutrient nitrogen. During the transition between depletion of medium nitrogen and the onset of ligninolytic activity, total free intracellular amino acids increased, then rapidly decreased; changes in glutamate concentration played a major role. Intracellular protein concentration fluctuated in a manner roughly converse to that of the concentration of free amino acids. Protein turnover was rapid (5–7%/h) during the transition period. Glutamate, glutamine, and histidine were the most effective of 14 nitrogenous compounds in suppressing ligninolytic activity after its onset. The suppressive effect was not mediated through carbon (glucose)-catabolite repression or by alterations in culture pH. Activities responsible for oxidation of lignin and the ligninrelated phenol, 4-hydroxy-3-methoxyacetophenone, responded similarly to added nitrogen. Synthesis of a secondary metabolite, veratryl alcohol, like lignin oxidation, was suppressed quite sharply by glutamate and significantly by NH ₄ ⁺ . Results indicate that nitrogen metabolism affects ligninolytic activity as a part of secondary metabolism, and suggest a role for glutamate metabolism in regulating this phase of culture development.Non-Standard Abbreviations DMS 2,2-dimethylsuccinate - GLC gas-liquid chromatography - TCA trichloroacetic acid     

Spirochaeta caldaria sp. nov., a thermophilic bacterium that enhances cellulose degradation by Clostridium thermocellum

Two strains of obligately anaerobic, thermophilic spirochetes were isolated from cyanobacterial mat samples collected at freshwater hot springs in Oregon and Utah, USA. The isolates grew optimally between 48° and 52°C, and did not grow at 25° or 60°C. Both strains fermented various pentoses, hexoses, and disaccharides. Amino acids or cellulose did not serve as fermentable substrates for growth. H₂, CO₂, acetate, and lactate were end products of d-glucose fermentation. On the basis of physiological characteristics, guanine + cytosine content of DNA, and comparisons of 16S ribosomal RNA sequences, it was concluded that the two isolates were representatives of a novel species of Spirochaeta for which the name Spirochaeta caldaria is proposed. One of the two strains was grown in coculture with a thermophilic cellulolytic bacterium (Clostridium thermocellum) in a medium containing cellulose as the only fermentable substrate. In the coculture cellulose was broken down at a faster rate than in the clostridial monoculture. The results are consistent with the suggestion that interactions between cellulolytic bacteria and non-cellulolytic spirochetes enhance cellulose breakdown in natural environments in which cellulose-containing plant material is biodegraded.     

Application of solid waste from anaerobic digestion of poultry litter in Agrocybe aegerita cultivation: mushroom production, lignocellulolytic enzymes activity and substrate utilization

The degradation and utilization of solid waste (SW) from anaerobic digestion of poultry litter by Agrocybe aegerita was evaluated through mushroom production, loss of organic matter (LOM), lignocellulolytic enzymes activity, lignocellulose degradation and mushroom nutrients content. Among the substrate combinations (SCs) tested, substrates composed of 10–20% SW, 70–80% wheat straw and 10% millet was found to produce the highest mushroom yield (770.5 and 642.9 g per 1.5 kg of substrate). LOM in all SCs tested varied between 8.8 and 48.2%. A. aegerita appears to degrade macromolecule components (0.6–21.8% lignin, 33.1–55.2% cellulose and 14–53.9% hemicellulose) during cultivation on the different SCs. Among the seven extracellular enzymes monitored, laccase, peroxidase and CMCase activities were higher before fruiting; while xylanase showed higher activities after fruiting. A source of carbohydrates (e.g., millet) in the substrate is needed in order to obtain yield and biological efficiency comparable to other commercially cultivated exotic mushrooms.     

Bioconversion of agricultural lignocellulosic wastes through the cultivation of the edible mushrooms Agrocybe aegerita,Volvariella volvacea and Pleurotus spp.

Ten selected wild and commercial strains of Pleurotus ostreatus,Pleurotus eryngii,Pleurotus pulmonarius, Agrocybe aegerita andVolvariella volvacea were cultivated on three agricultural wastes, i.e. wheat straw (WS), cotton waste (CW) and peanut shells (PS). All species demonstrated significantly higher colonization rates on WS and CW than on PS. WS supported faster growth of A. aegerita and Pleurotus spp., whereas V. volvacea performed better on CW. Comparison of growth rates on composted and non-composted WS and CW substrates revealed that in the latter case faster colonization was achieved, particularly for Pleurotus spp. However, one commercial strain of V. volvacea presented higher growth rates when the composted CW medium was used. Furthermore, earliness in the fructification of P. ostreatus, P. pulmonarius and V. volvacea strains was promoted in CW substrates, while WS favoured earliness of P. eryngii and A. aegerita. Similarly, high sporophore yields were obtained by P. ostreatus and P. pulmonarius on both wastes, whereas WS enhanced yield and basidioma size of P. eryngii and A. aegerita strains and CW production of V. volvacea. The substrates cellulose:lignin ratios were found to be positively correlated to mycelial growth rates and to mushroom yield of P. ostreatus and P. pulmonarius; in addition, positive correlation was also detected for carbon:nitrogen ratio and mushroom yield in P. eryngii and A. aegerita and between cellulose content and mushroom yield for V. volvacea strains.     

Effect of biological pretreatment with Trametes hirsuta yj9 on enzymatic hydrolysis of corn stover

In this study, a newly isolated Trametes hirsuta yj9 was used to pretreat corn stover in order to enhance enzymatic digestibility. T. hirsuta yj9 preferentially degraded lignin to be as high as 71.49% after 42-day pretreatment. Laccase and xylanase was the major ligninolytic and hydrolytic enzyme, respectively and filter paper activity (FPA) increased gradually with prolonged pretreatment time. Sugar yields increased significantly after pretreatment with T. hirsuta yj9, reaching an enzymatic digestibility of 73.99% after 42 days of pretreatment. Scanning electron microscopy (SEM) showed significant structural changes in pretreated corn stover, the surface of pretreated corn stover became increasingly coarse, the gaps between cellulose fibers were visible, and many pores were developed. Correlation analysis showed that sugar yields were inversely proportional to the lignin contents, less related to cellulose and hemicellulose contents.     

Newspaper as a substrate for cellulolytic landfill bacteria

Five cellulolytic bacterial isolates ( Clostridium and Eubacterium spp.) from a methane-producing landfill were examined to determine their ability to utilize newspaper as a substrate for growth. Solubilization was poor with even the most actively cellulolytic bacteria. The major factor causing the low activity seemed to be that as much as 24% of the newspaper was composed of the high molecular weight polymer lignin, which exerts a protective effect on the attack of otherwise susceptible polymers. The presence of ink on heavily printed paper also reduced the rate of cellulose solubilization. Although the ink did not appear directly toxic to the bacteria it masked the surface of the paper, covering the cellulose fibres and preventing bacterial adhesion to the substrate. The action of the cellulolytic isolates was also strongly inhibited below the optimum growth temperature of 37°C.     

Mutational and kinetic analysis of basic amino acid transport in the cyanobacterium Synechocystis sp. PCC 6803

Two nitrogen-deregulated mutants of Phanerochaete chrysosporium, der8-2 and der8-5, were isolated by subjecting wild type conidia to gamma irradiation, plating on Poly-R medium containing high levels of nitrogen, and identifying colonies that are able to decolorize Poly-R. The mutants showed high levels of ligninolytic activity (¹⁴C-synthetic lignin ¹⁴CO₂), and lignin peroxidase, manganese peroxidase and glucose oxidase activities in both low nitrogen (2.4 mM) and high nitrogen (24 mM) media. The wild type on the otherhand displayed these activities in low nitrogen medium but showed little or no activities in high nitrogen medium. Fast protein liquid chromatographic analyses showed that the wild type as well as the der mutants produce three major lignin peroxidase peaks (designated L1, L2 and L3) with lignin peroxidase activity in low nitrogen medium. Furthermore, in low nitrogen medium, mutant der8-5 produced up to fourfold greater lignin peroxidase activity than that produced by the wild type. In high nitrogen medium, the wild type produced no detectable lignin peroxidase peaks whereas the mutants produced peaks L1 and L2, but not L3, and a new lignin peroxidase protein peak designated LN. Mutants der8-2 and der8-5 also produced high levels of glucose oxidase, an enzyme known to be associated with secondary metabolism and an important source of H₂O₂ in ligninolytic cultures, both in low and high nitrogen media. In contrast, the wild type produced high levels of glucose oxidase in low nitrogen medium and only trace amounts of this enzyme in high nitrogen medium. The results of this study indicate that the der mutants are nitrogen-deregulated for the production of a set of secondary metabolic activities associated with lignin degradation such as lignin peroxidases, manganese peroxidases and glucose oxidase.     

Endoglucanase Activity of Compost-Dwelling Fungus Paecilomyces inflatus is Stimulated by Humic Acids and Other Low Molecular Mass Aromatics

Summary Paecilomyces inflatus isolated from municipal waste compost was found to have cellulolytic activity in several solid and liquid media. This study was done to reveal the multifarious effects of municipal waste compost on endoglucanase activity of P. inflatus. The highest enzyme activities under the conditions of solid-state fermentation were measured in authentic compost samples compared with wood, straw and bran substrates. In surface liquid cultures glucose, cellobiose, xylan, Avicel cellulose, carboxymethylcellulose (CM-cellulose), starch and citrus pectin were used as carbon sources. All carbon sources supported the growth of P. inflatus. However, only CM-cellulose, cellobiose and pectin noticeably stimulated endoglucanase (EG) activity. Further stimulation of EG activity was obtained in cultures containing 1% CM-cellulose as a carbon source by supplementation with low-molecular mass aromatic compounds vanillin, veratric acid and benzoic acid, and with soil humic acid (SHA). SHA and veratric acid were found to be the most efficient elicitors of the cellulolytic activity. P. inflatus was able to utilize nitrate and ammonium as pure nitrogen sources in media containing cellulose.     

Production of acetyl esterase during wood biodegradation byCoriolus versicolor

A role of acetyl esterase in wood biodegradation byCoriolus versicolor was examined by the assay of enzyme production and the chemical analysis of decayed wood meal of Japanese beech (Fagus crenata). Enzyme assay demonstrated that the degradation proceeded in two stages and acetyl esterase production was correlated with the cellulolytic and xylanolytic enzyme production in the second stage, not with the production of phenol-oxidizing enzymes. From the results of chemical analysis, acetyl and xylose contents in wood meal were observed to decrease simultaneously in the second stage. In contrast, rapid decrease of lignin was recognized during the initial three wk of incubation, and it was closely related with the production of phenol-oxidizing enzymes in the first stage. These results show that acetyl esterase ofC. versicolor participates in the degradation of acetylxylan and acts with the cellulolytic and xylanolytic systems, not with the ligninolytic system.     

Novel Penicillium cellulases for total hydrolysis of lignocellulosics

The (hemi)cellulolytic systems of two novel lignocellulolytic Penicillium strains (Penicillium pulvillorum TUB F-2220 and P. cf. simplicissimum TUB F-2378) have been studied. The cultures of the Penicillium strains were characterized by high cellulase and β-glucosidase as well moderate xylanase activities compared to the Trichoderma reesei reference strains QM 6a and RUTC30 (volumetric or per secreted protein, respectively). Comparison of the novel Penicillium and T. reesei secreted enzyme mixtures in the hydrolysis of (ligno)cellulose substrates showed that the F-2220 enzyme mixture gave higher yields in the hydrolysis of crystalline cellulose (Avicel) and similar yields in hydrolysis of pre-treated spruce and wheat straw than enzyme mixture secreted by the T. reesei reference strain. The sensitivity of the Penicillium cellulase complexes to softwood (spruce) and grass (wheat straw) lignins was lignin and temperature dependent: inhibition of cellulose hydrolysis in the presence of wheat straw lignin was minor at 35 °C while at 45 °C by spruce lignin a clear inhibition was observed. The two main proteins in the F-2220 (hemi)cellulase complex were partially purified and identified by peptide sequence similarity as glycosyl hydrolases (cellobiohydrolases) of families 7 and 6. Adsorption of the GH7 enzyme PpCBH1 on cellulose and lignins was studied showing that the lignin adsorption of the enzyme is temperature and pH dependent. The ppcbh1 coding sequence was obtained using PCR cloning and the translated amino acid sequence of PpCBH1 showed up to 82% amino acid sequence identity to known Penicillium cellobiohydrolases.     

Production of laccase and manganese peroxidase by<Emphasis Type="Italic"> Fomes sclerodermeus</Emphasis> grown on wheat bran

The aim of this work was to study the growth and production of ligninolytic enzymes by Fomes sclerodermeus using a natural medium based on wheat bran as the principal substrate in a solid-state fermentation. Growth was monitored by measuring the chitin content in the substrate. The maximum rate of growth was observed between days 7 and 18. A 38% total dry-weight loss of the substrate was measured after 28 days of cultivation. Differential hydrolysis of the substrate revealed that cellulose was more extensively degraded than lignin. In the 28-day incubation period, the losses of cellulose and lignin were 38 and 15%, respectively. No lignin peroxidase activity was found in any of the media tested. The maximum manganese-dependent peroxidase activity recorded was 6.3 U g⁻¹ at 14 days, while the maximum laccase activity was 270 U g⁻¹ at 28 days post-inoculation. Addition of commonly used inducers such as copper or manganese did not produce a further increase in the enzyme activities, nor did addition of glucose, asparagine, or malt extract. Electronic Publication