Solid-state fermentation of sugarcane bagasse with Flammulina velutipes and Trametes versicolor

The mushroom Flammulina velutipes and the white-rot fungus Trametes versicolor were cultivated separately on sugarcane bagasse for 40 days. Trametes versicolor produced laccase and manganese-peroxidase activities, showing a simultaneous degradation of lignin and holocellulose. However, only phenoloxidase activity was found with Flammulina velutipes. A preferential degradation of lignin was detected in F. velutipes, which exhibited a greater reduction in the ratio of weight loss to lignin loss than T. versicolor. A decrease in the syringyl/guaiacyl ratio observed with both fungi indicated the preferential degradation of non-condensed (syringyl-type) lignin units. An increase in the relative abundance of aromatic carboxylic acids suggested that the oxidative transformation of lignin unit side-chains was occurring. This was more noticeable with Flammulina velutipes than with T. versicolor.     

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.     

Biodelignification of Lemon Grass and Citronella Bagasse by White-Rot Fungi

Twelve white-rot fungi were grown in solid-state culture on lemon grass (Cymbopogon citratus) and citronella (Cymbopogon winterianus) bagasse. The two lignocellulosic substrates had 11% permanganate lignin and a holocellulose fraction of 58%. After 5 to 6 weeks at 20°C, nine fungi produced a solid residue from lemon grass with a higher in vitro dry matter enzyme digestibility than the original bagasse; seven did the same for citronella. The best fungus for both substrates was Bondarzewia berkeleyi; it increased the in vitro dry matter enzyme digestibility to 22 and 24% for lemon grass and citronella, respectively. The increases were correlated with weight loss and lignin loss. All fungi decreased lignin contents: 36% of the original value for lemon grass and 28% for citronella. Practically all fungi showed a preference for hemicellulose over cellulose.     

Selective Degradation of Wood Components by White-Rot Fungi

In order to find naturally occurring white-rot fungi which preferentially degrade lignin. 25 different species of such fungi were cultivated on pine wood blocks and on kraft lignin agar plates with and without cellulose. Due to differences in phenol oxidase reactions on the kraft lignin agar plates, the 25 fungi could be divided into two groups, 1 and 2, which also differed in other properties. The three Group I fungi Sporotrichum pulverulentum, Phanerochaete sp. L1 and Polyporus dichrous produced high levels of endo-l,4-β-glucanase and cellobiose:quinone oxidoreductase in shaking cellulose flasks and a low level of phenol oxidase in standing wood meal flasks, The four fungi Merulius tremellosus, Phlebia radiata, Pycuoporus cinnabarinus and Pleurotus ostreatus from Group 2, on the other hand, produced low levels of endo-1,4-β-glucanase and cellobiose:.quinone oxidoreductase in the cellulose. flasks and a high level of phenol oxidase in the wood meal flasks. Analyses of pine wood blocks degraded by the above-mentioned fungi in the presence of either malt extract, asparagine or NH₄H₂PO₄ revealed that malt extract gave good lignin degradation. In the presence of this nutrient source. P. cinnabarinus, at 3.4% weight loss, even degraded 12.5% lignin without loss of cellulose or mannan. No common degradation pattern was, however, obtained using mall extract, asparagine or NH₄H₂PO₄, It is suggested that while-rot fungi, which preferentially degrade lignin, may be found among Group 2 fungi producing large amounts of phenol oxidases.     

血红密孔菌高产漆酶菌株的筛选及其对烟梗的生物降解

白腐菌是目前已知的唯一能将木质素彻底降解的微生物,而漆酶在木质素分解过程中起着重要的作用,被广泛应用于农作物秸秆或甘蔗渣等多种类型生物质的生物预处理和生物降解。本研究利用白腐菌产漆酶发酵培养基对30株血红密孔菌Pycnoporus sanguineus菌株进行筛选,得到了多株漆酶高产菌株,并研究了血红密孔菌发酵粗酶液和菌丝对烟梗的生物降解条件。研究结果表明:血红密孔菌及其产生的漆酶表现出了对烟梗木质素较强的生物降解能力。在漆酶浓度为40U/mL、温度30℃、pH4.5的条件下处理24h,烟梗中木质素的降解率可达到50.4%,纤维素和半纤维素的降解率分别为17.5%和17.3%;漆酶浓度为5U/mL、温度30℃、pH4.5的条件下处理48h,木质素降解率可达到65.1%。血红密孔菌菌丝也表现出对烟梗较好的生物降解效果,接种培养7d后烟梗中木质素降解率可达30%以上,21d后木质素的降解率可达79.1%,而纤维素和半纤维素的降解率仅为20%和12%左右。本研究不但为生物质材料的生物预处理和生物降解提供了优质的白腐菌及漆酶资源,还为通过烟梗的生物预处理提高烟草梗丝和卷烟品质提供了重要参数,具有一定的应用前景。     

Succession and activity of microorganisms in stored bagasse

Summary Fresh sugarcane bagasse was fermented under defined conditions and investigated regarding a microbial succession during fermentation, in view of the enzyme activities of microorganisms against the main bagasse components: sucrose, pectin, hemicellulose, cellulose, and lignin.Altogether, 400 pure cultures of microorganisms were obtained from 8 g bagasse during 6.5 days of storage. This flora consists of bacteria (74%), actinomycetes (6%), yeasts (13%), and fungi (7%). The yeasts dominate in early fermentation, followed by bacteria, and then by actinomycetes and fungi.This succession coincides with the enzymic activities of the isolated organisms during fermentation. At first, residual sugar is consumed predominantly by the yeasts. Then the bacteria degrade the pectin, the hemicellulose, and in parts, the cellulose. Later, the actinomycetes and the fungi imperfecti attack the hemicellulose, the cellulose, and, partly, the lignin within the bagasse fiber.These results are corroborated by investigations using bagasse from bulk storage.     

Solid state fermentation and fractionation of oat straw by Basidiomycetes

Summary Seventee white-rot and brown-rot fungi were screened for their ability to fractionate the lignocellulose structure of oat straw through the preferential attack of lignin or cellulose. Fermentations were carried out under solid-state conditions with 25 g quantities of straw. The fermented straw was analyzed for weight loss, Klason lignin loss and cellulase digestion. All the fungi attacked both lignin and carbohydrate fractions causing 3–28% weight losses and 26–34 g/100 g enzymatic digestibility. Polyporus tulipiferae, Phanerochaete chrysosporium and Polyporus sp. were tested for the effects of various nitrogen, phosphate and carbon levels, incubation temperatures and incubation time. The three fungi had different responses to these factors.     

Degradation of toxaphene by Bjerkandera sp. strain BOL13 using waste biomass as a cosubstrate

The white-rot fungus Bjerkandera sp. strain BOL13 was capable of degrading toxaphene when supplied with wood chips, wheat husk or cane molasses as cosubstrates in batch culture experiments. Approximately 85% of toxaphene was removed when wheat husk was the main substrate. The production of lignin peroxidase was only stimulated when wheat husk was present in the liquid medium. Although xylanase was always detected, wheat husk supported the highest xylanase production. A negligible amount of β-glucosidase and cellulase were found in the batch culture medium. To the best of our knowledge, this is the first reported case of toxaphene degradation by white-rot fungi.     

Growth of higher fungi on wheat straw and their impact on the digestibility of the substrate

Summary The influence of the growth of three higher fungi on the composition of wheat straw was investigated. Pleurotus pulmonarius, P. sajor-caju and Lentinus edodes grew very well on lignocellulosic substrates, breaking down a considerable amount of lignin. The initial lignin concentration of straw was halved after 12 weeks of fungal growth, doubling the enzymic digestibility. Together with lignin, the higher fungi consumed half of the amount of hemicellulose (i.e. 15%), leaving cellulose fairly intact, which should remain as an energy source for ruminants.     

甘蔗渣纤维素降解菌株的筛选与鉴定

甘蔗渣是制糖工业的主要副产物。筛选甘蔗渣纤维素降解菌株对甘蔗渣乙醇产业具有重要的意义。以甘蔗渣为原料,通过分离和纯化得到14株菌株,对其进行纤维素刚果红平板染色实验和滤纸崩解实验,最终获得3株可以生产纤维素酶的菌株02-2-2、21-1-2和40-1-1。酶活性测定结果表明,菌株40-1-1的酶活力在培养3 d后达到最高,为27.26 U/mg。通过形态学和分子生物学鉴定,菌株02-2-2为枝顶孢属(Acremonium sp.),菌株21-1-2和40-1-1为光滑短梗霉属(Acrophialophora sp.)。研究筛选的菌株将为开展甘蔗渣纤维素降解利用提供参考。     

A biorefinery model for the production of oil and biomethane using castor plants

Castor (Ricinus communis L.) is an important oilseed crop worldwide whose inedible oil is widely used in the industrial, pharmaceutical, and agricultural sectors. Castor plants show high conversion potential for use as biorefining feedstocks. The present study was conducted to investigate the effects of two nitrogen fertilization levels (0 and 120 kg N ha⁻¹) on seed and oil yield. From a biorefinery perspective, the residual biomass of seed processing was analyzed in terms of fiber composition and biomethane production carrying out a biological pretreatment using two white-rot fungi (Pleurotus ostreatus and Irpex lacteus). Nitrogen fertilization resulted in an increase in seed and oil yields and a difference in capsule husk composition. Fungal pretreatment of capsule husks showed promising effects on anaerobic digestion, increasing the biomethane yield compared to untreated biomass. The highest lignin degradation and the lowest cellulose loss during pretreatment were obtained with I. lacteus, and this fungal pretreatment resulted in the highest biomethane yield (103.2 NmL g⁻¹ volatile solids) for the fertilized biomass.     

Biodelignification of wheat straw by different fungal associations

Seven strains of fungi were tested individually as well as in different combinations to determine their lignin degrading ability using wheat straw as natural substrate. When tested individuallyPhanerochaete chrysosporium caused a maximum loss in total organic matter (26.45%) as well as in the lignin component (28.93%). The associations between different groups: white-rot plus white-rot, white-rot plus brown-rot and white-rot plus soft-rot fungi revealed that in certain combinations the ligninolysis was enhanced to variable extent.Deadalea flavida plusP. chrysosporium was the best association to bring about a lignin loss of 36.27%.     

Cellulase stimulation during biodegradation of lignocellulosic residues at increased biomass loading

Microbial degradation of lignocellulosic biomass is primarily affected by the composition and structure of biomass, as well as enzyme activities that are influenced by the presence of in-process degradation products. This study focuses on the latter, and demonstrates that cellulase activity of Neurospora discreta is stimulated in the presence of in-process soluble lignin degradation products. Two types of biomass - cocopeat and sugarcane bagasse, with contrasting lignin content and cellulose structure were tested at two biomass loadings each. At the higher biomass loading, cocopeat showed the highest amount of hydrolyzed cellulose and cellulase activity, despite its low cellulose content and recalcitrant cellulose structure. A strong positive correlation was revealed between the amount of in-process degraded lignin and cellulase activity, indicating a stimulatory effect on cellulase, which contradicts most previous literature. Furthermore, the causal relationship between the amount of degraded lignin and cellulase activity was established in a model system of commercial cellulase and standard soluble lignin. This work could pave the way for using biomass loading as a process lever to enhance cellulose hydrolysis in microbial conversion of lignocellulosic biomass.     

Radiation and fermentation treatment of cellulosic wastes

The effect of radiation pasteurization of sugar cane bagasse and rice straw and fermentation using various strains of fungi were studied for upgrading of cellulosic wastes. The initial contamination by fungi and aerobic bacteria both in bagasse and straw was high. The doses of 30 kGy for sterilization and 8 kGy for elimination of fungi were required. Irradiation effect showed that rice straw contained comparatively radioresistant microorganisms. It was observed that all the fungi (Hericium erinacium, Pleurotus djamor, Ganoderma lucidum, Auricularia auricula, Lentinus sajor-caju, Coriolus versicolor, Polyporus arcularius, Coprinus cinereus) grow extending over the entire substrates during one month after inoculation in irradiated bagasse and rice straw with 3% rice bran and 65% moisture content incubated at 30°C. Initially, sugar cane bagasse and rice straw substrates contained 39.4% and 25.9% of cellulose, 22.9% and 26.9% of hemicellulose, and 19.6% and 13.9% of lignin + cutin, respectively. Neutral detergent fibre (NDF) values decreased significantly in sugar cane bagasse fermented byG. lucidum, A. auricula andP. arcularius, and in rice straw fermented by all the 8 strains of fungi. Acid detergent fibre (ADF) values also decreased in bagasse and rice straw fermented by all the fungi.P. arcularius, H. erinacium, G. lucidum andC. cinereus were found to be the most effective strains for delignification of sugar cane bagasse.     

Temporal Alterations in the Secretome of the Selective Ligninolytic Fungus Ceriporiopsis subvermispora during Growth on Aspen Wood Reveal This Organism's Strategy for Degrading Lignocellulose

The white-rot basidiomycetes efficiently degrade all wood cell wall polymers. Generally, these fungi simultaneously degrade cellulose and lignin, but certain organisms, such as Ceriporiopsis subvermispora, selectively remove lignin in advance of cellulose degradation. However, relatively little is known about the mechanism of selective ligninolysis. To address this issue, C. subvermispora was grown in liquid medium containing ball-milled aspen, and nano-liquid chromatography-tandem mass spectrometry was used to identify and estimate extracellular protein abundance over time. Several manganese peroxidases and an aryl alcohol oxidase, both associated with lignin degradation, were identified after 3 days of incubation. A glycoside hydrolase (GH) family 51 arabinofuranosidase was also identified after 3 days but then successively decreased in later samples. Several enzymes related to cellulose and xylan degradation, such as GH10 endoxylanase, GH5_5 endoglucanase, and GH7 cellobiohydrolase, were detected after 5 days. Peptides corresponding to potential cellulose-degrading enzymes GH12, GH45, lytic polysaccharide monooxygenase, and cellobiose dehydrogenase were most abundant after 7 days. This sequential production of enzymes provides a mechanism consistent with selective ligninolysis by C. subvermispora.     

Studies of growth conditions in wood for three white-rot fungi and their cellulaseless mutants

White-rot fungi, which have the ability to degrade all the wood components including lignin, are of great interest in biotechnological processes based on wood and other lignocellulosic materials. It was demonstrated earlier that enough lignin can be degraded to cause a decrease in the energy demand for production of thermomechanical pulp if wood chips are pretreated by cellulaseless mutants of white-rot fungi. This paper concerns the growth conditions in wood for three white-rot fungi and their cellulaseless mutants in order to determine optimal conditions for such pretreatment processes. The pH and temperature optima have been determined as well as the growth rate in wood. The results show that the growth rate in wood. at least for Cel 44 (a cellulaseless mutant of Sporotrichum pulverulentum), is not the rate-limiting step in delignification. From different mixtures of urea and NH₄H₂PO₄ the optimal nitrogen source was determined for the mutants. The optimal C/N ratio was found to vary between 160/1 and 400/1. It is suggested that the lower the C/N ratio, the faster the growth. It was also demonstrated that both water- and acetone-extractable substances in wood supported the growth of cellulaseless mutants. When some glucose was added to the wood, the weight loss caused by Cel 44 increased. All these observations support earlier findings that lignin in wood cannot be degraded by white-rot fungi unless a more easily metabolizable carbon source is used simultaneously.     

The effect of copper on the growth of wood-rotting fungi and a blue-stain fungus

The effect of copper (II) ions on the growth of three brown-rot fungi, six white-rot fungi and one blue-stain fungus in solid medium was evaluated. The fungi were grown in malt extract agar with different concentrations of copper added, and the radial growth rate was determined. At the end of the incubation period, the mycelial biomass and the media pH were determined. The white-rot and blue-stain fungus grew up to 3 mM and 6 mM copper, respectively and the brown-rot fungi were the only ones that grew up to 10 mM, with higher growth rates than those shown by the other fungi. In general, the brown-rot fungi produced greater acidification in the culture media than the white-rot fungi and blue-stain fungus, and the acidification increased when the amount of copper was increased. The biomass production for the different species, in the absence or presence of copper, was not related to the radial growth rate, and the fungal species that produced the greatest biomass amounts did not correspond to those that presented the highest growth rates. The brown-rot fungi Wolfiporia cocos and Laetiporus sulfureus and blue-stain fungus Ophiostoma sp. demonstrated greater tolerance to high copper concentrations in solid medium than the white-rot fungi, determined as radial growth rate. On the other hand, the highest biomass producers in solid medium with copper added were the white-rot fungi Ganoderma australe and Trametes versicolor and the brown-rot fungus Gloeophyllum trabeum.     

Comparative secretome of white-rot fungi reveals co-regulated carbohydrate-active enzymes associated with selective ligninolysis of ramie stalks

In the present research, Phanerochaete chrysosporium and Irpex Lacteus simultaneously degraded lignin and cellulose in ramie stalks, whereas Pleurotus ostreatus and Pleurotus eryngii could depolymerize lignin but little cellulose. Comparative proteomic analysis of these four white-rot fungi was used to investigate the molecular mechanism of this selective ligninolysis. 292 proteins, including CAZymes, sugar transporters, cytochrome P450, proteases, phosphatases and proteins with other function, were successfully identified. A total of 58 CAZyme proteins were differentially expressed, and at the same time, oxidoreductases participated in lignin degradation were expressed at higher levels in P. eryngii and P. ostreatus. Enzyme activity results indicated that cellulase activities were higher in P. chrysosporium and I. lacteus, while the activities of lignin-degrading enzymes were higher in P. eryngii and P. ostreatus. In addition to the lignocellulosic degrading enzymes, several proteins including sugar transporters, cytochrome P450 monooxygenases, peptidases, proteinases, phosphatases and kinases were also found to be differentially expressed among these four species of white-rot fungi. In summary, the protein expression patterns of P. eryngii and P. ostreatus exhibit co-upregulated oxidoreductase potential and co-downregulated cellulolytic capability relative to those of P. chrysosporium and I. lacteus, providing a mechanism consistent with selective ligninolysis by P. eryngii and P. ostreatus.     

Phenolic removal of olive-mill dry residues by laccase activity of white-rot fungi and its impact on tomato plant growth

We studied the influence of the laccase activity of two white-rot fungi on the toxic effect of water-soluble substances from dry residues of olives (ADOR) on tomato plants. Pycnoporus cinnabarinus and Coriolopsis rigida decreased the phenol content of ADOR to 73% after 15 days. P. cinnabarinus and C. rigida produced laccase activity after 5 and 15 days, respectively, and the highest activity in both fungi was detected at 20 days. The treatment of ADOR with these white-rot fungi decreased the phytotoxicity of this residue on tomato plants. A close relationship was found between the amount of laccase produced, the decrease in phenol content of ADOR by the saprobic fungi, decrease of phytotoxicity of ADOR, and the increase in dry weight of tomato plants. These results show that phenol removal by the laccase activity of white-rot fungi can be important in the elimination of phytotoxic substances present in olive-mill dry residues.     

Lignin biodegradation and ligninolytic enzyme studies during biopulping of Acacia mangium wood chips by tropical white rot fungi

White rot fungi are good lignin degraders and have the potential to be used in industry. In the present work, Phellinus sp., Daedalea sp., Trametes versicolor and Pycnoporus coccineus were selected due to their relatively high ligninolytic enzyme activity, and grown on Acacia mangium wood chips under solid state fermentation. Results obtained showed that manganese peroxidase produced is far more compared to lignin peroxidase, suggesting that MnP might be the predominating enzymes causing lignin degradation in Acacia mangium wood chips. Cellulase enzyme assays showed that no significant cellulase activity was detected in the enzyme preparation of T. versicolor and Phellinus sp. This low cellulolytic activity further suggests that these two white rot strains are of more interest in lignin degradation. The results on lignin losses showed 20–30% of lignin breakdown at 60 days of biodegradation. The highest lignin loss was found in Acacia mangium biotreated with T. versicolor after 60 days and recorded 26.9%, corresponding to the percentage of their wood weight loss recorded followed by P. coccineus. In general, lignin degradation was only significant from 20 days onwards. The overall percentage of lignin weight loss was within the range of 1.02–26.90% over the biodegradation periods. Microscopic observations conducted using scanning electron microscope showed that T. versicolor, P. coccineus, Daedalea sp. and Phellinus sp. had caused lignin degradation in Acacia mangium wood chips.