秸秆降解放线菌GC的筛选及其应用基础研究

运用循环流化技术,从土壤样品中筛选出1株具有单独降解秸秆能力的菌株GC,考察了该菌的生长特性及产纤维素酶和木质素酶能力,验证了该菌对小麦秸秆的处理效果。结果表明,该菌为放线菌的左式链霉菌(Streptomyces drozdowiczii);可在LB等基础培养基中快速繁殖;纤维素内切酶活和滤纸酶活分别可达67.57 U/mL和19.69 U/mL,并且具备木质素降解能力;该菌单独处理小麦秸秆20 d的秸秆失重率为11.52%;处理产物含多种石油烃、有机醇和植物甾醇等,表明该菌在秸秆等农业面源污染物的资源化利用方面具有良好的开发应用前景。     

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

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

Effect of different gas environments on bench-scale solid state fermentation of oat straw by white-rot fungi

Three white-rot fungi, Phanerochaete chrysosporium, Polyporus tulipiferae, and Polyporus sp. A336 were grown on 100-g amounts of chopped oat straw in gassed 4.5 L (diameter 16 cm, height 23 cm) solid-state reactors for two weeks. The different gas atmospheres were regulated by (1) air diffusion through foam plugs, (2) intermittent or continuous air flow, (3) intermittent oxygen, 50 or 100% continuous oxygen flow, and (4) continuous 10% carbon dioxide in oxygen flow. The fermented straw was analyzed for total weight loss, Klason lignin loss, and enzymatic (cellulase) hydrolysis. P. chrysosporium grown on straw in continuous oxygen at 35 degrees C caused a 41% weight loss and 33.5% hydrolysis was obtained when the pretreated straw was hydrolyzed with cellulase enzyme. P. tulipiferae caused a 27% weight loss and 34.3% cellulase hydrolysis in the straw at 30 degrees C. Polyporus sp. A336 selectively degraded lignin of the straw and under intermittent oxygen resulted in an 18% weight loss and 33.6% cellulase hydrolysis at 35 degrees C. When the straw was supplemented with 10% xylose (straw basis) and was continuously gassed with 50% oxygen, Polyporus sp. A336 produced a 14.5% weight loss and 38.7% cellulase hydrolysis. Oxygen and carbon dioxide exchange rates were measured for some of these bench-scale fermentations.     

Lignin solubilisation by Thermomonospora mesophila

Summary Thermomonospora mesophila degraded [¹⁴C]lignin-labelled wheat lignocellulose to yield high molecular weight water-soluble products and a small amount of ¹⁴CO₂. Solubilisation of [¹⁴C]lignin was found to be extracellular and inducible by growth on lignocellulose (straw) and hemicellulose (xylan), but was not correlated with xylanase or cellulase production.The acid-precipitable product of straw degradation by T. mesophila was found to be a complex of lignin, pentose-rich carbohydrate and protein with some similarity to humic acids. Solid-state ¹³C-NMR spectra of the dried product were generally similar to those of chemically extracted milled straw lignin but showed an increased content of carbonyl groups.The relationship between degradation and solubilisation of lignin is discussed and a role suggested for actinomycetes in humification and the exploitation of lignocellulose bioconversion.     

白腐菌及黑曲霉所产的纤维素复合酶对稻草秸秆的生物降解

研究了白腐菌及纤维素复合酶对稻草秸秆的协同生物降解。结果表明,利用黄孢原毛平革菌固态发酵稻草秸秆的过程中,LiP和MnP的最大活力可以达到28.3U/g和12.6U/g,同时,秸秆中的木质素能被有效降解,但纤维素、半纤维素降解率较低。添加黑曲霉所产的纤维素复合酶能有效地促进秸秆腐熟程度。在接入白腐菌培养10天后,每克稻草添加3 IU纤维素酶液并酶解48h可以使稻草秸秆中纤维素降解53.8%,半纤维素降解57.8%,木质素降解44.5%,干物质损失46.3%。此时细胞壁出现大范围破损,整个组织变得松散,秸秆完全腐熟。     

Effect of chemical treatments on the degradability of cotton straw by rumen microorganisms and by fungal cellulase

Three different chemical treatments—sulfur dioxide, ozone, and sodium hydroxide—were applied on cotton straw, and the effect on cell-wall degradability was assessed by using rumen microorganism and Trichoderma reesei cellulase. Sulfur dioxide (applied at 70°C for 72 h) did not change the lignin content of cotton straw but reduced the concentration of hemicellulose by 48%. Ozone exerted a dual effect, both on lignin (a 40% reduction) and hemicellulose (a 54% decrease). The treatment with NaOH did not solublize cell-wall components. The in vitro organic matter digestibility with rumen fluid of cotton straw was increased significantly by ozone and SO₂ treatments, by 120% and 50%, respectively, but not by NaOH. T. reesei cellulase was applied on the chemically pretreated cotton straw at a low level (6 filter paper U/g straw, organic matter), and the release of reducing sugars was determined. The highest level of reducing sugars (30.6 g/100 g organic matter) was obtained with the O₃-cellulase combination, which solubilized 64% of the cellulose and 88% of the hemicellulose. the SO₂- and the NaOH-pretreated cotton straw were hydrolyzed by T. reesei cellulase to the same extent (21 g reducing sugars/100 g organic matter). The rumen fluid digestibility of the enzymatic ally hydrolyzed straw was not increased further over the effect already obtained with the chemical pretreatments. However, the fermentability of the combined treatments was increased markedly. In the O₃-cellulase-treated cotton straw, 83% of the rumen fluid digestible material consisted of highly fermentable components. Although ozone proved to be the most potent pretreatment for enzymic saccharification in this study, the absolute result was modest. The limited effect of the combined O₃-cellulase treatment was probably associated with the pretreatment limitations, but not with the enzyme level. Based on the differential response of the chemically treated cotton straw to attack by rumen microorganisms on the one hand, and by T. reesei cellulase on the other hand, a hypothesis has been suggested as to the location of lignin and hemicellulose in the cellwall unit of cotton straw.     

Screening of a fungus capable of powerful and selective delignification on wheat straw

Aims: To screen and characterize a novel fungus with powerful and selective delignification capability on wheat straw. Methods and Results: A fungus capable of efficient delignification under solid‐state fermentation (SSF) conditions on wheat straw was screened. After 5 days of incubation, 13·07% of the lignin was removed by fungal degradation, and 7·62% of the holocellulose was lost. Furthermore, 46·53% of the alkali lignin was removed after 2 days of liquid fermentation. The fungus was identified as Fusarium concolor based on its morphology and an analysis of its 18S rDNA gene sequence. The molecular weight distribution of lignin was evaluated by gel permeation chromatography. Enzyme assay indicated that the fungus produced laccase, cellobiose dehydrogenase, xylanase and cellulase during the incubation period. Intracellular lignin peroxidase, manganese peroxidase and laccase were produced during liquid fermentation. Conclusions: We have successfully screened a fungus, F. concolor, which can efficiently degrade the lignin of wheat straw, with slight damage to the cellulose, after 5 days of SSF. Significance and Impact of the Study: The newly isolated strain could be used in pretreatment of lignocellulose materials prior to biopulping, bioconversion into fuel and substrates for the chemical industry.     

Biodegradation of paddy straw obtained from different geographic locations by means of <Emphasis Type="Italic">Phlebia</Emphasis> spp. for animal feed

Various cereal straws are used as feed by supplementing the green forage or other feed stuffs. An experiment was designed to see the effect of different geographic locations and climatological conditions on biochemical constituents, fungal degradation and in vitro digestibility of paddy straw. Paddy straw (PS) obtained from three different geographic locations of India was subjected to solid state fermentation using four white rot fungi i.e. Phlebia brevispora, P. fascicularia, P. floridensis and P. radiata. Changes in the biochemical constituents like water soluble content, hemicellulose, cellulose, lignin, total organic matter, and in vitro digestibility of paddy straw was analyzed over a period of 60 days along with lignocellulolytic enzymes i.e. laccase, xylanase and carboxymethyl cellulase. All the fungi degraded the straw samples and enhanced the in vitro digestibility. The paddy straw, obtained from north western zone (NWZ) suffered a maximum loss (228 g/kg) of lignin by P. radiata, while a maximum enhancement of in vitro digestibility from 185 to 256 g/kg was achieved by P. brevispora, which also caused minimum loss in total organic matter (98 g/kg). In PS obtained from central eastern zone (CEZ) and north eastern zone (NEZ), a maximum amount of lignin (210 and 195 g/kg, respectively) was degraded by P. floridensis and resulted into a respective enhancement of in vitro digestibility from 172 to 246 g/kg and 188 to 264 g/kg. The study demonstrates that geographic locations not only affect the biochemical constituents of paddy straw but the fungal degradation of fibers, their in vitro digestibility and lignocellulolytic enzyme activity of the fungus may also vary.     

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.     

Characterization of enzymatic saccharification for acid-pretreated lignocellulosic materials with different lignin composition

The enzymatic saccharification of three different feedstocks, rice straw, bagasse and silvergrass, which had been pretreated with different dilute acid concentrations, was studied to verify how enzymatic saccharification was affected by the lignin composition of the raw materials. There was a quantitatively inverse correlation between lignin content and enzymatic digestibility after pretreatment with 1%, 2% and 4% sulfuric acid. The lignin accounted for about 18.8–21.8% of pretreated rice straw, which was less than the 23.1–26.5% of pretreated bagasse and the 21.5–24.1% of pretreated silvergrass. The maximum glucose yield achieved, under an enzyme loading 6.5 FPU g^?1 DM for 72 h, was close to 0.8 g glucose/g glucan from the enzymatic hydrolysis of the pretreated rice straw; this was twice that from bagasse and silvergrass. A decrease in initial rate of glucose production was observed in all cases when the raw materials underwent enzymatic saccharification with 4% sulfuric acid pretreatment. It is suggested that the higher acid concentration led to an inhibition of β-glucosidase activity. Fourier transform infrared (FTIR) spectroscopy further indicated the chemical properties of the rice straw and silvergrass become more hydrophilic after pretreatment using 2% of sulfuric acid, but the pretreated bagasse tended to become more hydrophobic. The hydrophilic nature of the pretreated solid residues may increase the inhibitive effects of lignin on the cellulase and this could become very important for raw materials such as silvergrass that contain more lignin.     

Evaluation of chemical pretreatment for enzymatic solubilization of rice straw

Summary Rice straw was treated with NaOH, peracetic acid (PA), and sodium chlorite (NaClO₂). Quantitative changes in the composition of the treated straw, crystallinity of the treated straw and extracted cellulose, and susceptibility of the treated straw to Trichoderma reesei cellulase were studied. The alkali treatment resulted in a remarkable decrease in hemicellulose as well as lignin. Consequently, the recovery of residual straw after NaOH treatment was lowest among the three chemical reagents evaluated. The treatment with PA or NaCIO₂ resulted in a slight loss in hemicellulose and cellulose in the straw. The three chemical treatments caused little or no breakdown of the crystalline structure of cellulose in the straw. The treated straw was solubilized with the culture filtrate of T. reesei. The degree of enzymatic solubilization relative to the amount of residual straw was 69% after treatment with 0.25 N NaOH, 42% after treatment with 20% PA, and 50% after treatments with NaClO₂ (twice). The degree of enzymatic solubilization relative to the amount of the untreated straw, however, was 30% after treatment with 0.25 N NaOH, 32% after treatment with 20% PA, and 37% after treatments with NaClO₂ (twice).     

Sorption of penicillium-cellulase on cellulose and lignin

Sorption of Penicillium cellulase onto cotton linters samples differing in physical structure and onto KLASON - lignin from spruce has been investigated by determining cellobiase, CMCL- and ‘linters-activity’ as well as protein content of a cellulase culture filtrate before and after contact with the sorbent at different temperature for different time intervals. CMCL- and cellobiase-activity were found to be reduced much less by the sorption process onto the cellulose samples than ‘linters-activity’ and protein content. Sorption increased in the order: untreated linters < mercerized linters < wet fibrillated (‘colloidal milled’) linters. With the KLASON -lignin also a considerable sorption of the cellulase system was observed. Results are discussed with respect to preferential sorption of enzyme components.     

Changes in biochemical constituents of paddy straw during degradation by white rot fungi and its impact on in vitro digestibility

Aims: To improve the digestibility of paddy straw to be used as animal feed by means of selective delignification using white rot fungi. Methods and Results: Solid state fermentation of paddy straw was carried out with some white rot fungi for 60 days. Different biochemical analyses, e.g. total organic matter (TOM) loss, hemicellulose loss, cellulose loss, lignin loss and in vitro digestibility, were carried out along with laccase, xylanase and carboxymethyl cellulase activity. The results were compared with that of a widely studied fungus Phanerochaete chrysosporium, which degraded 464 g kg^?1 TOM and enhanced the in vitro digestibility from 185 to 254 g kg^?1 after 60 days of incubation. Straw inoculated with Phlebia brevispora possessed maximum crude protein. Conclusions: All the tested white rot fungi efficiently degraded the lignin and enhanced the in vitro digestibility of paddy straw. Phlebia brevispora, Phlebia radiata and P. chrysosporium enhanced the in vitro digestibility almost to similar levels, while the loss in TOM was much lesser in P. brevispora and P. radiata when compared to P. chrysosporium. Significance and Impact of the Study: The study reflects the potential of P. brevispora and P. radiata as suitable choices for practical use in terms of availability of organic matter with higher protein value, selective ligninolysis and better digestibility.     

Lignocellulolytic Enzymes Produced by Volvariella volvacea, the Edible Straw Mushroom

Volvariella volvacea, commonly known as the straw or paddy mushroom, had the following growth characteristics: minimum temperature, 25°C; optimal temperature, 37°C; maximal temperature, 40°C; pH optimum 6.0. Optimal pH for cellulase production was 5.5. The optimal initial pH for cellulase production and mycelial growth was found to be 6.0. The pH and temperature optima for cellulolytic activity were 5.0 and 50°C, respectively. Maximal cellulolytic activity was obtained within 5 days in shake-flask culture. The cellulases were found to be partly cell free and partly cell bound during growth on microcrystalline cellulose. The endoglucanase activity was primarily extracellular, and β-glucosidase activity was found exclusively extracellularly. Weak cellulase activity was detected when cells were grown on cellobiose and lactose. V. volvacea could not digest the lignin portion of newspaper in shake-flask cultivation. Phenol oxidase, an important enzyme in lignin biodegradation, also was lacking in the cell-free filtrate. However, the organism oxidized phenolic compounds when it was cultured on agar plates containing commercial lignin.     

Fungal activities involved in lignocellulose degradation by Pleurotus

Summary During growth of Pleurotus on cotton straw both the straw in general and the lignin in particular were degraded. After 4 days of fungal growth, activity of laccase, catechol oxidase, peroxidase, and cellulase were detected. This activity, however, declined rapidly after 8–10 days of growth.Lignin degradation began after 10 days and reached a maximum after 21 days. It would seem that the preliminary action of laccase is a prerequisite for lignin degradation.The Pleurotus ostreatus strain P₃ had no detectable laccase activity and showed very poor ability to degrade cotton straw and lignin.Water extract of cotton straw was found to be a potent inducer of laccase in liquid medium and had an effect much stronger than several small phenolic compounds. The degradation of washed cotton straw and lignin from this straw was lower than native straw, so was laccase activity on this medium. High carbon dioxide concentrations encouraged straw degradation by P. ostreatus florida but severly limited lignin degradation. Other fungi including the known lignin degrader Phanarochaete chrysosporium were able to degrade up to 40% of cotton straw dry weight within 21 days of fungal growth. The percentage degradation of lignin, however, was very low (only 10% in 21 days). Pleurotus ostreatus florida was able to degrade up to 56% of the lignin within this time.After treatment with P. ostreatus florida almost four times as much glucose was released when the straw was treated with commercial cellulases, showing increased availability of cellulose.It is suggested that treatment with P. ostreatus florida may be used to enrich low value food materials for ruminant animals.     

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.     

Highly efficient solubilization of natural lignocellulosic materials by a commercial cellulase immobilized on various solid supports

Summary A commercial preparation of cellulase was immobilized on CNBr-sepharose, ConA-sepharose, and CNBr-glass beads. When filter paper was used as the substrate, the specific activity of the enzyme immobilized on ConA-sepharose was more than twice that of the soluble enzyme, while the activity of the enzymes immobilized on the other two substrates was either very slightly (CNBr-sepharose) or slightly (CNBr-glass beads) reduced. The immobilized enzymes showed alterations both in the Km and V max values: these were generally either slightly increased (Km) or reduced (V max). In addition, the immobilized enzymes were more resistant to inhibition both by glucose and cellobiose, they were all more stable than the soluble enzyme and solubilized three different natural lignocellulosic materials (alfa-alfa, wheat straw, and pine needles) to a much greater or significantly greater extext than the soluble enzyme: the ConA-sepharose cellulase was the most efficient. The possibility of reusing the immobilized enzyme was also tested. It was found that the ConA-sepharose cellulase could be reused five times with a final loss of activity that ranged between 30% and 50%.     

Kinetics of wheat straw solid-state fermentation with Trametes versicolor and Pleurotus ostreatus — lignin and polysaccharide alteration and production of related enzymatic activities

Summary The kinetics of straw solid-state fermentation (SSF) with Trametes versicolor and Pleurotus ostreatus was investigated to characterize the delignification processes by these white-rot fungi. Two successive phases could be defined during straw transformation, characterized by changes in respiratory activity, changes in lignin and polysaccharide content and composition, increase in in-vitro digestibility, and enzymatic activities produced by the fungi. Lignin composition was analysed after CuO alkaline degradation, and decreases in syringyl/guaiacyl and syringyl/p-hydroxyphenyl ratios and cinnamic acid content were observed during the fungal treatment. An increase in the phenolic acid yield, revealing fungal degradation of side-chains in lignin, was produced by P. ostreatus. The highest xylanase level was produced by P. ostreatus, and exocellulase activity was nearly absent from straw treated with this fungus. Lactase activity was found in straw treated with both fungi, but lignin peroxidase was only detected during the initial phase of straw transformation with T. versicolor. High levels of H₂O₂-producing aryl-alcohol oxidase occurred throughout the straw SSF with P. ostreatus. Offprint requests to: A. T. Martínez     

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.     

Enhanced cellulase production from Trichoderma reesei QM 9414 on physically treated wheat straw

Summary Trichoderma reesei QM 9414 was grown on wheat straw as the sole carbon source. The straw was pretreated by physical and chemical methods. The particle size of straw was less than 0.177 mm. Growth of T. reesei QM 9414 was maximal with alkali-pretreated straw whereas cellulase production was optimal when physically pretreated straw was used as substrate. Cellulase yields expressed as IU enzyme activity/g cellulose present in the cultures were considerably higher when alkali pretreatment of wheat straw was omitted. Cellulase yields of 666 IU/g cellulose for filter paper activity (FPA) are the highest described for cultures of T. reesei QM 9414 carried out in analogous conditions. Crystallinity index of the cellulose contained in wheat straw increased slightly after alkali pretreatment. This increase did not decrease cellulose accessibility to the fungus. Delignification of wheat straw was not necessary to achieve the best cellulase production.