Growth Inhibition of Phytopathogenic Fungi and Oomycetes by Basidiomycete Irpex lacteus and Identification of its Antimicrobial Extracellular Metabolites

In dual culture confrontation assays, basidiomycete Irpex lacteus efficiently antagonized Fusarium spp., Colletotrichum spp., and Phytophthora spp. phytopathogenic strains, with growth inhibition percentages between 16.7–46.3%. Antibiosis assays evaluating the inhibitory effect of soluble extracellular metabolites indicated I. lacteus strain inhibited phytopathogens growth between 32.0–86.7%. Metabolites in the extracellular broth filtrate, identified by UPLC-QTOF mass spectrometer, included nine terpenes, two aldehydes, and derivatives of a polyketide, a quinazoline, and a xanthone, several of which had antifungal activity. I. lacteus strain and its extracellular metabolites might be valuable tools for phytopathogenic fungi and oomycete biocontrol of agricultural relevance.     

In Vitro Bleaching of Hardwood Kraft Pulp by Extracellular Enzymes Excreted from White Rot Fungi in a Cultivation System Using a Membrane Filter

To clarify the role of excreted extracellular enzymes during long-term incubation in a pulp biobleaching system with white rot fungi, we developed a cultivation system in which a membrane filter is used; this membrane filter can prevent direct contact between hyphae and kraft pulp, but allows extracellular enzymes to attack the kraft pulp. Phanerochaete sordida YK-624 brightened the pulp 21.4 points to 54.0% brightness after a 5-day in vitro treatment; this value was significantly higher than the values obtained with Phanerochaete chrysosporium and Coriolus versicolor after a 7-day treatment. Our results indicate that cell-free, membrane-filtered components from the in vitro bleaching system are capable of delignifying unbleached kraft pulp. Obvious candidates for filterable reagents capable of delignifying and bleaching kraft pulp are peroxidase and phenoloxidase proteins. The level of secreted manganese peroxidase activity in the filterable components was substantial during strain YK-624 in vitro bleaching. A positive correlation between the level of manganese peroxidase and brightening of the pulp was observed.     

Production of thermostable pectinase and xylanase for their potential application in bleaching of kraft pulp

A very high level of alkalophilic and thermostable pectinase and xylanase has been produced from newly isolated strains of Bacillus subtilis and Bacillus pumilus respectively. Enzyme production for pectinase was carried out under SSF using combinations of cheap agricultural residues while xylanase was produced under submerged fermentation using wheat bran as substrate to minimize the cost of production of these enzymes Among the various substrates tested, the highest yield of pectinase production was observed by using combination of WB + CW (6592 U/g of dry substrate) supplemented with 4% yeast extract when incubated at 37 °C for 72 h using deionized water of pH 7.0 as moistening agent. The biobleaching effect of these cellulase free enzymes on kraft pulp was determined. Both xylanase and pectinase showed stability over a broad range of pH from 6 to 10 and temperature from 55 to 70 °C. The bleaching efficiency of the pectinase and xylanase on kraft pulp was maximum after 150 min at 60 °C using enzyme dosage of 5 IU/ml of each enzyme at 10% pulp consistency with about 16% reduction in kappa number and 84% reduction in permanganate number. Enzyme treated pulp when subjected to CDED₁D₂ steps, 25% reduction in chlorine consumption and upto 19% reduction in consumption of chlorine dioxide was observed for obtaining the same %ISO brightness. Also an increase of 22 and 84% in whiteness and fluorescence respectively and a decrease of approximately 19% in the yellowness of the biotreated pulp were observed by pretreatment of the pulp with our enzymatic mixture.     

Potential of xylanase from thermophilic Bacillus sp. XTR-10 in biobleaching of wood kraft pulp

An extracellular xylanase produced under optimal conditions by a thermophilic strain of Bacillus sp. XTR-10 was evaluated for its potential application in biobleaching of wood kraft pulp. Spectrophotometric analysis showed considerable release of lignin derived compounds and chromophoric material by the xylanase treated pulp samples. Xylanase was found to be effective in the liberation of reducing sugars in the pulp filtrates with increment in enzyme dose and reaction time. Eight hours pretreatment with 40 IU of xylanase/g of dry pulp resulted in 16.2% reduction of kappa number with 25.94% ISO increase in brightness as compared to the control. The same treatment slightly lowered the tensile strength and burst index, however. Enzyme pretreatment of the pulp saved 15% active chlorine charges in single step and 18.7% in multiple steps chemical bleaching with attainment of brightness at the level of the control. These results indicate the potential of enzymatic pretreatment of pulp for reduction in environmental discharge of hazardous waste from the pulp and paper industry.     

Effects of Kraft Pulp and Lignin on Trametes versicolor Carbon Metabolism

The white rot basidiomycete Trametes (Coriolus) versicolor can substantially increase the brightness and decrease the lignin content of washed, unbleached hardwood kraft pulp (HWKP). Monokaryotic strain 52J was used to study how HWKP and the lignin in HWKP affect the carbon metabolism and secretions of T. versicolor. Earlier work indicated that a biobleaching culture supernatant contained all components necessary for HWKP biobleaching and delignification, but the supernatant needed frequent contact with the fungus to maintain these activities. Thus, labile small fungal metabolites may be the vital biobleaching system components renewed or replaced by the fungus. Nearly all of the CO₂ evolved by HWKP-containing cultures came from the added glucose, indicating that HWKP is not an important source of carbon or energy during biobleaching. Carbon dioxide appeared somewhat earlier in the absence of HWKP, but the culture partial O₂ pressure was little affected by the presence of pulp. The presence of HWKP in a culture markedly increased the culture's production of a number of acidic metabolites, including 2-phenyllactate, oxalate, adipate, glyoxylate, fumarate, mandelate, and glycolate. Although the total concentration of these pulp-induced metabolites was only 4.3 mM, these compounds functioned as effective manganese-complexing agents for the manganese peroxidase-mediated oxidation of phenol red, propelling the reaction at 2.4 times the rate of 50 mM sodium malonate, the standard chelator-buffer. The presence of HWKP in a culture also markedly stimulated fungal secretion of the enzymes manganese peroxidase, cellulase, and cellobiose-quinone oxidoreductase, but not laccase (phenol oxidase) or lignin peroxidase.     

Lipase production by Aspergillus ibericus using olive mill wastewater

Olive mill wastewater (OMW) characteristics make it a suitable resource to be used as a microbial culture media to produce value-added compounds, such as enzymes. In this work, the ability of the novel species Aspergillus ibericus to discolor OMW and produce lipase was studied. An initial screening on plates containing an OMW-based agar medium and an emulsified olive oil/rhodamine-B agar medium was employed to select the strain A. ibericus MUM 03.49. Then, experiments in conical flasks with liquid OMW-based media showed that the fungus could growth on undiluted OMW, with a chemical oxygen demand (COD) of 97 ± 2 g/L, and to produce up to 2,927 ± 54 U/L of lipase. When pure OMW was used in the media, the maximum COD and color reduction achieved were 45 and 97 %, respectively. When OMW diluted to 10 % was used, A. ibericus was able to reduce phenolic and aromatic compounds by 37 and 39 %, respectively. Additionally, lipase production was found to be promoted by the addition of mineral nutrients. When the fermentations were scaled up to a 2-L bioreactor, A. ibericus produced up to 8,319 ± 33 U/L of lipase, and the maximum COD and color reduction were 57 and 24 %, respectively.     

Enhanced delignification of mixed wood pulp by <Emphasis Type="Italic">Aspergillus fumigatus</Emphasis> laccase mediator system

An environmentally sound biobleaching to get high quality paper pulp from mixed wood pulp was attempted employing laccase from Aspergillus fumigatus VkJ2.4.5 for lignin removal. Laccase treatment was performed in the presence of a mediator N-hydroxybenzotriazole (HBT, 1.5% w/w), resulting into notably higher level of delignification of the pulp. Enzyme at 10 Ug⁻¹ of pulp at 50°C, pH 6.0, for 2 h with a pulp consistency of 10% was found suitable for enabling maximum decrease in the kappa number. The kappa number and yellowness decreased by 14 and 4% whereas ISO brightness improved by 7%. The presence of a characteristic peak at 280 nm indicated the presence of lignin in the effluent during biobleaching. Analysis of FTIR spectra of residual lignin revealed characteristic modifications following enzymatic bleaching by laccase mediator system (LMS). Variations in morphology and crystallinity of pulp were evaluated by scanning electron microscopy and X-ray diffraction analysis.     

Optimisation of the biological pretreatment of wheat straw with white-rot fungi for ethanol production

The biological pretreatment of lignocellulosic biomass for the production of bioethanol is an environmentally friendly alternative to the most frequently used process, steam explosion (SE). However, this pretreatment can still not be industrially implemented due to long incubation times. The main objective of this work was to test the viability of and optimise the biological pretreatment of lignocellulosic biomass, which uses ligninolytic fungi (Pleurotus eryngii and Irpex lacteus) in a solid-state fermentation of sterilised wheat straw complemented with a mild alkali treatment. In this study, the most important parameters of the mechanical and thermal substrate conditioning processes and the most important parameters of the fungal fermentation process were optimised to improve sugar recovery. The largest digestibilities were achieved with fermentation with I. lacteus under optimised conditions, under which cellulose and hemicellulose digestibility increased after 21 days of pretreatment from 16 to 100 % and 12 to 87 %, respectively. The maximum glucose yield (84 %) of cellulose available in raw material was obtained after only 14 days of pretreatment with an overall ethanol yield of 74 % of the theoretical value, which is similar to that reached with SE.     

Promoting effects of organic medium supplements on the micropropagation of promising ornamental Daphne species (Thymelaeaceae)

Three Daphne species (Thymelaeaceae) were propagated in vitro using media enriched with natural ingredients including coconut water, pineapple pulp, arabinogalactan, chitosan, and conditioned medium containing exudates of the green alga Desmodesmus subspicatus. High vigor of proliferative shoots and enhanced rooting efficiency were obtained. The propagation rate for shoot cultures of Daphne caucasica and Daphne tangutica increased significantly when cultured in the presence of 10 ml?L^?1 coconut water or 10 ml?L^?1 pineapple pulp. Addition of 10 ml?L^?1 pineapple pulp, 10 ml?L^?1 coconut water, or 20% conditioned medium to the culture medium stimulated organogenesis in D. caucasica. The percentage of rooted shoots in this difficult-to-root species reached 80% in enriched medium. Daphne jasminea plants rooted efficiently on media without growth regulators but supplemented with 10 ml?L^?1 pineapple pulp or 10 ml?L^?1 coconut water. Plants of D. caucasica and D. jasminea were successfully acclimatized to greenhouse conditions. Biochemical evaluation of pineapple pulp using thin-layer chromatography revealed the absence of natural auxins. However, the low-molecular-weight fraction (<500 Da) obtained via dialysis significantly stimulated rhizogenesis in each species tested.     

A cellulase-supported two-phase in situ system for enhanced biosynthesis of paclitaxel in <Emphasis Type="Italic">Taxus</Emphasis> × <Emphasis Type="Italic">media</Emphasis> hairy roots

The effectiveness of two liquid-phase culture systems, in situ supported with perfluorodecalin (PFD), and elicited with methyl jasmonate (100 µM) and sodium nitroprusside (10 µM) (spiked with l-phenylalanine (100 µM) and sucrose (30 g/l) feeding), and additionally combined with cellulolytic enzyme application (Cellulase or Viscozyme^® L at doses 0.01%, 0.1%, 0.5%  or 1%), was investigated on the enhancement of paclitaxel release in Taxus × media harbouring taxadiene synthase transgene hairy root cultures. Neither elicitation nor in situ application of PFD significantly had an effect on root growth until enzyme addition; however, the hairy root response to the culture conditions varied depending on enzyme type and dosage. The highest paclitaxel total yield (intracellular?+?extracellular) was determined in the one-phase elicited culture systems without enzymes and amounted to 264.2 µg/flask (1 434.9?±?516.6 µg/g DW) and 29.6 µg/flask in root biomass and medium phase, respectively. Although the application of cellulolytic enzymes did not enhance the total paclitaxel production, they intensified paclitaxel release in a dose-depending manner. Among two examined cellulolytic enzyme treatments, Viscozyme^® L addition caused the highest paclitaxel release up to 59% of its total content when used at a concentration of 0.01%. Whereas in two-phase in situ systems, the combination of Viscozyme^® L at dosage of 0.5% and PFD-aerated, resulted in the highest extracellular paclitaxel concentration up to 20%.     

Studies on Milk Clotting Enzymes Produced by Basidiomycetes

In the course of screening tests of Basidiomycete proteolytic enzymes, it was observed that some strains produced milk clotting enzymes with fairly weak proteolytic activities.

When sucrose-polypeptone and sucrose-corn steep liquor media were used, only 6 strains out of 44 strains tested showed weak milk clotting activities. Cheddar cheese making with culture filtrates of these 6 strains revealed that the culture filtrates of 2 strains, Irpex lacteus Fr. and Fomitopsis pinicola (Fr.) Karst., were able to produce Cheddar cheese of good quality.

On the other hand, when sucrose-distillers solubles media were used, a lot of strains showed high proteolytic activity in addition to high milk clotting activity. The ratio of milk clotting to proteolytic activities (MCA/PA) was assumed to be an important index for the selection of organism, and F. pinicola and Coriolus consors (Berk.) Imaz. were selected as the strain with high MCA/PA ratio.

As the investigation on culture conditions of 3 strains mentioned above showed that F. pinicola and I. lacteus, were richly productive of milk clotting enzymes, the 2 strains except C. consors were used for further studies on cheese making.

Cheddar cheese making with crude enzymes revealed that cheese products produced by the enzyme of F. pinicola had a slightly bitter taste after 5 months’ ripening but that those produced by the enzyme of I. lacteus had good quality.     

Biobleaching of wheat straw pulp with recombinant laccase from the hyperthermophilic <Emphasis Type="Italic">Thermus thermophilus</Emphasis>

The recombinant laccase from Thermus thermophilus was applied to the biobleaching of wheat straw pulp. The best bleaching effect was when the pulp was treated with 3 U laccase g⁻¹ dry pulp at 90°C, pH 4.5, 8% consistency for 1.5 h. Under these conditions, the pulp brightness was increased by 3.3% ISO, and the pulp kappa number was decreased by 5.6 U. Enzymatic treatment improved the bleachability of wheat straw pulp but caused no damage to the pulp fibers. The use of enzyme-treated pulp saved 25% H₂O₂ consumption in subsequent peroxide bleaching without decreasing the final brightness. Pulp biobleaching in the presence of 5 mM ABTS further increased the pulp brightness by 1.5% ISO. This is the first report on the application of laccase from T. thermophilus in the pulp and paper sector.     

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.     

Fungal interaction between Trichoderma spp. and Pleurotus ostreatus on the enriched solid media with licorice Glycyrrhiza glabra root extract

The aim of this study is to investigate the antifungal activity of mycelia of Pleurotus ostreatus (white oyster mushroom) and licorice (Glycyrrhiza glabra) root extract against three undesirable fungi. They are Trichoderma spp., Trichoderma harzianum I and Trichoderma harzianum II which was tested on PSA (potato sucrose agar) medium enriched with licorice (Glycyrrhiza glabra) root extract (PSA-G media) using three concentrations (0.05, 0.10 and 0.20 g/L) in alone and dual cultures. Trichoderma spp. showed less mycelial growth of 8.75, 9.17 and 9.50 mm/day on PSA-G0.05, PSA-G0.1 and PSA-G0.2 respectively compared with 10.25 mm/day on fresh PSA (control) in dual culture. The best mycelial growth inhibition was recorded on PSA-G0.2 (14.97%) by T. harzianum II in alone culture opposite 63.72% in dual ones. The lower mycelial growth rate of T. harzianum I was 17.75 mm/day on PSA-G0.1 (0.10 g/L). In dual culture, overgrowth time of T. harzianum I had 5 days compared as approx. 6 days in alone culture. Generally, when the concentration of licorice extract increased, the mycelial growth rate of the undesirable fungi decreased. Also, all PSA-G media, especially PSA-G0.2, indicated low growth averages compared with the control (fresh PSA) against the pathogen while this concentration encourages growth of oyster mushroom. Also, this concentration reduced the density of sporulation of green molds; therefore, this concentration can be applied to reduce influence this pathogen in cultivation farm.     

Occurrence of Two Types of Cystathionine β-Cleavage Enzyme in Bacillus subtilis

Production of extracellular polysaccharidases by Irpex lacteus Fr. was studied in different culture conditions.

The presence of fatty acids such as linolic, linolenic, erucic, palmitic acids etc. caused remarkably to increase the production of ceîlulase (filter paper disintegrating activity, FD), laminarinase and xylanase. On the contrary, fatty acids had not any special effect on the production of cellulolytic enzymes such as Avicelase and CMCase and of plant tissue macerating enzymes (MA).

When two kinds of carbon sources, e.g., cellulose powder and potato pulp were mixed together and used as an inducer, polysaccharidase production investigated, with the exception of CMCase, increased higher than when the two substances were used separately.     

Strain variability of <Emphasis Type="Italic">Irpex Lacteus</Emphasis> basidiomycetes in the synthesis of specific milk-clotting proteinases

The ability to synthesize milk-clotting (rennet) proteinases was studied in eight strains of Irpex Lacteus basidiomycetes. It was found that the studied I. lacteus strains are characterized by different enzyme activities in their culture liquid. For I. lacteus strains 2425, 2426, and 2427, the maximum milk-clotting activity was detected during the exponential growth phase on the 15th day of cultivation on glucose–peptone medium. These I. lacteus strains are the most prospective milk-clotting enzyme producers for further research and practical application. I. lacteus strains 2421, 2422, 2423, 2424, and 2428 showed lower values of the enzyme activity and require additional research to determine the optimal culture conditions.     

Transformation of 2,4,6-trinitrotoluene by white rot fungus Irpex lacteus

Up to 200 mg 2,4,6-trinitrotoluene (TNT) l^–1 was removed within 12 h after adding it to a 5-day old culture of Irpex lacteus. The initial formation of hydroxylamino-dinitrotoluenes (2- and 4-OHAmDNT) from TNT was detected, followed by their successive transformation to aminodinitrotoluenes (2- and 4-AmDNT). Transformation of TNT to AmDNT via OHAmDNT was fast, but the next step was slow and seemed to be a rate-limiting step in TNT degradation. OHAmDNT isomers were also rapidly transformed by an in vitro enzymatic system. Both the mycelium and extracellular enzymes of I. lacteus were required for the TNT degradation.     

Production of xylanase by Aspergilli using alternative carbon sources: application of the crude extract on cellulose pulp biobleaching

The ability of xylanolytic enzymes produced by Aspergillus fumigatus RP04 and Aspergillus niveus RP05 to promote the biobleaching of cellulose pulp was investigated. Both fungi grew for 4–5 days in liquid medium at 40°C, under static conditions. Xylanase production was tested using different carbon sources, including some types of xylans. A. fumigatus produced high levels of xylanase on agricultural residues (corncob or wheat bran), whereas A. niveus produced more xylanase on birchwood xylan. The optimum temperature of the xylanases from A. fumigatus and A. niveus was around 60–70°C. The enzymes were stable for 30 min at 60°C, maintaining 95–98% of the initial activity. After 1 h at this temperature, the xylanase from A. niveus still retained 85% of initial activity, while the xylanase from A. fumigatus was only 40% active. The pH optimum of the xylanases was acidic (4.5–5.5). The pH stability for the xylanase from A. fumigatus was higher at pH 6.0–8.0, while the enzyme from A. niveus was more stable at pH 4.5–6.5. Crude enzymatic extracts were used to clarify cellulose pulp and the best result was obtained with the A. niveus preparation, showing kappa efficiency around 39.6% as compared to only 11.7% for that of A. fumigatus.     

The use of extracellular enzymes from Streptomyces albus ATCC 3005 for the bleaching of eucalyptus kraft pulp

The suitability of culture supernatant from Streptomyces albus ATCC 3005 for use in the biobleaching of eucalyptus kraft pulp was investigated. S. albus was found to grow on a minimal salts medium containing oat spelts xylan and yeast extract as the main carbon and nitrogen sources, respectively. Maximal extracellular xylanase and peroxidase production was detected after 120 h (11.97 U ml(-1)) and 72 h (0.58 U ml(-1)), respectively. Importantly, no cellulase activity could be detected. When the effect of pH on enzyme activity was examined, maximal xylanase and peroxidase activity was obtained at pH 6.5 and pH 9.9, respectively. The optimum hydrogen peroxide (H2O2) concentration for peroxidase activity was found to occur at 20 mM, with peroxidase remaining active at 100 mM H2O2 after 1 h incubation at 53 degrees C; the half-life of the enzyme at that temperature was estimated to be 33 min. Short-term (1 h) biobleaching of eucalyptus kraft pulp with culture supernatant from S. albus in the presence of H2O2 resulted in a significant reduction of kappa number (2.85 units) with no change in viscosity. These results suggest a potential application of cellulase-free culture supernatants from S. albus in biobleaching.