Mechanisms of coal solubilization by the deuteromycetes Trichoderma atroviride and Fusarium oxysporum

Three different mechanisms can be envisaged that are used by fungi to solubilize coal: the production of alkaline substances, the extrusion of chelators and, of special interest in the scope of biotechnology, the action of enzymes. Whether these mechanisms are operating separately or in various combinations has not yet been finally assessed. The two deuteromycetes Fusarium oxysporum and Trichoderma atroviride solubilize coal by synergistic effects of various different mechanisms depending on the cell metabolism. F. oxysporum seems to solubilize coal by increasing the pH of the mycelial surroundings and by the action of chelators induced during growth in glutamate-containing media (without involvement of enzymes). T. atroviride, on the other hand, appears to use, in addition to an alkaline pH and a high chelator activity, at least two classes of enzyme activity to attack coal: hydrolytic activity for coal solubilization and ligninolytic activity for degradation of humic acids. Received: 3 February 1998 / Received revision: 31 August 1998 / Accepted: 3 September 1998     

Extracellular cross-linking of maize arabinoxylans by oxidation of feruloyl esters to form oligoferuloyl esters and ether-like bonds

Primary cell walls of grasses and cereals contain arabinoxylans with esterified ferulate side chains, which are proposed to cross‐link the polysaccharides during maturation by undergoing oxidative coupling. However, the mechanisms and control of arabinoxylan cross‐linking in vivo are unclear. Non‐lignifying maize (Zea mays L.) cell cultures were incubated with l‐ [1‐³H]arabinose or (E)‐[U‐¹⁴C]cinnamate (radiolabelling the pentosyl and feruloyl groups of endogenous arabinoxylans, respectively), or with exogenous feruloyl‐[³H]arabinoxylans. The cross‐linking rate of soluble extracellular arabinoxylans, monitored on Sepharose CL‐2B, peaked suddenly and transiently, typically at ～9 days after subculture. This peak was not associated with appreciable changes in peroxidase activity, and was probably governed by fluctuations in H₂O₂ and/or inhibitors. De‐esterified arabinoxylans failed to cross‐link, supporting a role for the feruloyl ester groups. The cross‐links were stable in vivo. Some of them also withstood mild alkaline conditions, indicating that they were not (only) based on ester bonds; however, most were cleaved by 6 m NaOH, which is a property of p‐hydroxybenzyl–sugar ether bonds. Cross‐linking of [¹⁴C]feruloyl‐arabinoxylans also occurred in vitro, in the presence of endogenous peroxidases plus exogenous H₂O₂. During cross‐linking, the feruloyl groups were oxidized, as shown by ultraviolet spectra and thin‐layer chromatography. Esterified diferulates were minor oxidation products; major products were: (i) esterified oligoferulates, released by treatment with mild alkali; and (ii) phenolic components attached to polysaccharides via relatively alkali‐stable (ether‐like) bonds. Thus, feruloyl esters participate in polysaccharide cross‐linking, but mainly by oligomerization rather than by dimerization. We propose that, after the oxidative coupling, strong p‐hydroxybenzyl–polysaccharide ether bonds are formed via quinone‐methide intermediates.     

Degradation of low rank coal by Trichoderma atroviride ES11

A new isolate of Trichoderma atroviride has been shown to grow on low rank coal as the sole carbon source. T. atroviride ES11 degrades ∼82% of particulate coal (10 g l⁻¹) over a period of 21 days with 50% reduction in 6 days. Glucose (5 g l⁻¹) as a supplemented carbon source enhanced the coal solubilisation efficiency of T. atroviride ES11, while 10 and 20 g l⁻¹ glucose decrease coal solubilisation efficiency. Addition of nitrogen [1 g l⁻¹ (NH₄)₂SO₄] to the medium also increased the coal solubilisation efficiency of T. atroviride ES11. Assay results from coal-free and coal-supplemented cultures suggested that several intracellular enzymes are possibly involved in coal depolymerisation processes some of which are constitutive (phenol hydroxylase) and others that were activated or induced in the presence of coal (2,3-dihydrobiphenyl-2,3-diol dehydrogenase, 3,4-dihydro phenanthrene-3,4-diol dehydrogenase, 1,2-dihydro-1,2-dihydroxynaphthalene dehydrogenase, 1,2-dihydro-1,2-dihydroxyanthracene dehydrogenase). GC-MS analysis of chloroform extracts obtained from coal degrading T. atroviride ES11 cultures showed the formation of only a limited number of specific compounds (4-hydroxyphenylethanol, 1,2-benzenediol, 2-octenoic acid), strongly suggesting that the intimate association between coal particles and fungal mycelia results in rapid and near-quantitative transfer of coal depolymerisation products into the cell. An erratum to this article can be found at     

Influence of aromatic compounds on biodegradation of [14C]-labeled xylan and mannan by the white-rot fungus Phlebia radiata

Radiolabeled [¹⁴C]arabinoxylan from wheat meal and [¹⁴C]galactoglucomannan from red clover meal were prepared by using ¹⁴CO₂ as a precursor. Twice as much mannan was mineralized than xylan after 14 days of incubation with Phlebia radiata. Low-molecular-weight phenolic compounds structurally related to lignin increased during mineralization of both hemicellulose fractions. Veratryl alcohol increased degradation of arabinoxylan by approximately 28.5%, whereas veratric acid increased it by only 9.0%. Vanillic acid and ferulic acid also stimulated degradation by 16.6% and 34.7%, respectively. Veratryl alcohol and ferulic acid increased degradation of galactoglucomannan by approximately 75%. Veratraldehyde in both cases repressed the degradation process (23.6% arabinoxylan, 43.8% galactoglucomannan). These results indicate that the degradation of hemicelluloses, e.g., xylan and mannan, by P. radiata is enhanced by addition of aromatic compounds. Journal of Industrial Microbiology & Biotechnology (2002) 28, 168–172 DOI: 10.1038/sj/jim/7000221 Received 25 July 2001/ Accepted in revised form 23 October 2001     

The effect of lyophilization and storage time on the survival rate and hydrolytic activity of <Emphasis Type="Italic">Trichoderma</Emphasis> strains

The study evaluates the survivability and storage stability of seven Trichoderma strains belonging to the species: T. harzianum (1), T. atroviride (4), and T. virens (2) after the lyophilization of their solid state cultures on wheat straw. Biomass of Trichoderma strains was freeze-dried with and without the addition of maltodextrin. Furthermore, in order to determine the ability of tested Trichoderma strains to preserve selected technological features, the biosynthesis of extracellular hydrolases (cellulases, xylanases, and polygalacturonases) after a 3-month storage of lyophilizates was investigated. Strains of T. atroviride (except TRS40) and T. harzianum TRS85 showed the highest viability after lyophilization process (up to 100%). After 3 months of storage, T. atroviride TRS14 exhibited the highest stability (95.23%); however, the number of active conidia remained at high level of 10⁶–10⁷ cfu/g for all tested T. atroviride strains and T. harzianum TRS85. Interestingly, after a 3-month storage of lyophilized formulations, most of the tested Trichoderma strains exhibited higher cellulolytic and xylanolytic activities compared to the control, i.e., before freeze-drying process. The highest activities of these enzymes exhibited the following: T. atroviride TRS14–2.37 U/g and T. atroviride TRS25–21.47 U/g, respectively, whereas pectinolytic activity was weak for all tested strains, with the highest value of 0.64 U/g registered for T. virens TRS109.     

Solid substrate fermentation of lignite by the coal-solubilizing mould, Trichoderma atroviride, in a new type of bioreactor

Trichoderma atroviride CBS 349 is able to solubilize lignite. The mould was cultured under non-sterile conditions in a new type of bioreactor for solid substrate fermentation. German lignite (lithotype A, Bergheim) was used as complex solid substrate. Over 40 days 140 g of 1.5 kg lignite held in a 25 l-bioreactor was solubilized by the fungus.     

Compatibility testing and enhancing the pulp bleaching process by hydrolases of the newly isolated thermophilic Isoptericola variabilis strain UD-6

Abstract

In the present study, Isoptericola variabilis strain UD-6 isolated from alkaline hot spring of Unapdev, Maharashtra, India was assessed for its biobleaching activity by hydrolytic enzymes on rice straw pulp. Results of primary and secondary screening manifested that it was a multi-enzyme producer, competent to produce amylase, cellulase, mannanase, pectinase, and xylanase at 9.73, 4.11, 6.26, 8.42, and 6.61?IU?ml^?1 in fermentation conditions, respectively. Maximum activity of all enzymes was gained at thermal temperature (50–55?°C), alkaline condition (pH 8–9), under 5?mM KCl and 5?mM NaCl salt concentration. In compatibility testing, activities of all enzymes were spectacularly reduced when they utilized with chemicals of pulp bleaching. Results of rice straw pulp bleaching was effectual when pulp was initially bleached with mannanase, pectinase, and xylanase enzymes (Es) for 90?min and then with diluted chemicals (DC) for further 90?min instead of their separate use. Treatment of rice straw pulp with Es?+?DC, enhanced the release of reducing sugars, hydrophobic compounds, and phenolic compounds, whereas Kappa number was reduced. Overall, the results of the present study indicated that pre-bleaching of pulp with hydrolytic enzymes obtained from I. variabilis strain UD-6 helps to minimize chemicals used in the bleaching process and make it more sustainable for pulp and paper industries as well as for the environment.     

Synergistic effect of Aspergillus niger and Trichoderma reesei enzyme sets on the saccharification of wheat straw and sugarcane bagasse

Plant‐degrading enzymes can be produced by fungi on abundantly available low‐cost plant biomass. However, enzymes sets after growth on complex substrates need to be better understood, especially with emphasis on differences between fungal species and the influence of inhibitory compounds in plant substrates, such as monosaccharides. In this study, Aspergillus niger and Trichoderma reesei were evaluated for the production of enzyme sets after growth on two “second generation” substrates: wheat straw (WS) and sugarcane bagasse (SCB). A. niger and T. reesei produced different sets of (hemi‐)cellulolytic enzymes after growth on WS and SCB. This was reflected in an overall strong synergistic effect in releasing sugars during saccharification using A. niger and T. reesei enzyme sets. T. reesei produced less hydrolytic enzymes after growth on non‐washed SCB. The sensitivity to non‐washed plant substrates was not reduced by using CreA/Cre1 mutants of T. reesei and A. niger with a defective carbon catabolite repression. The importance of removing monosaccharides for producing enzymes was further underlined by the decrease in hydrolytic activities with increased glucose concentrations in WS media. This study showed the importance of removing monosaccharides from the enzyme production media and combining T. reesei and A. niger enzyme sets to improve plant biomass saccharification.     

Biological control of southern corn leaf blight by Trichoderma atroviride SG3403

Trichoderma atroviride SG3403 showed high biocontrol activity against southern corn leaf blight (SCLB; pathogen: Cochliobolus heterostrophus). T. atroviride SG3403 could cause death of C. heterostrophus race O hypha on plates. Spraying T. atroviride SG3403 conidia suspension over maize seedling leaves protected the corn from SCLB infection. Biocontrol effect lasted for 30 days in the field. Trichoderma strain was able to induce resistance response in corn leaves against pathogen infection. In corn leaves treated with T. atroviride SG3403, the enzyme activities of phenylalanine ammonia lyase (PAL) and superoxide dismutase (SOD) reached the highest at 24 h, enzyme activity of catalase (CAT) reached the highest at 36 h after inoculation of pathogen C. heterostrophus race O. RNA expression levels of Pal, Sod and Cat (which synthesis enzyme PAL, SOD and CAT) were also upregulated and corresponded to the enzyme activity at the same time point. Enzyme activities and corresponding genes expression induced by Trichoderma SG3403 was more obvious than that induced by pathogen only, which implies that T. atroviride SG3403 induced corn defense gene expression against pathogen infection. Thus, induced resistance mechanism was possibly involved in the biocontrol of SCLB by T. atroviride SG3403.     

Characterization and biosynthesis of non-degradable polymers in plant cuticles

The structure and monomeric composition of the highly aliphatic and non-saponifiable fraction of cutans isolated from the leaf cuticles of Agave americana L. and Clivia miniata Reg. have been elucidated. Spectroscopic Fourier transform infrared and ¹³C-nuclear magnetic resonance, calorimetric and X-ray diffraction studies, together with biopolymer analysis after exhaustive ozonolysis, showed that the cutan fraction consists of an amorphous three-dimensional network linked by ether bonds containing double bonds and free carboxylic acid functions. Data obtained from fatty acid sorption indicated that the new biopolymer investigated here has a highly hydrophobic character constituting an additional barrier biopolymer in those cuticles where it is present. Labelled [¹⁴C]linoleic acid was preferentially incorporated into the non-ester part of C. miniata leaf disks in comparison with the cutin fraction of the cuticular membrane. This indicates that the cis-pentadiene system of polyunsaturated fatty acids is involved in the formation of intramolecular linkages, mainly ether bonds, of the aliphatic biopolymer. Received: 29 June 1998 / Accepted: 17 September 1998     

Metabolism of organophosphorus insecticides

Summary The metabolism of ³²P-Malathion in Rhizobium leguminosarum and Rhizobium trifolii has been investigated. In addition to inorganic phosphates and/or thiophosphates, 5 hydrolytic metabolites could be identified. The carboxylic acid derivatives constituted the major portion (35–40% of the total metabolites output) suggesting the presence of powerful carboxyesterases in both Rhizobium spp. Malaoxon could not be detected in the media of both organisms.     

Increased accumulation of phenolic metabolites in groundnut (Arachis hypogaea L.) genotypes contribute to defense against Sclerotium rolfsii infection

Abstract

Fungal infections cause several metabolic changes to the plants, which can affect its physiology and survival in various ways. In the present study, we have analysed various phenolic compounds and activity of oxidative enzymes in healthy and Sclerotium rolfsii-infected groundnut genotypes. Increased phenolics content and higher activity of oxidative enzymes was observed in the tolerant genotype (CS 19, GG 16) followed by susceptible genotype (GG 20, TG 37A). Among the phenolic compounds tested, chlorogenic acid content has increased greatly in leaf, stem and root of infected tolerant genotypes compared to the respective controls. In vitro growth of S. rolfsii showed significant inhibition at concentrations 500 and 1000 µg/mL of phenolic compounds in the radial growth inhibition assay. These results have strongly suggested that, higher accumulation of chlorogenic acid could be an important factor in imparting resistance and protecting groundnut against S. rolfsii infection in tolerant genotypes.     

Effect of Carbon Source on Production of Lytic Enzymes by the Sclerotial Parasites Trichoderma atroviride and Coniothyrium minitans

Three isolates of Trichoderma atroviride and two isolates of Coniothyrium minitans known to efficiently degrade sclerotia of Sclerotinia sclerotiorum were cultured on minimal medium with sucrose, carboxymethyl cellulose (CMC), xylan, laminarin, colloidal chitin or powdered sclerotia as carbon source. The activity of endochitinase, endo‐β‐1,3‐glucanase, endoxylanase and endocellulase in culture filtrates was determined after 7 and 15 days of culture using dye‐labelled substrates. The strongest inducers of chitinase were colloidal chitin and sclerotia powder. Chitinase activity appeared to be faster induced in the isolates of T. atroviride than in the isolates of C. minitans, but the maximum level of activity present in culture filtrates of the two species was similar. With CMC and xylan as carbon source, concurrent production of the corresponding enzymes was observed for the Trichoderma isolates. The isolates of C. minitans produced cellulase on xylan but not on CMC, whereas xylanase was produced on both carbon sources. Laminarin induced the formation of glucanases in the three isolates of T. atroviride but not the isolates of C. minitans. However, in the sclerotia‐containing cultures of C. minitans glucanase activity was even higher than in the respective cultures of the Trichoderma isolates. During the 31‐day study period, the pattern of enzyme production in shake cultures containing sclerotia powder was very similar for the isolates of T. atroviride and C. minitans. Glucanase activity generally reached a peak 24 days after inoculation of the flasks, whereas the activity of chitinase, cellulase and xylanase remained fairly constant throughout the experiment.     

Generation and identification of DNA sequence flanking T-DNA integration site of Trichoderma atroviride mutants with high dichlorvos-degrading capacity

A protocol for efficient Agrobacterium tumefaciens-mediated transformation (ATMT) of biocontrol fungus Trichoderma atroviride strain T23 was developed to construct mutants with improved dichlorvos-degradation ability. A transformation frequency of 5 × 10⁻⁶ was achieved. Among 110 genetically stable T-DNA transformants of T. atroviride T23, two transformants, AMT-12 and AMT-28, confirmed by Southern blot analysis to have single-copy inserts of T-DNA, showed an increase in dichlorvos-degradation ability of more than 10% compared to that of the wild type, exhibited similar tolerance to the pesticide, but lower spore formation ability. Five transformants exhibited a reduction in degradation of more than 70%, exhibited wild-type spore formation, and tolerated up to 800 μg/mL of dichlorvos. The left-flanking sequence of the insertion site in AMT-12 was cloned as a 1845-bp fragment and shown to have 89% identity to the DNA from T. atroviride IMI 206040; however, the involvement of this DNA in dichlorvos degradation remains still to be determined. This study can promote both a more efficient isolation of DNA sequence flanking T-DNA integration site in T. atroviride mutants and a more rational utilization of these transformants in dichlorvos degradation.     

Productivity of hydrolytic enzymes by mycorrhizal mushrooms

To survey the potential for production of extracellular hydrolytic enzymes by mycorrhizal mushrooms, productivities of these exo-enzymes from mycelia on potato-dextrose liquid medium were determined.Tricholoma matsutake produced relatively high levels of CM-cellulase and avicelase activities in all test strains. It also produced higher activity of acid proteinase than neutral proteinase. Its xylanase activities seemed to be higher than those of the other carbohydrases. The productivities ofLyophyllum shimeji strains were at similar levels to those ofT. matsutake strains. CM-cellulase and avicelase activities ofL. shimeji were higher than those ofT. matsutake. Neutral proteinase inL. shimeji strains showed higher activity levels than acid proteinase. The relative productivities of hydrolytic enzymes between the groups of mycorrhizal mushrooms and wood-rotting mushrooms were also examined.T. matsutake andL. shimeji both produce many kinds of hydrolytic enzymes in their culture broth, and the potential for production of hydrolytic enzymes by mycorrhizal mushrooms was judged to be relatively high.     

Oxidative Stress and Hepatocellular Injury Induced by Oral Administration of Cr3+ in Chicken

This study aims to investigate the oxidative stress and hepatocellular injury induced by Cr³⁺ in chicken. Different doses of CrCl₃ solutions (50% LD₅₀, 25% LD₅₀, and 12.5% LD₅₀) and equivalent water were orally administered to chicken. Chicken liver samples were measured for the activities of antioxidant enzymes, the contents of glutathione, total antioxidant capacity (T‐AOC), malondialdehyde (MDA), and hydrogen peroxide to indirectly evaluate the oxidative stress in chicken liver. Results indicated that the oral administration of Cr³⁺ at high dose significantly increased (P < 0.05) the MDA levels after 28 days of exposure, with decreased T‐AOC, glutathione, and antioxidant enzymes activities. Low and medium doses groups show that T‐AOC, glutathione, and antioxidant enzymes activities increased after 14 days, then decreased gradually, but low and medium groups higher than control group, only high group lower than control group finally. These statistics and histopathological analysis suggest that high dose and long‐term exposure of Cr³⁺ induce oxidative stress and hepatocellular injury.     

White‐rot fungi combined with lignite granules and lignitic xylite to decolorize textile industry wastewater

The feasibility of using immobilized fungi to decolorize textile industry wastewater containing dyes was examined in experiments with: two species of white‐rot fungi (a Marasmius species from Indonesia, which produces copious biomass, and Trametes hirsuta, which produces high levels of laccase); two types of lignite products as adsorbents and solid substrates (lignitic xylite and lignite granules); and four simulated wastewaters, each containing a different kinds of reactive textile azo dye. The growth, extracellular enzyme production, dye degradation and dye absorption parameters afforded by each permutation of fungus, substrate and dye were then measured. Both fungal species grew poorly on xylite, but much better on lignite granules. Marasmius sp. produced up to 67 U/L laccase on lignite granules, but just 10 U/L on xylite, and no other detectable extracellular enzymes. T. hirsuta produced 1343 U/L laccase and up to 12 U/L unspecific peroxidase when immobilized on lignite granules, and 898 U/L laccase with 14 U/L unspecific peroxidase when immobilized on xylite. The amount of color lost from the dye solutions depended on both the type of dye and the enzyme levels in the fermenter.     

Carbohydrate-based heteronuclear complexes as topoisomerase Iα inhibitor: approach toward anticancer chemotherapeutics

Due to the critical role of cellular enzymes necessary for cell proliferation by deciphering topological hurdles in the process of DNA replication, topoisomerases have been one of the major targets in the anticancer drug development area. A need, therefore, arises for new metallodrugs that specifically recognizes DNA and inhibits the activity of topoisomerase enzymes, herein, we report the synthesis and characterization of new metal-based glycoconjugate entities containing heterobimetallic core Cu^II–Sn^IV (1) and Ni^II–Sn^IV (2) derived from N-glycoside ligand (L). The optimized structure of complex 1 and other significant vibrational modes have been explained using dispersion corrected B3LYP/DFT calculations. In vitro DNA binding profile of the L and both the complexes 1 and 2 were done by various biophysical studies. Complex 1 breaks pBR322 DNA via a hydrolytic means which was validated by T4 DNA enzymatic assay. To get a mechanistic insight of mode of action topoisomerase I (Topo I) inhibition assay was carried out. Also, we have taken the help of molecular modeling studies in accordance with experimental findings. In vitro cytotoxicity of the complex 1 was evaluated against a panel of cancer cells which exhibited remarkably good anticancer activity (GI₅₀ values <10 μg/ml). Moreover, intracellular localization of the complex 1 was visualized by confocal microscopy against HeLa cells.     

Enzymatic hydrolysis and simultaneous saccharification and fermentation of steam-pretreated spruce using crude Trichoderma reesei and Trichoderma atroviride enzymes

The aim of this study was to compare the performance of the enzymes produced by Trichoderma reesei Rut C30 and the good extracellular β-glucosidase-producing mutant Trichoderma atroviride TUB F-1663 to that of commercial preparations in the enzymatic hydrolysis and the simultaneous saccharification and fermentation (SSF) of steam-pretreated spruce (SPS).The concentrated TUB F-1663 enzyme was found to be the most efficient in the hydrolysis of washed SPS at 50 g/L water-insoluble solids (WIS) in terms of the glucose produced (18.5 g/L), even in comparison with commercial cellulases (14.1–16.7 g/L). The enzyme preparations were studied at low enzyme loadings (5 FPU/g WIS) in SSF to produce ethanol from SPS. The enzyme supernatant and whole fermentation broth of T. atroviride as well as the whole broth of T. reesei proved to be as efficient in SSF as the commercial cellulase mixtures (ethanol yields of 61–76% of the theoretical were achieved), while low ethanol yields (<40%) were obtained with the β-glucosidase-deficient T. reesei supernatant.Therefore, it seems, that instead of using commercial cellulases, the TUB F-1663 enzymes and the whole broth of Rut C30 may be produced on-site, using a process stream as carbon source, and employed directly in the biomass-to-bioethanol process.