Organic acid production by Basidiomycetes. 3. Cultural conditions for L-malic acid production

Cultural conditions were examined for the purpose of increasing yields of l-malic acid by the Basidiomycetes Schizophyllum commune and Merulius tremellosus, which have the ability to produce this acid as a main product in CaCO(3)-containing medium in shaken culture. The most favorable nitrogen sources selected were 0.3% (NH(4))(2)SO(4) and 0.18% NH(4)Cl. Effective combinations of inorganic salts in the medium were 0.1% KH(2)PO(4), 0.05% MgSO(4).7H(2)O, and 0.05% KCl, and suitable concentrations of glucose were 5 to 10%. Several nonionic surface-active agents promoted the filamentous mycelial growth of these strains and increased acid production. In particular, Tween 80 in 0.3% concentration markedly stimulated malic acid production by S. commune, and yields greater than 50% based on available glucose, were obtained after 10 to 14 days. Acid production by M. tremellosus was stimulated most with 0.5% Carbowax 4000 (polyethylene glycol), and the resultant yields were more than 40%.     

Increased production of extracellular laccase by the white rot fungus Coriolus versicolor MTCC 138

Summary The white rot fungus Coriolus versicolor MTCC 138 has been identified as an excellent producer of the industrially important enzyme laccase. The basal medium containing glucose gave laccase activity of 155 U/ml. Screening of different media components and their effects on laccase production by Coriolus versicolor was studied using one factor at a time and L₉ (3⁴) orthogonal array method. A two-fold increase (305 U/ml) in laccase production was observed using a combination of glucose and starch as carbon source and yeast extract as nitrogen source. This activity is very high as compared to most of the reported strains. Hence this strain was explored for enhancement in laccase. The formation of extracellular laccase could be considerably stimulated by the addition of copper in the optimized medium. Addition of 1 mM copper enhanced laccase activity to 460 U/ml. Laccase production was further enhanced using different aromatic inducers. Among different inducers used 2,5-xylidine was found to be the best, and gave maximum laccase activity of 820 U/ml with 1 mM concentration. Thus, this strain could be an efficient and attractive source for laccase production.     

Organic acids and penicillin production

Summary Fed-batch fermentations with Penicillium chrysogenum, strain S 3723, were fed with glucose as carbon source or with a mixture of glucose and an organic acid (citrate, lactate, propionate, succinate, formate, or caproate). Increased yields of penicillin-V were observed with, propionate, and formate, 50% higher than in fermentations without acid in the feed. The yield with succinate was improved to the level previously observed with acetate (25%).     

Lignin degradeation by white rot fungi

Abstract. The wood-degrading white-rot fungus Phanerochaete chrysosporium , has been the subject of intensive research in recent years and, based upon isolation of the extracellular enzyme ligninase, major advances have now been made toward elucidating the mechanism by which this fungus degrades lignin. From these developments, a model emerges which could explain the process by which wood-degrading fungi in general, attack lignin.     

Effect of synthetic and natural culture media on laccase production by white rot fungi

Laccase is among the major enzymes of white rot fungi involved in lignocellulose degradation. The present paper reports its production by two white rot fungi (Coriolus versicolor, Funalia trogii) under different nutritional conditions. Various synthetic culture media and natural culture medium (molasses wastewater) were tested. Enzyme production in various synthetic culture media, molasses wastewater (vinasse) culture medium and in the absence or presence of cotton stalk supplements showed that vinasse culture medium was a better laccase-inducer medium than the synthetic culture medium. Addition of cotton stalk to various media enhanced the enzyme production. The highest laccase activity was obtained in vinasse culture medium with cotton stalk.     

Effects of various media and supplements on laccase production by some white rot fungi

White rot fungi produce three main extracellular enzymes involved in ligninolysis; laccase, lignin peroxidase and manganese peroxidase. Though all white rot fungi do not produce all three enzymes, laccase occupies an important place in ligninolysis. The present paper reports its production by some white rot fungi; Daedalea flavida, Phlebia brevispora, Phlebia radiata and Polyporus sanguineus under different nutritional conditions. Of the various basal media tested, mineral salts malt extract broth proved to be the best medium for laccase production. Sugarcane bagasse proved to be the best laccase inducer among the various supplements added to different media.     

Optimization of manganese peroxidase production by the white rot fungus Bjerkandera sp. strain BOS55

Manganese dependent peroxidase (MnP) is the most ubiquitous peroxidase produced by white rot fungi. MnP is known to be involved in lignin degradation, biobleaching and in the oxidation of hazardous organopollutants. Bjerkandera sp. strain BOS55 is a nitrogen-unregulated white rot fungus which produces high amounts of MnP in the excess of N-nutrients due to increased biomass yield. Therefore, the strain is a good candidate for use in large scale production of this enzyme. The objective of this study was to optimize the MnP production in N-sufficient cultures by varying different physiological factors such as Mn concentration, culture pH, incubation temperature and the addition of organic acids. The fungus produced the highest level of MnP (up to 900 U 1⁻¹) when the Mn concentration was 0.2 to 1 mM, the pH value was 5.2, and the incubation temperature was 30°C. A noteworthy finding was that MnP was also produced at lower levels in the complete absence of Mn. The addition of organic acids like glycolate, malonate, glucuronate, gluconate, 2-hydroxybutyrate to the culture medium increased the peak titres of MnP up to 1250 U 1⁻¹. FPLC profiles indicated that the organic acids stimulated the production of all MnP isoenzymes present in the extracellular fluid of the fungus.     

Synthetic dye decolourization by white rot fungi

Synthetic dyes are integral part of many industrial products. The effluents generated from textile dyeing units create major environmental problems and issues both in public and textile units. Industrial wastewater treatment is one of the major problems in the present scenario. Though, the physical and chemical methods offer some solutions to the problems, it is not affordable by the unit operators. Biological degradation is recognized as the most effective method for degrading the dye present in the waste. Research over a period of two decades had provided insight into the various aspects of biological degradation of dyes. It is observed that the white rot fungi have a non-specific enzyme system, which oxidizes the recalcitrant dyes. Detailed and extensive studies have been made and process developed for treatment of dye containing wastewaters by white rot fungi and their enzyme systems. An attempt is made to summarize the detailed research contributions on these lines.     

Dibenzyl sulfide metabolism by white rot fungi

Microbial metabolism of organosulfur compounds is of interest in the petroleum industry for in-field viscosity reduction and desulfurization. Here, dibenzyl sulfide (DBS) metabolism in white rot fungi was studied. Trametes trogii UAMH 8156, Trametes hirsuta UAMH 8165, Phanerochaete chrysosporium ATCC 24725, Trametes versicolor IFO 30340 (formerly Coriolus sp.), and Tyromyces palustris IFO 30339 all oxidized DBS to dibenzyl sulfoxide prior to oxidation to dibenzyl sulfone. The cytochrome P-450 inhibitor 1-aminobenzotriazole eliminated dibenzyl sulfoxide oxidation. Laccase activity (0.15 U/ml) was detected in the Trametes cultures, and concentrated culture supernatant and pure laccase catalyzed DBS oxidation to dibenzyl sulfoxide more efficiently in the presence of 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonate) (ABTS) than in its absence. These data suggest that the first oxidation step is catalyzed by extracellular enzymes but that subsequent metabolism is cytochrome P-450 mediated.     

Reduction of superoxide production by mitochondria oxidizing NADH in the presence of organic acids

Effects of multiple substrates on oxygen uptake and superoxide production by mitochondria isolated from the pericarp tissue of green bell pepper (Capsicum annuum L.) were studied. Mitochondria isolated from peppers stored at 4 °C for 5 and 6 days had higher rates of oxygen uptake and were less sensitive to cyanide than mitochondria isolated from freshly harvested peppers. Succinate enhanced state 2 and state 4 rates of oxygen uptake with exogenous NADH in the absence of cytochrome path inhibitors, but not state 3 rates by mitochondria isolated from either freshly harvested or cold-stored bell peppers. The sensitivity of NADH oxidation to cyanide was reduced by both malate and succinate in mitochondria from cold-stored bell peppers, whereas only succinate was effective in mitochondria from freshly harvested peppers.Mitochondria isolated from both freshly harvested peppers and those stored at 4 °C for 5 and 6 days produced superoxide in the absence of exogenous substrates. Superoxide production by mitochondria from freshly harvested bell peppers increased when the mitochondria were supplied with malate, succinate or NADH, but only NADH enhanced superoxide production by mitochondria from cold-stored peppers. Both succinate and malate reduced the production of superoxide by mitochondria isolated from cold-stored bell peppers. Succinate and malate as second substrates also reduced the production of superoxide with NADH by mitochondria from both freshly harvested and cold-stored bell peppers. Malonate, a competitive inhibitor of succinate dehydrogenase, was inhibitory to oxygen uptake and to superoxide production.Mitochondria isolated from cold-stored bell peppers converted succinate to pyruvate at 25 °C at considerably higher rates than those of mitochondria from freshly harvested bell peppers. Since pyruvate has been shown to activate the alternative oxidase and the presence of pyruvate is essential for continued alternative oxidase activity, we suggest that pyruvate limits superoxide production by enhancing the flow of electrons through the alternative path. A direct scavenging of superoxide by succinate, malate and pyruvate, however, cannot be ruled out.     

Extracellular oxidative enzyme production and PAH removal in soil by exploratory mycelium of white rot fungi

Selected strains of three species of white rot fungi, Pleurotus ostreatus, Phanerochaete chrysosporium and Trametes versicolor, were grown in sterilized soil from straw inocula. The respective colonization rates and mycelium density values decreased in the above mentioned order. Three- and four-ringed PAHs at 50 ppm inhibited growth of fungi in soil to some extent. The activities of fungal MnP and laccase (units per g dry weight of straw or soil), extracted with 50 mM succinate-lactate buffer (pH 4.5), were 5 to 20-fold higher in straw compared to soil. The enzyme activities per g dry soil in P. ostreatus and T. versicolor were similar, in contrast to P. chrysosporium, where they were extremely low. Compared to the aerated controls, P. ostreatus strains reduced the levels of anthracene, pyrene and phenanthrene by 81–87%, 84–93% and 41–64% within 2 months, respectively. During degradation of anthracene, all P. ostreatus strains accumulated anthraquinone. PAH removal rates in P. chrysosporium and T. versicolor soil cultures were much lower.     

Effect of culture conditions on manganese peroxidase production and activity by some white rot fungi

The ligninolytic system of white rot fungi is primarily composed of lignin peroxidase, manganese peroxidase (MnP) and laccase. The present work was carried out to determine the best culture conditions for production of MnP and its activity in the relatively little-explored cultures of Dichomitus squalens, Irpex flavus and Polyporus sanguineus, as compared with conditions for Phanerochaete chrysosporium and Coriolus versicolor. Studies on enzyme production under different nutritional conditions revealed veratryl alcohol, guaiacol, Reax 80 and Polyfon H to be excellent MnP inducers. Electronic Publication     

Comparison of pyrene biodegradation by white rot fungi

Six strains of white rot fungi, isolated from soil in Korea, were evaluated as to their ability to biodegrade the 4-ring polycyclic aromatic hydrocarbon pyrene. While growing in a complex fungal medium, Irpex lacteus, Trametes versicolor KR11W, and Phanerochaete chrysosporium mineralized 15.6, 12.7 and 7.0% of the added 0.84 nmol of radioactive pyrene, respectively. In these cultures, 33–46% of the added pyrene was converted to water-soluble polar metabolites, and 22–40% was incorporated into fungal biomass. Pleurotus ostreatus mineralized only 2.5% of the added pyrene, while T. versicolor KR65W and Microporus vernicipes failed to evolve ¹⁴CO₂ from pyrene. The information obtained aids in strain selection for clean-up of polycyclic aromatic hydrocarbon contamination.     

Degradation of anthracene by selected white rot fungi

Abstract Approximately 60% of the originally supplied anthracene (AC) was degraded in ligninolytic stationary cultures of selected white rot fungi within 21 days. All the white rot fungi tested oxidized AC to anthraquinone (AQ). Unlike Phanerochaete chrysosporium and strain Px, with Pleurotus ostreatus, Coriolopsis polyzona and Trametes versicolor , AQ did not accumulate in the cultures, indicating that AQ was degraded further and its degradation did not appear to be a rate-limiting step. However, P. ostreatus and C. polyzona failed to degrade AQ in the absence of AC. P. ostreatus, T. versicolor and strain Px did not produce lignin peroxidase (ligninase) (LIP) under the test conditions but oxidized AC to AQ suggesting that white rot fungi produce enzyme(s) other than LIP capable of oxidizing compounds with high ionization potential like AC. Moreover, in the case of Ph. chrysosporium and C. polyzona , AC degradation started earlier than the production of LIP. Veratryl alcohol (VA) seemed to be playing a role in AC oxidation catalyzed by LIP in Ph. chrysosporium .     

Production of ligninolytic peroxidases by the white rot fungusCoriolopsis occidentalis

Summary Coriolopsis occidentalis degraded Kraft lignin and dioxan lignin from spruce and, under nitrogen limitation, produced extracellular ligninase, Mn²⁺-activated peroxidase and veratryl alcohol. Concentrated culture fluid cleaved the C–C bond in a -O-4 lignin model compound yielding veratraldehyde.     

Conidioma production of the white root rot fungus in axenic culture under near-ultraviolet light radiation

 Conidiomata of the white root rot fungus were produced in axenic culture under near-ultraviolet light radiation. Pieces of sterilized Japanese pear twigs were placed on 7-day-old oatmeal agar culture in plates. The plates were further incubated for 5 days and then illuminated by near-ultraviolet light. Synnemata developed on the twigs within 5 weeks in 19 of 20 isolates tested, and conidia were observed in 12 of the 19 isolates. The synnemata and conidia produced were morphologically identical to those of Dematophora necatrix. Received: October 29, 2001 / Accepted: March 11, 2002     

Biodegradation of pentachlorophenol by the white rot fungus Phanerochaete chrysosporium

Extensive biodegradation of pentachlorophenol (PCP) by the white rot fungus Phanerochaete chrysosporium was demonstrated by the disappearance and mineralization of [14C]PCP in nutrient nitrogen-limited culture. Mass balance analyses demonstrated the formation of water-soluble metabolites of [14C]PCP during degradation. Involvement of the lignin-degrading system of this fungus was suggested by the fact the time of onset, time course, and eventual decline in the rate of PCP mineralization were similar to those observed for [14C]lignin degradation. Also, a purified ligninase was shown to be able to catalyze the initial oxidation of PCP. Although biodegradation of PCP was decreased in nutrient nitrogen-sufficient (i.e., nonligninolytic) cultures of P. chrysosporium, substantial biodegradation of PCP did occur, suggesting that in addition to the lignin-degrading system, another degradation system may also be responsible for some of the PCP degradation observed. Toxicity studies showed that PCP concentrations above 4 mg/liter (15 microM) prevented growth when fungal cultures were initiated by inoculation with spores. The lethal effects of PCP could, however, be circumvented by allowing the fungus to establish a mycelial mat before adding PCP. With this procedure, the fungus was able to grow and mineralize [14C]PCP at concentrations as high as 500 mg/liter (1.9 mM).     

Effect of polycyclic aromatic hydrocarbons on laccase production by white rot fungus Pleurotus ostreatus D1

The effect of polycyclic aromatic hydrocarbons (PAHs) on the dynamics of laccase production by the fungus Pleurotus ostreatus D1 under conditions of submerged cultivation on Kirk's medium has been studied. It has been shown that phenanthrene, fluoranthene, pyrene, and chrysene actively induce this enzyme, whereas fluorene and anthrecene had a smaller effect. Addition of Mn2+ ions to cultivation medium elevates the laccase activity twofold and more in the presence of all the studied PAHs. Electrophoresis under nondenaturing conditions demonstrates induction of additional laccase species by xenobiotics. Ligninolytic peroxidase activities are undetectable under the conditions used.