Enhanced ligninolytic enzyme production and degrading capability of Phanerochaete chrysosporium and Trametes versicolor

Ligninolytic enzyme production by the white-rot fungi Phanerochaete chrysosporium and Trametes versicolor precultivated with different insoluble lignocellulosic materials (grape seeds, barley bran and wood shavings) was investigated. Cultures of Phanerochaete chrysosporium precultivated with grape seeds and barley bran showed maximum lignin peroxidase (LiP) and manganese-dependent peroxidase (MnP) activities (1000 and 1232 U/l, respectively). Trametes versicolor precultivated with the same lignocellulosic residues showed the maximum laccase activity (around 250 U/l). For both fungi, the ligninolytic activities were about two-fold higher than those attained in the control cultures. In vitro decolorization of the polymeric dye Poly R-478 by the extracellular liquid obtained in the above-mentioned cultures was monitored in order to determine the respective capabilities of laccase, LiP and MnP. It is noteworthy that the degrading capability of LiP when P. chrysosporium was precultivated with barley bran gave a percentage of Poly R-478 decolorization of about 80% in 100 s, whereas control cultures showed a lower percentage, around 20%, after 2 min of the decolorization reaction.     

Improving laccase production by employing different lignocellulosic wastes in submerged cultures of Trametes versicolor

Laccase production by the white-rot fungus Trametes versicolor (CBS100.29) grown in submerged cultures was studied. Addition of different insoluble lignocellulosic materials into the culture medium in order to enhance laccase production was investigated. The lignocellulosic materials were grape seeds, grape stalks and barley bran, selected because of their availability and low cost, since they are agro-industrial wastes abundant in most countries. Barley bran gave the highest activities, a maximum value of 639U/l, which was 10 times the value attained in the cultures without lignocellulosics addition. The decolourisation of a model dye, Phenol Red, by the ligninolytic fluids obtained in the above-mentioned cultures was investigated. Grape stalk and barley bran cultures showed the highest ability to decolourise the dye, attaining a percentage of decolourisation of around 60% in 72 h.     

Biodegradation of Grape Cluster Stems and Ligninolytic Enzyme Production by Phanerochaete chrysosporium during Semi‐Solid‐State Cultivation

The degradation undergone by grape cluster stems (woody component of vine bagasse), an agroindustrial waste, was investigated during the semi‐solid‐state cultivation of Phanerochaete chrysosporium BKM‐F‐1767 (ATCC 24725). For this, the content of lignin, cellulose and hemicellulose in grape cluster stems was determined before and after the enzymatic process. It was found that about 20% of Klason lignin, 48% of hemicellulose and 5% of cellulose were degraded during the process, being the ligninolytic enzymes (manganese‐dependent peroxidase and lignin peroxidase) produced by such cultures responsible for the degradation of grape cluster stems. In parallel, semi‐solid‐state cultures of P. chrysosporium grown on an inert support (cubes of nylon sponge), which is not susceptible to undergoing degradation during the enzymatic process, were used as reference cultures. In addition, the in vivo decolourisation of a model dye, the polymeric dye Poly R‐478, by both grape cluster stem and nylon cultures was studied in order to assess their degradative ability. A percentage of biological decolourisation higher than 90% after four days of dye addition was obtained using nylon sponge cultures, whereas grape cluster stem cultures led to a decolourisation of around 70% after eight days of dye incubation. The lower percentage of dye degradation achieved by the cultures grown on grape cluster stems was due to the enzymes produced, which were not only employed in the decolourisation of the dye but also in the degradation of the support, as indicated by the data mentioned above.     

In vivo decolourization of the polymeric dye poly R‐478 by corncob cultures of Phanerochaete chrysosporium

In this paper, the in vivo decolourization of the polymeric dye Poly R‐478 by semi‐solid‐state cultures of Phanerochaete chrysosporium BKM‐F‐1767 (ATCC 24725) was investigated, employing corncob as a support. In order to stimulate the ligninolytic system of the fungus, the cultures were supplemented with veratryl alcohol (2 mM) or manganese (IV) oxide (1 g/l). Maximum manganese‐dependent peroxidase (MnP) and lignin peroxidase (LiP) activities of around 2,000 U/l and 400 U/l were attained by the former, whereas the activities reached by the latter were of about 1,500 U/l and 200 U/l, respectively. Furthermore, laccase activity (around 150 U/l) was only detected in manganese (IV) oxide supplemented cultures. The polymeric dye Poly R‐478 (0.02 w/v) was added to three‐day‐old cultures. A percentage of biological decolourization of about 85% was achieved using cultures supplemented with veratryl alcohol, whereas MnO₂ cultures showed a rather lower percentage of around 58% after nine days of dye incubation. Moreover, a correlation between MnP activity and Poly R‐478 decolourization could be observed, indicating that this enzyme is mainly responsible for dye degradation. In the present work, the in vivo decolourizing capability of the ligninolytic complex secreted by P. chrysosporium was investigated under the above‐mentioned cultivation conditions, employing a model compound, such as the polymeric dye Poly R‐478.     

Ligninolytic enzyme production and the ability of decolourisation of Poly R-478 in packed-bed bioreactors by Phanerochaete chrysosporium

The production of ligninolytic enzymes by the fungus Phanerochaetechrysosporium BKM-F-1767 (ATCC 24725) in packed-bed tubular bioreactors, operating in semi-solid-state conditions, was studied. Three types of carriers were assayed: cubes of polyurethane foam, cubes of nylon sponge and chopped corncob, in order to determine the more suitable one to produce ligninolytic enzymes by this fungus. The cultivations were carried out in discontinuous and in continuous mode. For discontinuous cultivation, maximum individual manganese-dependent peroxidase (MnP) activities of 1593, 1371 and 346 U/l were achieved in the bioreactors filled with cubes of nylon sponge, cubes of polyurethane foam and with corncob, respectively. On the other hand, lignin peroxidase (LiP) activities about 100 U/l were found in the two former and around 200 U/l in the latter. Moreover, laccase, was detected in all cultures, with average values of 30 U/l. Nonetheless, continuous mode cultivation led to lower ligninolytic enzyme activities than those produced in discontinuous, except in the case of the corncob. Furthermore, the decolourisation of the dye Poly R-478 by the above-mentioned cultures was investigated. The percentage of biological decolourisation reached was about 70% in the bioreactor filled with cubes of nylon sponge whereas it was rather low in the others (around 30%).     

Influence of Several Activators on the Extracellular Laccase Activity and In vivo Decolourization of Poly R‐478 by Semi‐Solid‐State Cultures of Trametes versicolor

The effect of several laccase activity activators,such as ethanol (novel activator), veratryl alcohol, melanin production and aeration level, on the laccase production by Trametes versicolor (CBS100.29) was investigated. The microorganism was cultivated on nylon sponge, functioning as a physical support on which the mycelium was bound. The cultures with veratryl alcohol showed maximum laccase and manganese‐dependent peroxidase (MnP) activities of 238 U/l and 125 U/l, respectively. The laccase activity found is about two times higher than that attained in the control cultures. On the contrary, MnP activity did not appear to be influenced by the addition of this alcohol. Ethanol‐supplemented cultures led to maximum laccase and MnP activity levels of about 102 U/l and 101 U/l, respectively. These activities were approx. 40% lower than those achieved in the reference cultures. The decolourization of the polymeric dye Poly R‐478 by the above‐mentioned cultures was also investigated. A percentage of biological decolourization of around 90% was achieved with control and veratryl alcohol‐supplemented cultures, whereas with ethanol‐supplemented cultures a slightly lower percentage of around 85% was reached after seven days of dye incubation.     

Effect of carbon,nitrogen sources and inducers on ligninolytic enzyme production by Morchella crassipes

The effect of different carbon, nitrogen sources and inducers on growth and ligninolytic activity by Morel mushroom Morchella crassipes was investigated. The maximum growth was observed in mineral salts broth containing glucose as the carbon source and sodium nitrate as the nitrogen source. Among the inducers, chemical inducers inhibited the growth whereas in natural substrates, growth was not affected much. Manganese peroxidase and lignin peroxidase activity were not detected in the medium with different carbon and nitrogen sources, whereas laccase activity varied depending on carbon source (0.7–3.48 U/ml). Among the inducers, natural inducers resulted in an increase in the enzyme activities. Maximum laccase activity was observed in rice straw (12. 6 U/ml) followed by ABTS (11.6 U/ml); Manganese peroxidase activity was maximum in rice straw (14.32 U/l) wheat straw (12.16 U/l) and phenol red (15 U/l) as the inducers, whereas for Lignin peroxidase activity, rice straw (22 U/l), wheat straw (16 U/l) and veratrylalcohol (20 U/l) served as the best inducers.     

Ligninolytic enzymes from corncob cultures of Phanerochaete chrysosporium under semi-solid-state conditions

The effects of adding some inducers of lignolytic activity to semi-solid-state cultures of Phanerochaete chrysosporium BKM-F-1767 (ATCC 24725) were investigated. The inducers assayed were veratryl alcohol and solid manganese (IV) oxide. The microorganism was cultured on corncob, which functioned both as physical support and source of nutrients. Supplementing the cultures with veratryl alcohol created the situation where manganese-dependent peroxidase (MnP) and lignin peroxidase (LiP) activities of approximately 1,500 U/l and 200 U/l, respectively, could be attained. These activities were considerably higher than those obtained in the reference cultures (about 5 and 4-fold). In the same way, the addition of manganese (IV) oxide led to MnP and LiP activity levels of about 2,000 U/l and 300 U/l, respectively. These activities were also notably above (about 6 and 5-fold, respectively) those achieved in the reference cultures. Moreover, laccase activity (around 200 U/l) was only detected in veratryl alcohol or manganese (IV) oxide supplemented cultures.     

Enzymes from spent mushroom substrate of <Emphasis Type="Italic">Pleurotus sajor</Emphasis>-<Emphasis Type="Italic">caju</Emphasis> for the decolourisation and detoxification of textile dyes

The potential of ligninolytic enzymes, including lignin peroxidase (LiP) as the main enzyme from the spent mushroom substrate of Pleurotus sajor-caju was evaluated for the decolourisation of five dyes from azo and anthraquinone dye groups. Among the azo dyes, reactive black 5 and reactive orange 16 were 84.0 and 80.9% decolourised respectively, after 4 h of incubation with 45 U of LiP as compared to 32.1% decolourisation of disperse blue 79. Among the anthraquinone dyes, disperse red 60 was decolourised to 47.2% after 4 h of incubation with 45 U of LiP as compared to 5.9% decolourisation of disperse blue 56. Increasing the LiP concentration and incubation time had a positive effect on the decolourisation of anthraquinone dyes as compared to azo dyes. A 67.9% decolourisation of synthetic textile waste-water was achieved after 4 h of incubation with 25 U of LiP. Increasing the incubation time significantly increased (P < 0.05) the decolourisation of synthetic textile waste-water. Further, there was a 52.4% reduction in the toxicity of synthetic textile waste-water treated with 55 U of LiP for 4 h. However, only 35.7% reduction in toxicity was achieved when the synthetic textile waste-water was treated with 55 U of LiP for 24 h. In this study, it was shown that the spent mushroom substrate of P. sajor-caju could be a cheap source of ligninolytic enzymes for the decolourisation of dyes in textile industry wastewaters.     

Amelioration of ligninolytic enzyme production by Phanerochaete chrysosporium in airlift bioreactors

Maximum activities of manganese-dependent peroxidase (MnP) and lignin peroxidase (LiP) in free cultures of Phanerochaete chrysosporium (ATCC 24725) were 258 U l^–1 and 103 U l^–1, respectively, in an airlift bioreactor. Immobilisation of the fungus on an inert carrier as well as several design modifications of the bioreactor employed gave MnP activities around 500–600 U l^–1 during 9 days' operation. The continuous operation of the latter led to MnP and LiP activities about 140 U l^–1 and 100 U l^–1, respectively, for two months, without operational problems. Furthermore, the extracellular liquid secreted decolourised the polymeric dye Poly R-478 about 56%.     

Degradation of dye-containing textile effluent by the agaric white-rot fungus Clitocybula dusenii

Raw mixed-dye wastewater from a textile dye-producing plant was partly decolorized by the agaric white-rot fungus, Clitocybula dusenii. The fungus had higher Mn peroxidase (MnP) and laccase activities when grown with dye effluent than in control cultures. The activity of MnP increased commensurately with the proportion of the raw dye wastewater in the medium (control: 20 U l^–1; 10% v/v effluent: 67 U l^–1; 25% v/v effluent: 130 U l^–1; and 33% v/v effluent: 180 U l^–1). Maximal decolorization rates were achieved over 20 d at 28 °C using four-fold diluted dye-containing effluent on a 5 d pre-grown mycelium.     

Irpex lacteus, a white rot fungus applicable to water and soil bioremediation

Growth parameters, ligninolytic enzyme activities and ability to degrade polycyclic aromatic hydrocarbons by the fungus Irpex lacteus were characterized and compared with those of other white rot fungi capable of rapid decolorization of poly R-478 and Remazol Brilliant Blue R dyes. I. lacteus was able to grow on mineral and complex media and efficiently colonized sterile and non-sterile soil by exploratory mycelium growing from a wheat straw inoculum. In shallow stationary cultures growing on high nitrogen mineral medium containing 45 mM ammonium as nitrogen source, the fungus produced lignin peroxidase (LIP), Mn-dependent peroxidase (MnP) and laccase simultaneously, the respective maximal activities of 70, 970 and 36 U/l being attained around day 18. Growing in nitrogen-limited medium (2.4 mM ammonium), no LIP was formed and levels of MnP and laccase decreased significantly. During growth in sterile soil, the fungus synthesized LIP and laccase but not MnP. I. lacteus efficiently removed three- and four-ringed PAHs from liquid media and artificially spiked soil. The variety of ligninolytic enzymes, robust growth, capability of soil colonization and resistance to inhibitory action of soil bacteria make I. lacteus a suitable fungal organism for use in bioremediation. Received: 30 March 2000 / Accepted: 19 May 2000     

Production of laccase and manganese peroxidase by Pleurotus pulmonarius in solid-state cultures and application in dye decolorization

The production of ligninolytic enzymes (laccase and Mn-dependent peroxidase) by the white-rot fungus Pleurotus pulmonarius (FR.) Quélet was studied in solid-state cultures using agricultural and food wastes as substrate. The highest activities of laccase were found in wheat bran (2,860?±?250 U/L), pineapple peel (2,450?±?230 U/L), and orange bagasse (2,100?±?270 U/L) cultures, all of them at an initial moisture level of 85 %. The highest activities of Mn peroxidase were obtained in pineapple peel cultures (2,200?±?205 U/L) at an initial moisture level of 75 %. In general, the condition of high initial moisture level (80–90 %) was the best condition for laccase activity, while the best condition for Mn peroxidase activity was cultivation at low initial moisture (50–70 %). Cultures containing high Mn peroxidase activities were more efficient in the decolorization of the industrial dyes remazol brilliant blue R (RBBR), Congo red, methylene blue, and ethyl violet than those containing high laccase activity. Also, crude enzymatic extracts with high Mn peroxidase activity were more efficient in the in vitro decolorization of methylene blue, ethyl violet, and Congo red. The dye RBBR was efficiently decolorized by both crude extracts, rich in Mn peroxidase activity or rich in laccase activity.     

A packed-bed fungal bioreactor for the continuous decolourisation of azo-dyes (Orange II)

The degradation of an azo dye, Orange II, by immobilised Phanerochaete chrysosporium in a continuous packed bed bioreactor for periods longer than 30 days has been carried out. Nearly complete decolourisation (>95%) was achieved when working at a high dye load rate of 0.2 g l⁻¹ d⁻¹, a temperature of 37 °C, a hydraulic retention time (HRT) of 24 h and applying oxygen gas in a pulsed flow. These conditions allowed Manganese peroxidase (MnP) production and the subsequently Orange II decolourisation. A correlation between residual MnP activity in the effluent and decolourisation was established. Apparently, for decolourisation to be effective, a minimum MnP activity was required, no substantial increase in efficiency at MnP activities higher than 10 U 1 ⁻¹ was observed. The treatment caused, the breakdown of the chromophoric group as well as the cleavage of the aromatic ring.     

Different proportions of laccase isoenzymes produced by submerged cultures of Trametes versicolor grown on lignocellulosic wastes

The white-rot fungus Trametes versicolor grown in submerged culture produced two laccase isoenzymes, LacI and LacII. Addition of insoluble lignocellulosic materials into the culture medium increased the total laccase activity. The proportion of laccase isoenzymes also changed depending on the lignocellulosic material employed, with ratios of activity LacII/LacI from 0.9 (barley straw) to 4.4 (grape stalks). Besides, this proportion played an important role in the dye decolourisation.     

Decolourisation of reactive textile dyes Drimarene Blue X3LR and Remazol Brilliant Blue R by <Emphasis Type="Italic">Funalia trogii</Emphasis> ATCC 200800

Decolourisation of reactive dyes Drimarene Blue X3LR and Remazol Brilliant Blue R by white rot fungi Funalia trogii was studied under static conditions. The effect of various conditions such as mycelial age, initial dye and glucose concentrations on decolourisation were also investigated. Decolourisation activity of F. trogii was compared with Phanerochaete chrysosporium known as test microorganism. It was found that 7-day-old cultures were more effective than 5-day-old cultures of F. trogii for decolourisation of these dyes. Decolourisations by F. trogii of both dyes were increased with glucose concentration decreasing. In contrast, decolourisations by P. chrysosporium were decreased. F. trogii decolourised 92–98% of both dyes within 4–10 h. However, P. chrysosporium partially decolourised (11–20%) these dyes during 10days incubation period under the same conditions.     

Cellulolytic enzymes of a thermotolerantAspergillus terreus strain and their action on cellulosic substrates

A thermotolerant fungal strainAspergillus terreus produced high activities of cellulolytic enzymes when grown in shake flasks for 8 days at 40°C or 14 days at 28°C in medium containing 2.5% (w/v) cellulose powder and 1% (w/v) wheat bran. There was little difference between the final activities of endo-(1,4)--glucanase (ca. 14.4 U/ml); filter paper activity (ca. 1.3 U/ml) and -glucosidase (ca. 10 U/ml). Endoglucanase had maximum activity at 60°C and pH 3.8; the other two enzymes were optimal at 60°C and pH 4.8. The maximum hydrolysis of different cellulosic substrates (about 50%) was obtained within 48 h when 1.1 U/ml of filter paper cellulase activity were employed to saccharify 100 mg alkali-treated cotton, filter paper, bagasse, and rice straw at 50°C and pH 4.8. The major end-product, glucose, was produced from all substrates, with traces of cellobiose and other larger oligosaccharides being present in rice straw hydrolysates.     

Decolourisation of Remazol Brilliant Blue R via a novel Bjerkandera sp. strain

A novel strain of Bjerkandera sp. (B33/3), with particularly high decolourisation activities upon Poly R-478 and Remazol Brilliant Blue R (RBBR) dyes, was isolated. The role of the ligninolytic extracellular enzymes produced by this strain on decolourisation of RBBR was studied in some depth. The basis of decolourisation is an enzyme-mediated process, in which the main enzyme responsible is a recently described peroxidase with capacity for oxidation of manganese, as well as veratryl alcohol and 2,6-dimethoxyphenol in a manganese-independent reaction.     

Carbon and nitrogen sources influence the ligninolytic enzyme activity of <Emphasis Type="Italic">Trametes versicolor</Emphasis>

Among carbon sources studied, cellobiose and mannitol provided the highest laccase (Lac) activity (648 and 742 U1^-1, respectively) of Trametes versicolor 775 while glucose gave maximum manganese peroxidase (MnP) and peroxidase activities (44 and 114U1^-1, respectively). Citrus fruit peel as growth substrate enhanced Lac activity 7-fold when compared to the medium with cellobiose, whereas grape vine sawdust increased MnP and peroxidase activity up to 148 and 677U1^-1, respectively.     

Color removal ability of <Emphasis Type="Italic">Phanerochaete chrysosporium</Emphasis> in relation to lignin peroxidase and manganese peroxidase produced in molasses wastewater

Decolorization of molasses wastewater (MWW) from an ethanolic fermentation plant by Phanerochaete chrysosporium was studied. By diluting MWW properly (10%v/v) and incubating it with an appropriate concentration of the spores (2.5 × 10⁶/ml), extensive decolorization occurred (75%) on day 5 of the incubation. The colour removal ability was found to be correlated to the activity of ligninolytic enzyme system: lignin peroxidase (LiP) activity was 185 U/l while manganese peroxidase (MnP) activity equaled 25 U/l. Effects of some selected operating variables were studied: manganese(II), veratryl alcohol (VA), glucose as a carbon source and urea and ammonium nitrate, each as a source of nitrogen. Results showed that the colour reduction and LiP activity were highest (76% and 186 U/l, respectively) either when no Mn(II) was added or added at the lowest level tested (0.16 mg/l to provide 0.3 mg/l). Activity of MnP was highest (25 U/l) when Mn(II) added to the diluted MWW at the highest level (100 ppm) while activity of LiP was lowest (7.1 U/l) at this level of added Mn(II). The colour reduction in the presence of the added VA was shown to be little less than in its absence (70 vs. 75%). When urea as an organic source of nitrogen for the fungus, was added to the MWW, the decolorizing activity of P. chrysosporium decreased significantly (15 vs. 75%) and no activities were detected for LiP and MnP. Use of ammonium nitrate as an inorganic source of nitrogen did not show such a decelerating effects, although no improvements in the metabolic behavior of the fungus (i.e., LiP and MnP activities) deaccelerating was observed. Effects of addition of glucose was also discussed.