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.     

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.     

Ligninolytic Enzymes of the White Rot Basidiomycete Trametes trogii

The white rot fungus Trametes trogii strain BAFC 463 produced laccase, manganese peroxidase, lignin peroxidase and cellobiose dehydrogenase, as well as two hydrogen peroxide‐producing activities: glucose oxidizing activity and glyoxal oxidase. In high‐N (40 mM N) cultures, the titres of laccase, MnP and GLOX were 27 (6.55 U/ml), 45 (403.00 mU/ml)and 8 (32,14 mU/ml) fold higher, respectively, than those measured in an N‐limited medium. This is consistent with the fact that the ligninolytic system of T. trogii is expressed constitutively. Lower activities of all the enzymes tested were recorded upon decreasing the initial pH of the medium from 6.5 to 4.5. Adding veratryl alcohol improved GLOX production, while laccase activity was stimulated by tryptophan. Supplying Tween 80 strongly reduced the activity of both MnP and GLOX, but increased laccase production. The titre of MnP was affected by the concentration of Mn in the culture medium, the highest levels were obtained with 90 μM Mn (II). LiP activity, as CDH activity, were detected only in the mediumsupplemented with sawdust. In this medium, laccase production reached a maximum of 4.75 U/ml, MnP 747.60 mU/ml and GLOX 117.11 mU/ml. LiP, MnP and GLOX activities were co‐induced, attaining their highest levels at the beginning of secondary metabolism, but while MnP, laccase, GLOX and CDH activities were also present in the primary growth phase, LiP activity appears to beidiophasic. The simultaneous presence of high ligninolytic and hydrogen peroxide producing activities in this fungus makes it an attractive microorganism for future biotechnological applications.     

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.     

Parameter optimization for production of ligninolytic enzymes using agro-industrial wastes by response surface method

Lignin and manganese peroxidase (LiP, MnP) and laccase production by Phanerocheate chrysosporium was optimized by response surface methodology for brewery waste and apple pomace. The effect of moisture, copper sulphate, and veratryl alcohol (VA) concentrations on enzyme production was studied. Moisture and VA had significant positive effect on MnP and LiP production and the viability of P. chrysosporium (p < 0.05) and copper sulphate produced a negative effect. However, moisture and copper sulphate had a significant positive (p < 0.05) effect on laccase production, but VA had an insignificant positive effect (p < 0.05). Higher values of MnP, LiP and viability of P. chrysosporium on apple pomace (1287.5 U MnP/gds (units/gram dry substrate), 305 U LiP/gds, and 10.38 Log 10 viability) and brewery waste (792 U MnP/gds and 9.83 Log 10 viability) were obtained with 80% moisture, 3 mmol/kg VA, and 0.5 mmol/kg copper. LiP production in brewery waste (7.87 U/gds) was maximal at 70% moisture, 2 mmol/kg VA, and 1 mmol/kg copper. Higher production of laccase in apple pomace (789 U/gds) and brewery waste (841 U/gds) were obtained with 80% moisture, 3 mmol/kg VA, and 1.5 mmol/kg copper. Thus, moisture along with VA and copper sulphate was pertinent for the production of ligninolytic enzymes and increased cell viability.     

Effect of the different parts of the corn cob employed as a carrier on ligninolytic activity in solid state cultures by P. chrysosporium

Outside and inside corn cob were used to study ligninolytic enzymes produced by Phanerochaete chrysosporium BKM-F-1767 (ATCC 24725) during solid state fermentation conditions. In a previous work by employing a mixture of outside and inside corn cob, we achieved a maximum MnP activity of 96?U l^?1 but LiP activities were low. In the present work we determined which part of the corn cob is more suitable in order to obtain high ligninolytic activities. We could find MnP activities about 300?U l^?1 by employing inside corn cob as a carrier and a maximum LiP activity of 24?U l^?1. ?In a subsequent experiment, using inside corn cob as a carrier, we could considerably improve ligninolytic enzymes production, by supplementing the medium with Tween 80 (0.5% v/v). We obtained a maximum MnP activity of 384?U l^?1and a maximum LiP activity of 155?U?l^?1.     

Lignin and manganese peroxidase activity in extracts from straw solid substrate fermentations

Manganese and lignin peroxidase (MnP, LiP) activities were measured in straw extracts from cultures of Phanerochaete chrysosporium. Out of six MnP substrates, the MBTH/DMAB (3-methyl-2-benzothiazolinone hydrazone/3-(dimethylamino)benzoic acid), gave the highest MnP activity. Detection of LiP activity as veratryl alcohol oxidation was inhibited by phenols in the straw culture extracts. Appropriate levels of veratryl alcohol and peroxide (4 mM and 0.4 mM, respectively), and a restricted sample volume (not larger than 10%) were necessary to detect activity.     

Manganese peroxidase production by Bjerkandera sp. BOS55

The white-rot fungus Bjerkandera sp. BOS55 has been suggested as a good alternative for the production of ligninolytic enzymes, specially Manganese peroxidase (MnP), by its potential ability to degrade complex compounds. However, the application of this fungus requires the complete knowledge of the fermentation pattern in submerged cultures, conditions similar to those existing in industrial size reactors. For this purpose, the nutritional and environmental factors enabling high ligninolytic activity were studied. According to the results, under limitation and sufficiency of nitrogen, there is a threshold concentration for nitrogen from which MnP is produced. However, under nitrogen excess, the ligninolytic stage of the fungus was coincident with growth, with no apparent substrate limitation according to existing levels of carbon and nitrogen. Concerning carbon concentration, MnP synthesis took place independently of glucose concentration, this indicating that carbon limitation does not seem to be the triggering factor for MnP secretion. Other two environmental factors were studied: oxygenation and agitation, but no significant effect on MnP production was observed, a quite different aspect from the behaviour of other known fungi like Phanerochaete chrysosporium.     

Effects of temperature on the production of manganese peroxidase and lignin peroxidase byPhanerochaete chrysosporium

Growth temperature played an important role in the appearance, maximum level and ratio of manganese peroxidase (MnP) and lignin peroxidase (LIP) activities in the cultures ofPhanerochaete chrysosporium. While at higher temperatures (39, 33, and 28°C) both enzymes were produced (with LIP being the major one) at 23°C MnP was dominant. At 18°C, of the two ligninolytic peroxidases only MnP activity was detected. Decrease of proteolytic activity at lower temperatures probably contributed to the retention of MnP and LIP activities.     

Potential of ligninolytic enzymatic complex produced by white‐rot fungi from genus Trametes isolated from Bulgarian forest soil

Because of the crucial role of ligninolytic enzymes in a variety of industrial processes, the demand for a new effective producer has been constantly increasing. Furthermore, information on enzyme synthesis by autochthonous fungal strains is very seldom found. Two fungal strains producing ligninolytic enzymes were isolated from Bulgarian forest soil. They were identified as being Trametes trogii and T. hirsuta. These two strains were assessed for their enzyme activities, laccase (Lac), lignin peroxidase (LiP) and Mn‐dependent peroxidase (MnP) in culture filtrate depending on the temperature and the type of nutrient medium. T. trogii was selected as the better producer of ligninolytic enzymes. The production process was further improved by optimizing a number of parameters such as incubation time, type of cultivation, volume ratio of medium/air, inoculum size and the addition of inducers. The maximum activities of enzymes synthesized by T. trogii was detected as 11100 U/L for Lac, 2.5 U/L for LiP and 4.5 U/L for MnP after 14 days of incubation at 25°C under static conditions, volume ratio of medium/air 1:6, and 3 plugs as inoculum. Among the supplements tested, 5% glycerol increased Lac activity to a significant extent. The addition of 1% veratryl alcohol had a positive effect on MnP.     

Effect of carbon and nitrogen limitation on lignin peroxidase and manganese peroxidaseproduction by Phanerochaete flavido-alba

The ligninolytic enzymes lignin peroxidase (LiP) and manganese dependent peroxidase(MnP), were detected in extracellular fluids of Phanerochaete flavido-alba FPL 106507cultures under carbon or nitrogen limitation. MnP activities were found to be higher than LiPactivities under all growth conditions tested. Higher titres of both peroxidases were obtainedunder carbon limitation in excess nitrogen. Isoelectric points (pIs) observed after FPLC and IEFof concentrated extracellular fluids revealed more acidic pIs for LiP enzymes obtained innitrogen-limited cultures than those in carbon-limited cultures. However, the change in thelimiting growth factor does not significantly affect MnP pIs.     

Effect of culture conditions on the production of ligninolytic enzymes by white rot fungi Phanerochaete chrysosporium (ATCC 20696) and separation of its lignin peroxidase

The present work was carried out to determine the optimum culture conditions of Phanerochaete chrysosporium (ATCC 20696) for maximizing ligninolytic enzyme production. Additionally, separation of its lignin peroxidase was conducted. After experiments, an optimized culture medium/condition was constructed (per liter of Kirk’s medium): dextrose 10 g, ammonium tartrate 0.11 g, Tween-80 0.5 g, MnSO₄ 7 mg, and veratryl alcohol 0.3 g in 10 mM acetic acid buffer pH 4.5. Under the optimized experimental condition, both lignin peroxidase (LiP) and manganese peroxidase (MnP) were detected and reach the highest yield at 30°C on the 8th day culture. Salt precipitation methods was used in the extraction and purification processes. Results show that salt precipitation with 60% (NH₄)₂SO₄ yielded the best result, especially toward LiP. Enzyme separation was conducted and two fractions with LiP activity. LiP1 and LiP2 were produced using three columns sequentially: desalting column, Q FF ion exchange column and Sepharyl S-300 HR gel filtration. LiP1 and LiP2 had been purified by 9.6- and 7.6-fold with a yield of 22.9% and 18.6%, respectively. According to the data of sodium dodecyl sulfate polyacrilamide gel electrophoresis (SDS-PAGE), the molecular weights of the enzymes are 38 kDa and 40 kDa, respectively.     

Effect of copper ions on the ligninolytic enzyme complex and the antioxidant enzyme activity in the white-rot fungus Trametes trogii 46

White-rot fungi of the Phylum Basidiomycota are quite promising in ligninolytic enzyme production and the optimization of their synthesis is of particular significance. The aim of this study was to investigate the effect of enhanced concentration of copper (Cu) ions (25–1000 μg/ml) on the activity of the ligninolytic enzyme complex (laccase, Lac; lignin peroxidase, LiP; Mn-peroxidase, MnP) in Trametes trogii 4₆, as well as the changes in the antioxidant cell response. All concentrations tested reduced significantly in growth and glucose consumption. Cu ions affected the ligninolytic enzyme activity in a dose dependent manner. Concentrations in the range of 25–100 μg/ml strongly stimulated Lac production (a 5–6-fold increase compared to the control). LiP activity was also induced by Cu, with the peak value being recorded following exposure to 50 μg/ml metal ions. In contrast, the addition of Cu ions had a positive effect on MnP activity at a concentration higher than 100 μg/ml. The maximum enzyme level was achieved at 1000 μg/ml. The results obtained on superoxide dismutase and catalase activities indicated that exposure of T. trogii 4₆ mycelia to Cu ions promoted oxidative stress. Both enzyme activities were co-ordinately produced with Lac and LiP but not co-ordinately with MnP.     

>Decolourisation and depolymerisation of solubilised low-rank coal by the white-rot basidiomycete Phanerochaete chrysosporium

Peroxidases secreted by the white-rot basidiomycete Phanerochaete chrysosporium can oxidise a wide range of recalcitrant compounds including lignin and aromatic xenobiotics. Since low-rank coals such as brown coal and lignite retain structural features of the parent lignin, we investigated the possibility that P. chrysosporium is capable of acting on a brown coal, with the production of useful low-molecular-mass compounds. In nitrogen-limiting liquid medium containing 0.03% solubilised Morwell brown coal, P. chrysosporium was found to convert about 85% of the coal after 16 days incubation to a form not recoverable by alkali-washing and acid-precipitation. The modal molecular mass of the residual coal macromolecules was reduced from the initial 65kDa to 32 kDa. Extensive bleaching of the coal coincided with the presence of extracellular lignin peroxidase (LiP) and manganese-dependent peroxidase (MnP), although both LiP and MnP activity were lower in cultures containing coal. These reductions are accounted for by interference with the enzyme assays by solubilised coal and by binding of MnP to precipitated coal. LiP was about eight times more sensitive than MnP to inhibition by solubilised coal. In nitrogen-sufficient medium containing solubilised coal, neither coal modification nor LiP activity were observed, suggesting that LiP is an essential component of the bleaching process.     

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%.     

Manganese peroxidase production in submerged cultures by free and immobilized mycelia of Nematoloma frowardii

The agaric basidiomycete Nematoloma frowardii has been suggested as a good alternative for production of the extracellular ligninolytic enzyme, manganese-dependent peroxidase (MnP). Some cultural and environmental factors influencing the enzymatic activity in shaken flasks and aerated fermenter cultures were evaluated to improve the yields of the process. A low nitrogen medium (1.36 mM N added as ammonium tartrate), containing 16 g/l glucose (C/N ratio=65.3), 2mM Mn2+ and inoculated with immobilized polyurethane foam mycelium, made it possible to obtain a MnP yield of 2304 nkat/l in 8 days. Under these operational conditions, the enzyme productivity in the immobilized cells of N. frowardii was 1.4 times higher than that obtained with the free fungus. In the procedure with the reusable immobilized mycelium (semi-continuous culture) as many as three subsequent 10 day batches could be fermented by using the same carrier with no loss of MnP activity.     

Production of laccase, manganese peroxidase and lignin peroxidase by Brazilian marine-derived fungi

Marine-derived fungi are a potential for the search of new compounds with relevant features. Among these, the ligninolytic enzymes have potential applications in a large number of fields, including the environmental and industrial sectors. This is the work aimed to evaluate the enzymatic activities of three marine-derived fungi (Aspergillus sclerotiorum CBMAI 849, Cladosporium cladosporioides CBMAI 857 and Mucor racemosus CBMAI 847) under different carbon sources and salinity conditions by using statistical experimental design. MnP, LiP and laccase were detected when these fungi were cultured in malt extract, however when grown on basal medium containing glucose and wheat bran LiP was not detected and yet an increase in MnP and laccase was observed. Statistical analysis through surface responses was performed and results showed high values of MnP and laccase activities under 12.5% and 23% (w/v) salinity, highlighting the potential use of these fungi for industrial applications and in bioremediation of contaminated sites having high salt concentrations. The highest values for LiP (75376.34 UI L⁻¹), MnP (4484.30 IU L⁻¹) and laccase (898.15 UI L⁻¹) were obtained with the fungus M. racemosus CBMAI 847 and it is the first report concerning ligninolytic enzymes production by a zygomycete from this genus.     

Extracellular ligninolytic enzyme production by Phanerochaete chrysosporium in a new solid-state bioreactor

The production of manganese-dependent peroxidase (MnP) and lignin peroxidase (LiP) by the fungus Phanerochaete chrysosporium (ATCC 24725) in a new bioreactor, the Immersion Bioreactor, which grows cells under solid-state conditions, was studied. Maximum MnP and LiP activities were 987 U l^–1 and 356 U l^–1, respectively. The polymeric dye, Poly R-478, was degraded at 2.4 mg l^–1 min^–1 using the extracellular culture filtrate.     

EXTRACELLULAR LIGNINOLYTIC ENZYMES PRODUCTION BY Pleurotus eryngii ON AGROINDUSTRIAL WASTES

Pleurotus eryngii (DC.) Gillet (MCC58) was investigated for its ligninolytic ability to produce laccase (Lac), manganese peroxidase (MnP), aryl alcohol oxidase (AAO), and lignin peroxidase (LiP) enzymes through solid-state fermentation using apricot and pomegranate agroindustrial wastes. The reducing sugar, protein, lignin, and cellulose levels in these were studied. Also, the production of these ligninolytic enzymes was researched over the growth of the microorganism throughout 20 days, and the reducing sugar, protein, and nitrogen levels were recorded during the stationary cultivation at 28 ± 0.5°C. The highest Lac activity was obtained as 1618.5 ± 25 U/L on day 12 of cultivation using apricot. The highest MnP activity was attained as 570.82 ± 15 U/L on day 17 in pomegranate culture and about the same as apricot culture. There were low LiP activities in both cultures. The maximum LiP value detected was 16.13 ± 0.8 U/L in apricot cultures. In addition, AAO activities in both cultures showed similar trends up to day 17 of cultivation, with the highest AAO activity determined as 105.99 ± 6.3 U/L on day 10 in apricot cultures. Decolorization of the azo dye methyl orange was also achieved with produced ligninolytic enzymes by P. eryngii using apricot and pomegranate wastes.     

Degradation of the herbicide bentazon as related to enzyme production by Phanerochaete chrysosporium in two solid substrate fermentation systems

Enzyme production and degradation of the herbicide bentazon by Phanerochaete chrysosporium growing on straw (solid substrate fermentation, SSF) and the effect of nitrogen and the hydraulic retention time (HRT) were studied using a small bioreactor and batch cultures. The best degradation of bentazon was obtained in the low nitrogen treatments, indicating participation of the ligninolytic system of the fungus. The treatments that degraded bentazon also had manganese peroxidase (MnP) activity, which seemed to be necessary for degradation. Pure MnP (with Mn(II) and H₂O₂) did not oxidize bentazon. However, in the presence of MnP, Mn(II) and Tween 80, bentazon was slowly oxidized in a H₂O₂-independent reaction. Bentazon was a substrate of pure lignin peroxidase (LiP) and was oxidized significantly faster (22,000–29,000 times) as compared to the MnP-Tween 80 system. Although LiP was a better enzyme for bentazon oxidation in vitro, its role in the SSF systems remains unclear since it was detected only in treatments with high nitrogen and high HRT where no degradation of bentazon occurred. Inhibition of LiP activity may be due to phenols and extractives present in the straw.