Phenoloxidase-producing halotolerant fungi from olive brine wastewater

The main aim of this study was to assess the ability of producing extracellular phenoloxidases (EPO) of fungi isolated from olive brine wastewater (OBW), the effluent from the debittering process of table olives. Five out of twenty isolates displayed EPO-producing ability as assessed by selective agar plate assays and were all halotolerant. Among them, Citeromyces matritensis (syn. Candida globosa) and Aspergillus fumigatus concomitantly produced laccase and Mn-peroxidase (MnP) activities. Candida boidinii and Candida bombi, instead, only produced laccase and Candida diddensiae only released MnP. Both C. matritensis and A. fumigatus, grown in shaken cultures on OBW, maintained their EPO-producing ability and removed phenols by 52.3 and 82.3%, respectively, after 10 d incubation in the non-supplemented effluent. These results might suggest the possible use of these strains in the treatment of other saline phenol-rich effluents, such as pickling and tannery wastewater.     

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     

Simple and easy method for the determination of fungal growth and decolourative capacity in solid media

A technique was developed for studying the biodegradative ability of white rot fungi in different solid media. This technique enables the gravimetric determination of fungal growth (increase of biomass) and the spectrometric measurement of fungal decolourization ability (both by the determination of the production of the extracellular enzyme manganese-dependent peroxidase (MnP) and by the rate of decolourization of dyes). Bjerkandera sp., strain BOS55, was grown in different solid media. Its growth rate, decolourization of solophenil blue 2BL (azoic dye), neutral red (eurhodin dye), methyl green and crystal violet (triphenylmethane dyes) and the production of MnP were determined. Application of this technique enabled a spectrometric quantification of enzymatic activity. Assays indicate that greater amounts of MnP were present in agar plate cultures of Bjerkandera sp. than in liquid cultures.     

Liquid state fermentation of apple pomace sludge for the production of ligninolytic enzymes and liberation of polyphenolic compounds

Ligninolytic enzyme production and polyphenolic compound extraction by liquid-state culture of Phanerochaete chrysosporium ATCC 24275 was investigated by employing apple pomace sludge and synthetic medium. Different physico-chemical and biological parameters namely viscosity, zeta potential and particle size, viability and enzyme production were investigated. The ligninolytic enzyme production was higher in apple pomace sludge (45 U/l of laccase, 220 U/l of MnP and 6.5 U/l of LiP) than in synthetic medium (17 U/l of laccase, 37 U/l of MnP and 6 U/l). These maximal activities were found during the stationary and decline phase. It was also found that enzyme production was strongly correlated with P. chrysoporium viability in both synthetic medium and apple pomace sludge. Moreover, physico-chemical parameters, such as particle size, zeta potential and viscosity were strongly correlated to the viability of P. chrysosporium and to the ligninolytic enzyme production. An increase in polyphenol content extracted by acetone (383–720 mg GAE/l) was observed during fermentation of apple pomace and it was found that the polyphenol content extracted by ethanol increased ～1.5 fold until 67 h of fermentation and later it decreased. It was found that antioxidant activity increased to 35% and eventually decreased based on the change in the polyphenol content.     

Continuous Acid Blue 45 decolorization by using a novel open fungal reactor system with ozone as the bactericide

To maintain long-term lignin-degrading enzyme production under non-sterile conditions was a key to the technical application of white rot fungi in wastewater treatment. In this work, a novel open fungal reactor system with ozone as the bactericide, and using immobilized Phanerochaete chrysosporium, was built and operated continuously to produce the manganese peroxidase and decolorize the Acid Blue 45. The results showed that an average of 84% Acid Blue 45 decolorization, the manganese peroxidase production with its activity ranging from 63 U L⁻¹ to 5 U L⁻¹, was achieved during about 25 days system continuous operation. The contaminating bacteria in the reactor can be controlled at a level of 4.65 × 10⁴ CFU ml⁻¹ that did not adversely affect the fungal activity. The result of this study provides a new practical way for future design and operation of white-rot fungi reactor under non-sterile conditions.     

Lignin degradation by white rot fungi on spruce wood shavings during short-time solid-state fermentations monitored by near infrared spectroscopy

Due to their outstanding capability of degrading the recalcitrant biomacromolecule lignin, white rot fungi have been attracting interest for several technological applications in mechanical pulping and wood surface modification. However, little is known about the time course of delignification in early stages of colonisation of wood by these fungi. Using a Fourier transform near infrared (FT-NIR) spectroscopic technique, lignin loss of sterilised spruce wood shavings (0.4–2.0 mm particle size) that had been degraded by various species of white rot fungi could be monitored already during the first 2 weeks. The delignification kinetics of Dichomitus squalens, three Phlebia species (Phlebia brevispora, Phlebia radiata and Phlebia tremellosa), three strains of Ceriporiopsis subvermispora as well as the white rot ascomycete Hypoxylon fragiforme and the basidiomycete Oxyporus latemarginatus were determined. Each of the fungi tested was able to reduce the lignin content of spruce wood significantly during the first week. The amount of delignification achieved by the selected white rot fungi after 2 weeks ranged from 7.2% for C. subvermispora (FPL 105.752) to 2.5% for P. radiata. Delignification was significant (P = 95%) already after 3 days treatment with C. subvermispora and P. tremellosa. Activities of extracellular ligninolytic enzymes (laccase, manganese peroxidase and/or lignin peroxidase), expressed by each of the tested fungi, were determined. Lignin was degraded when peroxidase activity was detected in the fungal cultures, but only a low level of correlation between enzyme activities and the extent of delignification was found.     

Aislamiento y evaluación de la actividad enzimática de hongos descomponedores de madera (Quindío,Colombia)

White rot fungi (Ascomycota and Basidiomycota) were collected on fallen trunks with different decay stages, in a subandean forest (La Montaña del Ocaso nature reserve), and it was evaluated their ligninolitic activity. They were cultured on malt extract agar. Then it was performed semiquantitative tests for laccase and cellobiose dehydrogenase (CDH) activity using ABTS and DCPIP as enzymatic inducers. Based on the results of these tests, the fungi with higher activities from trunks with different decay stages were selected: Cookeina sulcipes (for stage 1), a fungus from the family Corticiaceae (for stage 2), Xylaria polymorpha (for stage 3) and Earliella sp. (for stage 4). A fermentation was performed at 28 °C, during 11 days, in a rotatory shaker at 150 rpm. Biomass, glucose, proteins and enzyme activities measurements were performed daily. The fungi that were in the trunks with decay states from 1 to 3, showed higher laccase activity as the state of decay increased. A higher DCH activity was also associated with a higher. Also, there was a positive relationship between both enzymes' activities. Erliella was the fungus which presented the highest biomass production (1140,19 g/l), laccase activity (157 UL^?1) and CDH activity (43,50 UL^?1). This work is the first report of laccase and CDH activity for Cookeina sulcipes and Earliella sp. Moreover, it gives basis for the use of these native fungi in biotechnological applications and the acknowledgment of their function in the wood decay process in native forest.     

A new semi-selective medium for Fusarium graminearum,F. proliferatum,F. subglutinans and F. verticillioides in maize seed

Zea mays L., known also as corn and maize, is the most important crop according to the amount of tonnes produced each year. Fungi cause significant destruction of maize in the field as well as during storage rendering the grain unsuitable for human consumption by decreasing its nutritional value and by producing mycotoxins that are detrimental to both human and animal health. Fusarium species are widely distributed and are amongst the most frequently isolated fungal species by plant pathologists. Due to the fact that the Fusarium species involved in maize ear rot vary in fungicide sensitivity, pathogenicity as well as in their capability to produce mycotoxins, accurate quantification and identification is of paramount significance. Currently no method has been developed to test for Fusarium species in maize seed that has been validated and published by the International Seed Testing Association (ISTA). Malachite green agar 2.5 ppm (MGA 2.5) is a potent selective medium for isolation and enumeration of Fusarium spp. In this study, eight different media compositions, potato dextrose agar (PDA), PDA + malachite green oxalate, corn meal agar, 1/2 PDA + malachite green oxalate, 1% malt agar, carnation leaf agar supplemented with potassium chloride (KCLA), malachite green agar (MGA 2.5) and MGA 2.5 + sterile carnation leaf pieces were compared using four Fusarium species (F. graminearum, F. proliferatum, F. subglutinans and F. verticillioides) and five commonly encountered saprophytic fungi (Aspergillus niger, Penicillium crustosum, P. digitatum, Trichoderma harzianum and Rhizopus stolonifer). The maize kernels were surface disinfected using three concentrations of sodium hypochlorite (0.5%, 1% and 1.5% NaOCl) and for different time intervals (1 min, 3 min, 5 min and 10 min). The effect of black-blue light (365 nm) on sporulation of the fungi was also investigated. Surface disinfection of maize seeds with 1% NaOCl for 5 min provided consistent results. PDA, 1/2 PDA, 1% malt agar and KCLA allowed profuse growth of the Fusarium species as well as saprophytes. Media that contained malachite green oxalate was most inhibitory to the radial colony growth of the saprophytes and the Fusarium species. The Fusarium species growing on these media formed underdeveloped morphological structures, thereby obscuring accurate identification. MGA 2.5 showed better hindering of the saprophytes in some instances. MGA 2.5 amended with sterile carnation leaf pieces was the most satisfactory medium in hindering the growth of the saprophytes while allowing adequate sporulation by the four Fusarium species to permit accurate identification. The media also resulted in higher F. verticillioides and lower saprophytic fungal isolation frequency when compared to the other media tested.     

Changes in oxidative enzyme activity during interspecific mycelial interactions involving the white-rot fungus Trametes versicolor

Interspecific fungal antagonism leads to biochemical changes in competing mycelia, including up-regulation of oxidative enzymes. Laccase, manganese peroxidase (MnP), manganese-repressed peroxidase (MRP) and lignin peroxidase (LiP) gene expression and enzyme activity were compared during agar interactions between Trametes versicolor and five other wood decay fungi resulting in a range of interaction outcomes from deadlock to replacement of one fungus by another. Increased laccase and Mn-oxidising activities were detected at all interaction zones, but there were few changes in activity in regions away from the interaction zone in T. versicolor mycelia compared to self-pairings. Whilst no LiP activity was detected in any pairing, low level LiP gene expression was detected. MnP activity was detected but not expression of MnP genes; instead, MRP could explain the observed activity. No relationship was found between extent of enzyme activity increase and interaction outcome. Similarities between patterns of gene expression and enzyme activity are discussed.     

Response surface analysis for enzymatic decolorization of Congo red by manganese peroxidase

The enzymatic decolorization process of manganese peroxidase (MnP) is a complex system, which is greatly affected by the concentrations of H₂O₂, Mn²⁺, dye and enzyme. This work aimed to study these factors and investigate the combined interactions between them by applying response surface methodology (RSM) for decolorization of Congo red with MnP from Schizophyllum sp. F17, meanwhile conventional one-factor-at-a-time analysis was carried out. Through the one-factor-at-a-time analysis the optimized H₂O₂, Mn²⁺, Congo red and MnP extract was 0.2 mM, 0.5 mM, 50 mg/l and 0.8 ml, respectively, and the maximum decolorization attained under such conditions was 24.2%. Response surface analysis was conducted through Box–Behnken design and a second-order polynomial model (R² = 0.8565) was generated to describe the combined effect and the interactions quantificationally. ANOVA analysis indicated that the interactions between H₂O₂ and MnP, between dye and MnP were significant; the optimum condition through RSM was found to be 0.35 mM H₂O₂, 0.5 mM Mn²⁺, 75 mg/l Congo red and 1.4 ml MnP extract, for maximum decolorization of 30.8%.     

Effect of growth substrate,method of fermentation,and nitrogen source on lignocellulose-degrading enzymes production by white-rot basidiomycetes

The exploration of seven physiologically different white rot fungi potential to produce cellulase, xylanase, laccase, and manganese peroxidase (MnP) showed that the enzyme yield and their ratio in enzyme preparations significantly depends on the fungus species, lignocellulosic growth substrate, and cultivation method. The fruit residues were appropriate growth substrates for the production of hydrolytic enzymes and laccase. The highest endoglucanase (111 U ml⁻¹) and xylanase (135 U ml⁻¹) activities were revealed in submerged fermentation (SF) of banana peels by Pycnoporus coccineus. In the same cultivation conditions Cerrena maxima accumulated the highest level of laccase activity (7,620 U l⁻¹). The lignified materials (wheat straw and tree leaves) appeared to be appropriate for the MnP secretion by majority basidiomycetes. With few exceptions, SF favored to hydrolases and laccase production by fungi tested whereas SSF was appropriate for the MnP accumulation. Thus, the Coriolopsis polyzona hydrolases activity increased more than threefold, while laccase yield increased 15-fold when tree leaves were undergone to SF instead SSF. The supplementation of nitrogen to the control medium seemed to have a negative effect on all enzyme production in SSF of wheat straw and tree leaves by Pleurotus ostreatus. In SF peptone and ammonium containing salts significantly increased C. polyzona and Trametes versicolor hydrolases and laccase yields. However, in most cases the supplementation of media with additional nitrogen lowered the fungi specific enzyme activities. Especially strong repression of T. versicolor MnP production was revealed.     

Fungal post-treatment of pulp mill effluents for the removal of recalcitrant pollutants

The objective of this work was to evaluate the post-treatment of an anaerobic recalcitrant effluent (anaerobically-treated weak black liquor, AnE) in an aerobic, upflow reactor packed with “biocubes” of Trametes versicolor immobilized onto small cubes of holm oak wood. The treated effluent (named anaerobic effluent; AnE) from an anaerobic fluidized bed reactor was fed to an up-flow aerobic fungal packed bed reactor (PBR). Two HRT were tested in this unit, namely 5 and 2.5 days; the PBR operated 60 days at 5-day HRT and 35 days at 2.5-day HRT. The aerobic packed bench scale reactor was a glass column 1.5 L total geometric volume containing 0.75 L biocubes of T. versicolor immobilized onto holm oak wood small cubes of 5 mm side. The reactor was operated at 25 °C. The pH of the AnE was adjusted to 4.5 before feeding; no carbohydrates or other soluble carbon source was supplemented. The fungal packed bed bioreactor averaged organic matter removals of 30% and 32% COD basis, during an experimental run of 60 days at 5-day HRT and 35 days at 2.5-day HRT, respectively. Colour and ligninoids contents were removed at higher percentages (69% and 54% respectively, average of both HRT). There was no significant difference between reactor performance at 5- and 2.5-day HRT, so, operation at 2.5-day HRT is recommended since reactor throughput is double. Activity of manganese peroxidase and laccase was found during the entire operation of the fungal PBR whereas lignin peroxidase activity practically disappeared in the second operation period. In general, enzyme activities were higher in the first period of operation (5-day HRT) than at 2.5-day HRT. To the best of our knowledge, this is one of the few works that demonstrated extended performance (3 months) of a fungal bioreactor for the treatment of a recalcitrant wastewater with no supplementation of glucose or other expensive, soluble carbohydrate.     

Enhanced decolorization of Solar brilliant red 80 textile dye by an indigenous white rot fungus Schizophyllum commune IBL-06

An indigenously isolated white rot fungus, Schizophyllum commune IBL-06 was used to decolorize Solar brilliant red 80 direct dye in Kirk’s basal salts medium. In initial screening study, the maximum decolorization (84.8%) of Solar brilliant red 80 was achieved in 7 days shaking incubation period at pH 4.5 and 30 °C. Different physical and nutritional factors including pH, temperature and fungal inoculum density were statistically optimized through Completely Randomized Design (CRD), to enhance the efficiency of S. commune IBL-06 for maximum decolorization of Solar brilliant red 80 dye. The effects of inexpensive carbon and nitrogen sources were also investigated. Percent dye decolorization was determined by a reduction in optical density at the wavelength of maximum absorbance (λ_max, 590 nm). Under optimum conditions, the S. commune IBL-06 completely decolorized (100%) the Solar brilliant red 80 dye using maltose and ammonium sulfate as inexpensive carbon and nitrogen sources, respectively in 3 days. S. commune IBL-06 produced the three major ligninolytic enzymes lignin peroxidase (LiP), manganase peroxidase (MnP) and lacaase (Lac) during the decolorization of Solar brilliant red 80. LiP was the major enzyme (944 U/mL) secreted by S. commune IBL-06 along with comparatively lower activities of MnP and Laccase.     

Isolation and partial characterization of phenol oxidases from Mangifera indica L. sap (latex)

Mango sap (latex), a by-product in mango industry, was separated into upper non-aqueous phase and lower aqueous phase. Aqueous phase contains very low protein (4.3 mg/ml) but contains high specific activities for peroxidase and polyphenol oxidase. The aqueous phase of sap was subjected to ion-exchange chromatography on DEAE-Sephacel. The bound protein was separated into three enzyme peaks: peak I showed peroxidase activity, peak II showed polyphenol oxidase activity and peak III showed activities against substrates of peroxidase as well as polyphenol oxidase. On native PAGE and SDS-PAGE, each peak showed a single band. Based on the substrate specificity and inhibitor studies peak III was identified as laccase. Although they showed variations in their mobility on native PAGE, these enzymes showed similar molecular weight of 100,000 ± 5000. These enzymes exhibited maximum activity at pH 6 however, polyphenol oxidase showed good activity even in basic pH. Peroxidase and polyphenol oxidase showed stability up to 70 °C while laccase was found to be stable up to 60 °C. Syringaldazine was the best substrate for laccase while catechol was the best for polyphenol oxidase. Thus, mango sap a by-product in mango industry is a good source of these phenol oxidases.     

Effect of nutrient nitrogen and manganese on manganese peroxidase and laccase production by Pleurotus sajor-caju

Pleurotus sajor-caju, strain Pl-27, produces manganese-dependent peroxidase (MnP) and laccase, but not lignin peroxidase, when grown on a defined medium with glucose as sole carbon source. MnP activity was detected in fungal cultures supplemented with both high (26 mM-N) and low (2.6 mM-N) nutrient nitrogen although higher specific activity values were recorded under the latter conditions. Conversely, laccase production was not influenced by nutrient nitrogen levels under the growth conditions adopted. Both the titre and time of appearence of MnP were also affected by the concentration of Mn in the culture medium with highest enzyme levels recorded in cultures supplemented with 15 ppm Mn. Two MnP and five laccase isoforms were identified by FPLC and gel electrophoresis.     

Dephenolisation of olive mill wastewater using adapted Trametes versicolor

Olive mill wastewater (OMW) is an effluent of the olive oil extraction process. The large volumes involved, along with the high phenolic content and chemical oxygen demand, cause major environmental problems. The presence of phenolics limits the effectiveness of aerobic or anaerobic treatment of this wastewater. In most of the studies performed on OMW, the concentration of phenolics is reduced by diluting the OMW prior to biological treatment, which leads to an increase in waste volume. Therefore, the aim of this work was to investigate the possibility of reducing the phenolic content without dilution and without any addition of nutrients or pretreatment by using the white-rot fungi Trametes versicolor FPRL 28A INI. Through an adaptation process, the fungus was able to remove 78% of total phenolics in shake flask experiments and 39% in static culture using undiluted OMW medium. In continuously stirred tank reactor (CSTR) conditions, 70% of total phenolics removal was achieved. Analysis with GC–MS showed that all simple phenolics disappeared from the medium after the 8th day of cultivation at an 0.25 vvm aeration rate. The maximum activities of phenol degrading enzymes laccase and manganese peroxidase (MnP) obtained under these conditions were 762.14 ± 42.11 and 97.80 ± 8.11 U l^?1 respectively.     

The selective visualization of lignin peroxidase,manganese peroxidase and laccase,produced by white rot fungi on solid media

A visual method for the selective screening of lignin degrading enzymes, produced by white rot fungi (WRF), was investigated by the addition of coloring additives to solid media. Of the additives used in the enzyme production media, guaiacol and RBBR could be used for the detection of lignin peroxidase (LiP), manganese peroxidase (MnP) and laccase. Syringaldazine and Acid Red 264 were able for the detection of both the MnP and laccase, and the LiP and laccase, respectively, and a combination of these two additives was able to detect each of the ligninases produced by the WRF on solid media.     

Cyanobacterial biomass as N-supplement to agro-waste for hyper-production of laccase from Pleurotus ostreatus in solid state fermentation

The potential application of dry biomass of a cyanobacterium Anacystis nidulans as a supplement in SSF for the production of laccase from Pleurotus ostreatus was evaluated. Experiments were carried out in solid culture using groundnut shell as a basic substrate supplemented with four independent nitrogen sources (ammonium sulphate, urea, yeast extract and dry powder of cyanobacteria). All the four supplements enhanced the enzyme yield, and yeast extract showed precedence over inorganic nitrogenous sources. However, when dry biomass of A. nidulans was used as an additive to groundnut shell (agricultural residues), it supported maximum cell growth (56.83 ± 5.56 mg/g dry substrate) and laccase production (49.21 ± 4.89 U/g dry substrate). Addition of 1 mM copper salt in the medium containing groundnut shell supplemented with yeast extract gave laccase activity of 32.64 ± 3.4 U/g dry substrate. When dry powder of cyanobacterial biomass was used as N-supplement, laccase production enhanced to 65.42 ± 6.48 U/g dry substrate. In addition to the enhancement to enzyme production inhibitory effects of high concentrations of copper was also diminished in the medium having dry cyanobacterial biomass. This study, forms the first report on the potential application of cyanobacterial biomass as an additive for production of laccase by Pleurotus ostraetus MTCC 1804 in solid state fermentation and has relevance in scale-up production of this fungal enzyme of commercial significance.     

Characterization and kinetic properties of the purified Trematosphaeria mangrovei laccase enzyme

The properties of Trematosphaeria mangrovei laccase enzyme purified on Sephadex G-100 column were investigated. SDS–PAGE of the purified laccase enzyme showed a single band at 48 kDa. The pure laccase reached its maximal activity at temperature 65 °C, pH 4.0 with K_m equal 1.4 mM and V_max equal 184.84 U/mg protein. The substrate specificity of the purified laccase was greatly influenced by the nature and position of the substituted groups in the phenolic ring. The pure laccase was tested with some metal ions and inhibitors, FeSO₄ completely inhibited laccase enzyme and also highly affected by (NaN₃) at a concentration of 1 mM. Amino acid composition of the pure enzyme was also determined. Carbohydrate content of purified laccase enzyme was 23% of the enzyme sample. The UV absorption spectra of the purified laccase enzyme showed a single peak at 260–280 nm.     

Extracellular Ligninolytic Enzymes in Bjerkandera adusta and Lentinus squarrosulus

Extracellular ligninolytic enzyme activities were determined in two white-rot fungi, Bjerkandera adusta and Lentinus squarrosulus. To investigate the activity of extracellular enzymes, cultures were incubated over a period of 20 days in nutrient rich medium (NRM) and nutrient poor medium under static and shaking conditions. Enzymatic activity was varied with media and their incubation conditions. The highest level of Aryl alcohol oxidase (AAO) was detected under shaking condition of both medium while Manganese peroxidase (MnP) activity was best in NRM under both conditions. AAO is the main oxidases enzyme in B. adusta while laccase plays important role in L. squarrosulus. MnP is the main peroxidase enzyme in both varieties.