Bio-remediation of colored industrial wastewaters by the white-rot fungi Phanerochaete chrysosporium and Pleurotus ostreatus and their enzymes

The effect of Phanerochaete chrysosporium and Pleurotus ostreatus whole cells and their ligninolytic enzymes on models of colored industrial wastewaters was evaluated. Models of acid, direct and reactive dye wastewaters from textile industry have been defined on the basis of discharged amounts, economic relevance and representativeness of chemical structures of the contained dyes. Phanerochaete chrysosporium provided an effective decolourization of direct dye wastewater model, reaching about 45% decolourization in only 1 day of treatment, and about 90% decolourization within 7 days, whilst P. ostreatus was able to decolorize and detoxify acid dye wastewater model providing 40% decolourization in only 1 day, and 60% in 7 days. P. ostreatus growth conditions that induce laccase production (up to 130,000 U/l) were identified, and extra-cellular enzyme mixtures, with known laccase isoenzyme composition, were produced and used in wastewater models decolourization. The mixtures decolorized and detoxified the acid dye wastewater model, suggesting laccases as the main agents of wastewater decolourization by P. ostreatus. A laccase mixture was immobilized by entrapment in Cu-alginate beads, and the immobilized enzymes were shown to be effective in batch decolourization, even after 15 stepwise additions of dye for a total exposure of about 1 month.     

Production of extracellular enzymes by two Pleurotus species using banana pseudostem biomass

The degradation of lignocellulosic biomass of banana pseudoste was investigated during solid state fermentation (SSF) by P. ostreatus and P. sajor-caju. Both organisms proved to be efficient degraders of banana pseudostem biomass. P.ostreatus degraded hemicellulose (40% of dry weight, d.w.) better than cellulose (17.5% of d.w.) and lignin (10% of d.w.). P. sajor-caju also degraded hemicellulose (31% of d.w.) better than cellulose (12.4% of d.w.) and lignin (6% of d.w.). In both cases, a preferential removal of hemicellulose during the initial growth period and a delayed degradation of lignin were observed. The kinetics of cellulolytic, hemicellulolytic and lignolytic enzyme production in liquid culture were also examined. The activities of CMCase and β-glucosidase were highest at 16 days of growth and avicelase activity was at its maximum after 24 days (CMCase - 1.1 IU/ml, β-glucosidase - 0.09 IU/ml in the case of P. ostreatus; CMCase - 1.0 IU/ml, β-glucosidase - 0.087 - IU/ml in the case of P. sajor-caju.). Xylanase and laccase activity reached their maximum after day 16 and day 24 of incubation, respectively. (Xylanase - 1.1 IU/ml and laccase 3.0 IU/ml in the case of P. ostreatus; xylanase - 1.0 IU/ml and laccase - 3.6 IU/ml in the case of P. sajor-caju.). The efficient degrading capacity of test fungi demonstrated their potential use in the conversion of banana pseudostem biomass into mycelial protein-rich fermented animal feed.     

Production of laccases by Pleurotus ostreatus in submerged fermentation in co‐culture with Trichoderma viride

Aims: To evaluate the production and stability of laccases by Pleurotus ostreatus in liquid co‐cultures with Trichoderma viride as a function of infection time and agitation rate. Methods and Results: Pleurotus ostreatus cultures were infected with T. viride spores at 30 and 48 h. Maximal laccase volumetric activity was seen after 48 h (control cultures) or 72 h (co‐cultures) of cultivation time. Only the cultures infected at 30 h showed an increased laccase volumetric activity compared to control cultures. After maximal laccase volumetric activity value was reached, a sharp decrease in it was observed in control cultures. Co‐cultures exhibited a comparatively lower loss of activity. The influence of P. ostreatus and/or T. viride on the stability of laccase volumetric activity and isoenzyme pattern was evaluated. Trichoderma viride induced changes in the laccase isoenzyme pattern. Agitated cultures increased biomass growth and specific productivity threefold and sevenfold, respectively, to the static cultures. Conclusions: The laccase volumetric activity is very likely the result of the balance between biosynthesis and degradation/biotransformation rates occurring during the cultures. The individual presence of P. ostreatus or T. viride in the culture negatively affected the volumetric laccase activity. Significance and Impact of the Study: The evaluation of culture parameters that could influence Trichoderma–basidomycetes interaction and laccase production during submerged fermentation has not been reported. This study showed how laccase production in co‐cultures of P. ostreatus and T. viride was influenced by the infection time and agitation/oxygenation conditions.     

Influence of PAHs on ligninolytic enzymes of the fungus <Emphasis Type="Italic">Pleurotus ostreatus</Emphasis> D1

Polycyclic aromatic hydrocarbons (PAHs), their derivatives, and their degradation products were assayed for the ability to enhance activities of ligninolytic enzymes (laccase and versatile peroxidase) of the fungus Pleurotus ostreatus D1. The activities of both laccase and versatile peroxidase were induced by the PAHs, their derivatives, and their degradation products. Laccase was produced mostly in the first 7–10 days, whereas the production of versatile peroxidase began after 5–7 days of cultivation. Non-denaturing PAGE showed the presence of additional forms of laccase and versatile peroxidase in the presence of the xenobiotics in the cultivation medium. The difference in the production time for these enzymes may reflect that laccases are involved in the first stages of PAHs degradation and that versatile peroxidase can be necessary for oxidation of some degradation products. This is the first report on versatile peroxidase induction by PAHs and their derivatives.     

Strategies for dephenolization of raw olive mill wastewater by means of <Emphasis Type="Italic">Pleurotus ostreatus</Emphasis>

The reduction of polyphenols content in olive mill wastewater (OMW) is a major issue in olive oil manufacturing. Although researchers have pointed out the potential of white-rot fungus in dephenolizing OMW, the results available in the literature mainly concern pretreated (sterilized) OMW. This paper deals with the reduction of polyphenols content in untreated OMW by means of a white-rot fungus, Pleurotus ostreatus. Dephenolization was performed both in an airlift bioreactor and in aerated flasks. The process was carried out under controlled non-sterile conditions, with different operating configurations (batch, continuous, biomass recycling) representative of potential industrial operations. Total organic carbon, polyphenols concentration, phenol oxidase activity, dissolved oxygen concentration, oxygen consumption rate, and pH were measured during every run. Tests were carried out with or without added nutrients (potato starch and potato dextrose) and laccases inducers (i.e., CuSO₄). OMW endogenous microorganisms were competing with P. ostreatus for oxygen during simultaneous fermentation. Dephenolization of raw OMW by P. ostreatus under single batch was as large as 70%. Dephenolization was still extensive even when biomass was recycled up to six times. OMW pre-aeration had to be provided under continuous operation to avoid oxygen consumption by endogenous microorganisms that might spoil the process. The role of laccases in the dephenolization process has been discussed. Dephenolization under batch conditions with biomass recycling and added nutrients proved to be the most effective configuration for OMW polyphenols reduction in industrial plants (42–68% for five cycles).     

Biodegradation Kinetics of Naphthalene in Soil Medium Using Pleurotus Ostreatus in Batch Mode with Addition of Fibrous Biomass as a Nutrient

The efficiency and kinetics of naphthalene biodegradation in a soil medium using Pleurotus ostreatus (a type of white rot fungus) in batch mode with and without the addition of oil palm fiber (OPF) as a nutrient are evaluated in this study. Three batches are considered in the biodegradation study: (i) control—spiked soil; (ii) spiked soil with fungus; and (iii) spiked soil with both fungus and OPF. Biodegradation is conducted over a period of 22 days for which soil naphthalene concentrations are determined with respect to microwave extraction and high-performance liquid chromatography (HPLC) analysis. The results indicate that inoculation with Pleurotus ostreatus significantly enhances soil naphthalene biodegradation to 84%, which is further enhanced upon the addition of OPF to 98% with respect to the degradation rate. The high carbon content in OPF (>40%) affords it the capacity to be a viable nutrient supplement for Pleurotus ostreatus, thereby enhancing the potential of Pleurotus ostreatus in the biodegradation of polycylic aromatic hydrocarbons (PAHs), and indicating the potential of OPF as a nutrient for PAH biodegradation. A relationship between OPF mass and the biodegradation rate constant has been determined to be linear according to the following equation: k = 0.0429 × OPF + 0.1291.     

Degradation of fluorene,anthracene, phenanthrene,fluoranthene, and pyrene lacks connection to the production of extracellular enzymes by Pleurotus ostreatus and Bjerkandera adusta

The degradation of polycyclic aromatic hydrocarbons (PAH) was studied in liquid cultures of Bjerkandera adusta and Pleurotus ostreatus during 7 weeks of cultivation. During only 3 days of incubation, B. adusta removed 56% and 38% of fluorene and anthracene, while P. ostreatus degraded 43% and 60% of these compounds; other PAH were degraded to a lower extent. Except for anthracene in cultures of P. ostreatus, all PAH were removed uniformly during the cultivation time but fluorene and anthracene were degraded faster than other PAH. Supplementation of liquid cultures with milled wood decreased the concentration of PAH in the solution and diminished the degradation of PAH. The fungi produced valuable activity of manganese-dependent peroxidase; laccase was secreted only by P. ostreatus and was strongly induced by the addition of milled wood. The production of the oxidative enzymes did not correlate directly to the metabolisation of PAH.     

Laccase production in solid‐state and submerged fermentation by Pleurotus ostreatus

A solid‐state fermentation (SSF) system for production of an industrially important enzyme laccase by Pleurotus ostreatus was developed by using potato dextrose yeast extract medium and polyurethane foam as a supporting material. The maximum laccase production in the SSF system was as high as 3×10⁵ U/L. Addition of inducers, such as copper and ferulic acid, further enhanced the laccase production in SSF. Moreover, the time required for the maximum laccase production was reduced to 6 days compared to 10 days reported earlier. The improvement achieved by the SSF system was investigated by comparing it to a submerged fermentation system (SmF), both experimentally and by using a standard theoretical model along with a parameter sensitivity analysis. Laccase production in SSF was found to be twice of that in SmF. One of the main reasons for higher laccase production in SSF compared to SmF was possibly due to the presence of higher proteolytic activity in SmF. Strong proteolytic activity in SmF presumably caused subsequent laccase degradation, which lowered the ultimate laccase production in SmF compared to SSF.     

Molecular breeding of a novel <Emphasis Type="Italic">Coprinopsis cinerea</Emphasis> strain possessing a heterologous laccase gene, <Emphasis Type="Italic">lccK</Emphasis>, driven by a constitutive promoter

Genomic DNA encoding the Pleurotus ostreatus LccK laccase was fused with the Coprinopsis cinerea β-tubulin promoter and terminator, and introduced into a C. cinerea strain. Linkage analysis, native PAGE separations, substrate specificity investigations and expression profiling indicated that C. cinerea transformants secrete P. ostreatus LccK, suggesting that the introns of the lccK gene are correctly spliced and the signal peptide for secretion is functional in C. cinerea. Transformants constitutively expressing laccase may be useful for the degradation of aromatic compounds.     

Enhanced biodegradation of methylhydrazine and hydrazine contaminated NASA wastewater in fixed-film bioreactor

The aerobic biodegradation of National Aeronautics and Space Administration (NASA) wastewater that contains mixtures of highly concentrated methylhydrazine/hydrazine, citric acid and their reaction product was studied on a laboratory-scale fixed film trickle-bed reactor. The degrading organisms, Achromobacter sp., Rhodococcus B30 and Rhodococcus J10, were immobilized on coarse sand grains used as support-media in the columns. Under continuous flow operation, Rhodococcus sp. degraded the methylhydrazine content of the wastewater from a concentration of 10 to 2.5 mg/mL within 12 days and the hydrazine from 0.8 to 0.1 mg/mL in 7 days. The Achromobacter sp. was equally efficient in degrading the organics present in the wastewater, reducing the concentration of the methylhydrazine from 10 to 5 mg/mL within 12 days and that of the hydrazine from 0.8 to 0.2 mg/mL in 7 days. The pseudo first-order rate constants of 0.137 day^-1 and 0.232 day^-1 were obtained for the removal of methylhydrazine and hydrazine, respectively, in wastewater in the reactor column. In the batch cultures, rate constants for the degradation were 0.046 and 0.079 day^-1 for methylhydrazine and hydrazine respectively. These results demonstrate that the continuous flow bioreactor afford greater degradation efficiencies than those obtained when the wastewater was incubated with the microbes in growth-limited batch experiments. They also show that wastewater containing hydrazine is more amenable to microbial degradation than one that is predominant in methylhydrazine, in spite of the longer lag period observed for hydrazine containing wastewater. The influence of substrate concentration and recycle rate on the degradation efficiency is reported. The major advantages of the trickle-bed reactor over the batch system include very high substrate volumetric rate of turnover, higher rates of degradation and tolerance of the 100% concentrated NASA wastewater. The results of the present laboratory scale study will be of great importance in the design and operation of an industrial immobilized biofilm reactor for the treatment of methylhydrazine and hydrazine contaminated NASA wastewater.     

Influence of cultivation conditions on pyrene degradation by the fungus Pleurotus Ostreatus D1

For the first time the dependence of completeness of pyrene degradation by the white-rot fungus Pleurotus ostreatus D1 on cultivation conditions was found. In Kirk’s medium about 65.6 ± 0.9% of the initial pyrene was metabolized after 3 weeks, with pyrene-4,5-dihydrodiol accumulating. This process was accompanied by laccase production only. In basidiomycetes rich medium, P. ostreatus D1 metabolized up to 89.8 ± 2.3% of pyrene within 3 weeks without pyrene-4,5-dihydrodiol accumulation throughout the time of cultivation. Phenanthrene and phthalic acid were identified as the metabolites produced from pyrene degradation under these conditions. Accumulation of phenanthrene with its subsequent disappearance was observed. One more metabolite probably was the product of phenanthrene degradation. Pyrene metabolism in basidiomycetes rich medium was accompanied first by laccase and tyrosinase production and later by versatile peroxidase production. The cell-associated activities of laccase, tyrosinase, and versatile peroxidase were found. The data obtained indicate that both enzymes (laccase and versatile peroxidase) are necessary for complete degradation of pyrene. Furthermore, both cell-associated and extracellular laccases can catalyse the first stages of pyrene degradation, and versatile peroxidase can be necessary for oxidation of the resulting metabolites.     

Efficient phenol degradation by laccase immobilized on functional magnetic nanoparticles in fixed bed reactor under high‐gradient magnetic field

Enzymatic degradation of emerging contaminants has gained great interest for the past few years. However, free enzyme often incurs high costs in practice. The immobilized laccase on the polyethylenimine (PEI)‐functionalized magnetic nanoparticles (Fe₃O₄–NH₂–PEI–laccase) was fabricated to efficiently degrade phenolic compounds continuously in a newly fixed bed reactor under a high‐gradient magnetic field. The degradation rate of continuous treatment in the bed after 18 h was 2.38 times as high as that of batch treatment after six successive operations with the same treatment duration. Under the optimal conditions of volume fraction of nickel wires mesh, flow rate of phenol solution, phenol concentration, and Fe₃O₄–NH₂–PEI–laccase amount, the degradation rate of phenol kept over 70.30% in 48 h continuous treatment. The fixed bed reactor filled with Fe₃O₄–NH₂–PEI–laccase provided a promising avenue for the continuous biodegradation of phenolic compounds for industrial wastewater in practice.     

Variability of enzymatic activities in ligninolytic fungi Pleurotus ostreatus and Lentinus tigrinus after protoplasting and UV-mutagenization

Summary Protoplast preparation and UV-irradiation of mycelial fragments were used to study the variability of production of laccase, peroxidase and manganese-dependent peroxidase (MnP) involved in lignin degradation in Pleurotus ostreatus and Lentinus tigrinus. After protoplasting, the variability of production of all enzymes increased substantially and was comparable to that of isolates after mutagenesis.     

Chrysene bioconversion by the white rot fungus <Emphasis Type="Italic">Pleurotus ostreatus</Emphasis> D1

The effect of cultivation conditions on chrysene bioconversion by the fungus Pleurotus ostreatus D1 was shown. Under the laccase production conditions, transformation of this polycyclic aromatic hydrocarbon occurs with accumulation of the quinone metabolite. Under both the laccase and versatile peroxidase production conditions, chrysene degradation occurs, with the stages leading to phthalic acid formation and its further utilization. The formation of phthalic acid as a metabolite of chrysene degradation by white rot fungi was revealed for the first time. The data obtained suggest that the laccase revealed on the mycelial surface and the extracellular laccase are probably involved at the initial stages of chrysene metabolism, whereas versatile peroxidase seems to be required for oxidizing the metabolites formed.     

Degradation of Polycyclic Aromatic Hydrocarbons by Crude Extracts from Spent Mushroom Substrate and its Possible Mechanisms

Biodegradation of polycyclic aromatic hydrocarbons (PAHs) by pure laccase has been reported, but the high cost limited its application in environmental bioremediation. Here, we reported a study about PAHs degradation by crude extracts (CEs) containing laccase, which were obtained by extracting four spent mushroom (Agaricus bisporus, Pleurotus eryngii, Pleurotus ostreatus, and Coprinus comatus) substrates. The results showed that anthracene, benzo[a]pyrene, and benzo[a]anthracene were top three degradable PAHs by CEs while naphthalene was most recalcitrant. The PAHs oxidation was enhanced in the presence of 2,2-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS). Laccase included in CE might play a major role in PAHs degradation. The maximum degradation rate of anthracene and benzo[a]pyrene was observed by using crude extracts from P. eryngii while the highest laccase activities were found in crude extracts from A. bisporus, moreover, crude extracts from P. eryngii, which contained less laccase activities, degraded more anthracene and benzo[a]pyrene than pure laccase with higher laccase activities. The lack of correlation between laccase activity and PAHs degradation rate indicated that other factors might also influence the PAHs degradation. Boiled CEs were added to determine the effect on PAHs degradation by laccase. The results showed that all four boiled CEs had improved the PAHs oxidation. The maximum improvement was observed by adding CEs from P. eryngii. It suggested that some mediators indeed existed in CEs and CEs from P. eryngii contained most. As a result, CEs from P. eryngii has the most application potential in PAHs bioremediation.     

Stability of immobilized laccase on Luffa Cylindrica fibers and assessment of synthetic hormone degradation

In this work were studied the pH, thermal, and storage stability of free and immobilized laccases. Enzymes were produced by Pleurotus ostreatus on potato dextrose (PD) broth and potato dextrose modified (PDM) broth, and immobilized using Luffa cylindrica fibers as support. Both free and immobilized enzymes were assessed on their respective enzymatic activities and for 17α-ethinylestradiol (EE2) degradation. The optimum pH conditions concerning laccase activity ranged from 3.6 to 4.6, while temperature ranged between 30?°C and 50?°C for both free and immobilized enzyme. Laccase produced using PD broth presented greater storage stability and thermal stability than that of PDM. Best EE2 removals were of 79.22% and 75.00% for the free and immobilized enzymes, respectively. Removal rates were assessed during 8?h at pH 5. The removal of 17α-ethinylestradiol was stabilized in the fourth cycle of use. Results imply that immobilization promoted stability towards pH and temperature variations, although media played a decisive role in the enzymatic activity. Both free and immobilized laccases of P. ostreatus were able to degrade EE2, whereas immobilized laccase in PDM medium presented possible reuse applicability, albeit removal was not optimal when compared to other reports.     

Variability of laccase activity in the white-rot basidiomycete Pleurotus ostreatus

The production of laccase in liquid cultures of the white-rot fungusPleurotus ostreatus was highly variable. During the first days of cultivation, the relative variability was as high as 80–100% and it decreased to 30% in the course of cultivation. The main source of variability was assumed to be the independent development of enzyme activity in individual cultures. Cultures with high laccase production showed also high production of the other ligninolytic enzyme—Mn-dependent peroxidase. The variability was probably due to the source of inoculum, deactivation of the enzyme in culture liquid and genetic variations among the cultures. Variability of laccase activities was lower during solid-state fermentation on wheat straw and during the growth in nonsterile soil.     

An Evaluation of Soil Colonisation Potential of Selected Fungi and their Production of Ligninolytic Enzymes for Use in Soil Bioremediation Applications

Initially sixteen fungi were screened for potential ligninolytic activity using decolourisation of a polymeric dye Poly R-478. From this, four fungi were selected, Trametes versicolor, Pleurotus ostreatus, Collybia sp., and an isolate (identified as Rhizoctonia solani) isolated from a grassland soil. Differences in the ligninolytic enzyme profiles of each of the fungi were observed. All of the four fungi tested produced MnP and laccase while the Collybia sp. and R. solani produced LiP in addition. Enzyme activity levels also varied greatly over the 21 days of testing with T. versicolor producing levels of MnP and laccase three to four times greater than the other fungi. The four fungi were then tested for their ability to colonise sand, peat (forest) and basalt and marl mixed till (field) soils through visual measurement and biomass detection in soil microcosms. Trametes versicolor and the Collybia sp. failed to grow in any of the non-sterilised soils whereas the R. solani and P. ostreatus isolates grew satisfactorily. Primers were␣designed to detect MnP and laccase genes in P.␣ostreatus and RTPCR was used to detect that these genes are expressed in forest and field soils.     

Study of laccase production by Pleurotus ostreatus in a 5 l bioreactor and application of the enzyme to determine the antioxidant concentration of human plasma

Aims: To achieve high laccase production from Pleurotus ostreatus in a bench top bioreactor and to utilize the enzyme for determination of the total antioxidant concentration (TAC) of human plasma. Methods and Results: Laccase production by P. ostreatus studied in a benchtop bioreactor was as high as, 874·0 U ml^?1 in presence of copper sulfate. The enzyme was used to replace metmyoglobin and hydrogen peroxide for the estimation of TAC in human plasma. The trolox equivalent antioxidant concentrations determined by the laccase‐based method and metmyoglobin method ranged from 1·63 ± 0·011 to 1·80 ± 0·006 mmol l^?1 and from 1·41 ± 0·004 to 1·51 ± 0·008 mmol l^?1 plasma, respectively. Conclusions: Pleurotus ostreatus produced high amount of extracellular laccase in a benchtop bioreactor. The enzyme can be used to assay TAC of blood plasma without the interference encountered with the hydrogen peroxide and metmyoglobin mediated assay method. Significance and Impact of the Study: Laccase production by P. ostreatus obtained in this study was the highest among all reported laccase producing white‐rot fungi. Moreover, an accurate laccase‐based assay method was developed for detection of TAC in human plasma.