Activity and elution profile of laccase during biological decolorization and dephenolization of olive mill wastewater

The performance and enzymatic strategy exhibited by basidiomycete Euc-1, a laccase producing strain, was investigated during the biodegradation of olive mill wastewater (OMW). This strain yielded better decolorization of solidified OMW than Phanerochaete chrysosporium and removed 90% of phenols (initial concentration=800 mg l(-1)), 73% of color (initial A465=4.4), and 45% of chemical oxygen demand in batch cultures containing OMW. Since partial phenol removal occurred before the detection of enzymatic activity, no plausible correlation could be established between them. In contrast, decolorization occurred only after the detection of laccase activity and coincided with its production over time. Two laccase fractions (Lac1 and Lac2) were separated by chromatography. OMW strongly induced Lac2 that was almost absent in defined liquid medium. Furthermore, Lac2 was the main laccase fraction in the presence of OMW. This study pointed out that basidiomycete Euc-1 and its ligninolytic system could be a useful tool for the bioremediation of wastewater generated in the process of olive oil extraction.     

Elimination of Endocrine Disrupting Chemicals using White Rot Fungi and their Lignin Modifying Enzymes: A Review

The ability of white rot fungi (WRF) and their lignin modifying enzymes (LMEs), i.e. laccase and lignin‐ and manganese‐dependent peroxidase, to treat endocrine disrupting chemicals (EDCs) is extensively reviewed in this paper. These chemicals cause adverse health effects by mimicking endogenous hormones in receiving organisms. The alkylphenolic EDCs nonylphenol, bisphenol A and triclosan, the phthalic acid esters dibutylphthalate, diethylphthalate and di‐(2‐ethylhexyl)phthalate, the natural estrogens estrone, 17β‐estradiol, estriol and 17α‐ethynylestradiol and the phytoestrogens genistein and β‐sitosterol have been shown to be eliminated by several fungi and LMEs. WRF have manifested a highly efficient removal of EDCs in aqueous media and soil matrices using both LME and non LME‐systems. The ligninolytic system of WRF could also be used for the elimination of several EDCs and the associated hormone‐mimicking activity. The transformation of EDCs by LMEs and WRF is supported by emerging knowledge on the physiology and biochemistry of these organisms and the biocatalytic properties of their enzymes. Due to field reaction conditions, which drastically differ from laboratory conditions, further efforts will have to be directed towards developing robust and reliable biotechnological processes for the treatment of EDC‐contaminated environmental matrices.     

Improving the bioremediation of phenolic wastewaters by Trametes versicolor

The successful bioremediation of a phenolic wastewater by Trametes versicolor was found to be dependent on a range of factors including: fungal growth, culture age and activity and enzyme (laccase) production. These aspects were enhanced by the optimisation of the growth medium used and time of addition of the pollutant to the fungal cultures. Different media containing 'high' (20 g/L), 'low' (2 g/L) and 'sufficient' (10 g/L) concentrations of carbon and nitrogen sources were investigated. The medium containing both glucose and peptone at 10 g/L resulted in the highest Growth Related Productivity (the product of specific yield and micro) of laccase (1.46 Units of laccase activity)/gram biomass/day and was used in all further experiments. The use of the guaiacol as an inducer further increased laccase activity 780% without inhibiting growth; similarly the phenolic effluent studied boosted activity almost 5 times. The timing of the addition of the phenolic effluent was found to have important consequences in its removal and at least 8 days of prior growth was required. Under these conditions, 0.125 g phenol/g biomass and 0.231 g o-cresol/g biomass were removed from solution per day.     

Screening of fungal isolates and properties of Ganoderma applanatum intended for olive mill wastewater decolourization and dephenolization

AIMS: To investigate different autochthonous isolates of wood-rotting fungi for the removal of both colour and phenolic compounds from olive mill wastewaters (OMW). METHODS AND RESULTS: The isolates Bjerkandera adusta Ba-100, Fomes fomentarius Ff-106, Ganoderma applanatum Ga-20, Irpex lacteus Il-3, Trametes versicolor Tv-101 and Tv-103 were preliminarily screened for their OMW-decolourizing potential on potato dextrose agar supplemented with different OMW concentrations. A further screening of batch cultures under different agitation speeds, to test the effect of shear stress, resulted in the selection of isolate G. applanatum Ga-20. Batch cultures grown in OMW-based medium exhibited strong laccase induction and significant decrease in the values of phenols, colour and chemical oxygen demand. Concomitant onset of laccase activity and colour removal was observed, and apart from laccase, neither lignin peroxidase nor manganese-dependent peroxidase activities were detected. Moreover, the depletion of aromatic compounds with high and low apparent molecular mass was observed by chromatographic analysis. CONCLUSIONS: Isolate G. applanatum Ga-20 exhibited interesting properties for its use in bioremediation of OMW, namely high removal of recalcitrant phenolic compounds and strong colour abatement. SIGNIFICANCE AND IMPACT OF THE STUDY: For the first time, the white-rot fungus G. applanatum proves to be effective for the decolourization and dephenolization of OMW.     

Influence of nutrients on enhancing laccase production by Botryosphaeria rhodina MAMB-05.

The physiological requirements needed to enhance the production of laccases by the ascomycete Botryosphaeria rhodina MAMB-05 in submerged cultivation were examined under non-induced and induced (veratryl alcohol, VA) conditions. Under non-induced conditions (-VA), the initial pH, C:N ratio, and inorganic N source did not influence laccase production, in contrast to Tween 80, soybean oil, and copper, which significantly increased laccase production, and proline and urea, which suppressed laccase formation. In addition, Tween 60 could serve as the sole carbon source for the production of these enzymes. Under VA-induced conditions of fungal growth, factors such as inoculum type, time-point of addition of inducer, initial pH, C:N ratio, and type of N source, influenced the production of laccases; however, unlike the non-induced conditions, proline and urea did not act as suppressors. Each of these physiological conditions exerted different effects on biomass production. The nutritional conditions examined for B. rhodina MAMB-05 are discussed in relation to their influence on fungal growth and laccase production.     

Enzymatic grafting of natural phenols to flax fibres: Development of antimicrobial properties

Unbleached flax fibres for paper production were treated with laccase from Pycnoporus cinnabarinus and low molecular weight phenols (syringaldehyde - SA, acetosyringone - AS and p-coumaric acid - PCA) to evaluate the potential of this treatment to biomodify high cellulose content fibres. After the enzymatic treatment with the phenols, an increase in kappa number was found, probably due to a covalent binding of the phenoxy radicals on fibres. Grafting was more evident in pulps treated with PCA (an increase of 4 kappa number points with respect to the laccase control was achieved). Paper handsheets from treated pulps showed antimicrobial activity against the bacteria tested: Staphylococcus aureus, Pseudomonas aeruginosa and Klebsiella pneumoniae. An important reduction on microbial count was obtained after incubation of liquid cultures of the bacteria with grafted handsheets. AS and PCA grafted fibres showed a high antibacterial activity on K. pneumoniae, getting a nearly total growth inhibition. AS fibres also caused a high reduction in bacterial population of P. aeruginosa (97% reduction). Optical properties of handsheets from treated pulps were also determined, showing a brightness decrease and increase in coloration, evaluated by CIE L*a*b* system, caused by the laccase induced grafting of the phenols. The results suggest that these low molecular weight phenols, covalently bound to the flax fibres by the laccase treatment, can act as antimicrobial agents and produce handsheets with antimicrobial activity.     

Effect of Nutritional Parameters on Laccase Production by the Culinary and Medicinal Mushroom, <Emphasis Type="Italic">Grifola frondosa</Emphasis>

Extracellular laccase in cultures of Grifola frondosa grown in liquid culture on a defined medium was first detectable in the early/middle stages of primary growth, and enzyme activity continued to increase even after fungal biomass production had peaked. Laccase production was significantly increased by supplementing cultures with 100–500 μM Cu over the basal level (1.6 μM Cu) and peak levels observed at 300 μM Cu were 7-fold higher than in unsupplemented controls. Decreased laccase activity similar to levels detected in unsupplemented controls, as well as an adverse effect on fungal growth, occurred with further supplementation up to and including 0.9 mM Cu, but higher enzyme titres (2- to 16-fold compared with controls) were induced in cultures supplemented with 1–2 mM Cu²⁺. SDS-PAGE combined with activity staining revealed the presence of a single protein band (M _r 70 kDa) exhibiting laccase activity in control culture fluids, whereas an additional distinct laccase protein band (M _r 45 kDa) was observed in cultures supplemented with 1–2 mM Cu. Increased levels of extracellular laccase activity, and both laccase isozymes, were also detected in cultures of G. frondosa supplemented with ferulic, vanillic, veratric and 4-hydroxybenzoic acids, and 4-hydroxybenzaldehyde. Using 2,2′-azino-bis(ethylbenzothiazoline-6-sulfonate) (ABTS) as substrate, the optimal temperature and pH values for laccase activity were 65°C and pH 2.2, respectively, and the enzyme was relatively heat stable. In solid-state cultures of G. frondosa grown under conditions adopted for industrial-scale mushroom production, extracellular laccase levels increased during the substrate colonization phase, peaked when the substrate was fully colonized, and then decreased sharply during fruit body development.     

Phenolic removal in olive oil mill wastewater by strains of Pleurotus spp. in respect to their phenol oxidase (laccase) activity

The ability of several Pleurotus spp. strains to remove phenolic compounds from an olive oil mill wastewater (OMW) was studied. All strains tested in this work were able to grow in OMW without any addition of nutrients and any pre-treatment, except sterilization. High laccase activity was measured in the growth medium, while 69-76% of the initial phenolic compounds were removed. The black color of OMW became yellow-brown and brighter as the strains grew. The lowest phenolic concentrations were reached after 12/15 days. A decrease of the phytotoxicity, as described by the parameter Germination Index, was noticed in the OMW treated with some Pleurotus spp strains, although this decrease was not proportional to the phenolic removal. A new parameter, namely Phenol-toxicity Index, was considered in the present paper. Using this parameter it was found that the remaining phenolics and/or some of the oxidation products of the laccase reaction in the treated OMW were more toxic than the original phenolic compounds.     

Effect of nutritional parameters on laccase production by the culinary and medicinal mushroom, Grifola frondosa

Summary Extracellular laccase in cultures of Grifola frondosa grown in liquid culture on a defined medium was first detectable in the early/middle stages of primary growth, and enzyme activity continued to increase even after fungal biomass production had peaked. Laccase production was significantly increased by supplementing cultures with 100–500 (M Cu over the basal level (1.6 mM Cu) and peak levels observed at 300 mM Cu were ∼ ∼7-fold higher than in unsupplemented controls. Decreased laccase activity similar to levels detected in unsupplemented controls, as well as an adverse effect on fungal growth, occurred with further supplementation up to and including 0.9 mM Cu, but higher enzyme titres (2- to 16-fold compared with controls) were induced in cultures supplemented with 1–2 mM Cu²⁺. SDS-PAGE combined with activity staining revealed the presence of a single protein band (M _r ∼ ∼70 kDa) exhibiting laccase activity in control culture fluids, whereas an additional distinct second laccase protein band (M _r␣∼ ∼45 kDa) was observed in cultures supplemented with 1–2 mM Cu. Increased levels of extracellular laccase activity, and both laccase isozymes, were also detected in cultures of G. frondosa supplemented with ferulic, vanillic, veratric and 4-hydroxybenzoic acids, and 4-hydroxybenzaldehyde. The optimal temperature and pH values for laccase activity were 65 °C and pH 2.2 (using 2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonate) {ABTS} as substrate), respectively, and the enzyme was relatively heat stable. In solid-state cultures of G. frondosa grown under conditions adopted for industrial-scale mushroom production, extracellular laccase levels increased during the substrate colonization phase, peaked when the substrate was fully colonized, and then decreased sharply during fruit body development.     

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.     

Fruiting of Agaricus bisporus

Several enzymes were assayed in extracts from mycelium-colonised compost during growth and fruiting of Agaricus bisporus (Lange) Imbach. Comparison of changes of enzyme levels in axenic and nonaxenic cultures and in cultures of non-fruiting strains indicated that they were associated directly with the fungal mycelium. Large changes were found in the amounts of laccase and cellulase which were correlated with fruit body development. Laccase concentration increased during mycelial growth and then declined rapidly at the start of fruiting. Cellulase activity could be detected throughout growth but increased at fruiting. No such changes were observed in xylanase, alkaline protease, laminarinase and acid and alkaline phosphatases. Activities of laccase and cellulase were measured in axenic cultures arrested at various stages of fruiting development. Such cultures showed that the changes in concentration of laccase and cellulase were associated with the enlargement of fruit bodies.     

Rhizosphere dynamics during phytoremediation of olive mill wastewater

The potential of phytoremediation as a treatment option for olive mill wastewater (OMW) was tested on five perennial tree species. Cupressus sempervirens and Quercus ilex proved tolerant to six-month OMW treatment followed by six-month water irrigation, whereas Salix sp. and Laurus nobilis and, later, Pinus mugo suffered from phytotoxic effects. Test plants were compared to controls after treatment and irrigation, by monitoring biochemical and microbiological variations in the rhizosphere soil. OMW-treated soils were exposed to 50-fold higher phenols concentrations, which, irrespective of whether the respective plants were OMW-resistant or susceptible, were reduced by more than 90% by the end of the irrigation cycle, owing to significantly increased laccase, peroxidase and β-glucosidase activities, recovery/acquisition of bacterial culturability and transitory development of specialized fungal communities sharing the presence of Geotrichum candidum. Of all results, the identification of Penicillium chrysogenum and Penicillium aurantiogriseum as dominant rhizosphere fungi was distinctive of OMW-tolerant species.     

Laccase from Trametes hirsuta Grown on Paper Cuttings: Application to Synthetic Dye Decolorization at Different pH Values

The potential of paper cuttings to produce laccase from Trametes hirsuta grown under solid‐state conditions was investigated. In addition, cultures were also grown on barley bran, a support commonly used in solid‐state fermentation (SSF), for comparison. Paper cutting cultures showed a maximum individual laccase activity of 7695 U/L on day 9. In addition, the ability to decolorize two structurally different dyes (Indigo Carmine and Lissamine Green B) by the extracellular liquid from both paper and barley bran cultures at pH values between 2 and 11 was analyzed. Laccase‐containing enzyme preparations from both cultures decolorized the dyes tested at pH values between 4 and 7 and, in addition, the laccase‐containing enzyme preparation from paper cutting cultures was also able to decolorize the dyes tested at alkaline pH values. This is a very interesting and novel result, since no decolorization by fungal laccases has been reported until recently at pH values higher than pH 7.     

Roles of two white-rot basidiomycete fungi in decolorisation and detoxification of olive mill waste water

A Phanerochaete chrysosporium strain was isolated from Moroccan olive mill waste water (OMW) and its ability to degrade OMW in different culture conditions was investigated and compared to that of Pleurotus ostreatus. The results indicated that Ph. chrysosporium isolate is more efficient than Pl. ostreatus in decolorising and detoxifying OMW in the presence of added nutrients. Ph. chrysosporium is able to remove more than 50% of the colour and phenols from OMW within 6 days of incubation, whereas Pl. ostreatus needs more than 12 days to reach similar results in the same conditions. Many factors affecting the treatment of diluted OMW (20%) by Ph. chrysosporium were studied, including the effects of added nutrients, initial pH, temperature and inoculated biomass. Once the optimisation of 20% OMW biodegradation process had been set up, higher OMW concentrations (50%) were tested. The results show that the fungus is capable of reducing all parameters analysed (colour A395, phenol content and chemical oxygen demand) by at least 60%, after only 9 days of growth.     

Roles of Lignin Peroxidase and Manganese Peroxidase from Phanerochaete chrysosporium in the Decolorization of Olive Mill Wastewaters

The relative contributions of lignin peroxidase (LiP) and manganese peroxidase (MnP) to the decolorization of olive mill wastewaters (OMW) by Phanerochaete chrysosporium were investigated. A relatively low level (25%) of OMW decolorization was found with P. chrysosporium which was grown in a medium with a high Mn(II) concentration and in which a high level of MnP (0.65 (mu)M) was produced. In contrast, a high degree of OMW decolorization (more than 70%) was observed with P. chrysosporium which was grown in a medium with a low Mn(II) concentration but which resulted in a high level of LiP activity (0.3 (mu)M). In this culture medium, increasing the Mn(II) concentration resulted in decreased levels of OMW decolorization and LiP activity. Decolorization by reconstituted cultures of P. chrysosporium was found to be more enhanced by the addition of isolated LiP than by the addition of isolated MnP. The highest OMW decolorization levels were obtained at low initial chemical oxygen demands combined with high levels of extracellular LiP. These data, plus the positive effect of veratryl alcohol on OMW decolorization and LiP activity, indicate that culture conditions which yield high levels of LiP activity lead to high levels of OMW decolorization.     

Laccase-mediated detoxification of phenolic compounds.

The ability of a polyphenoloxidase, the laccase of the fungus Rhizoctonia praticola, to detoxify phenolic pollutants was examined. The growth of the fungus could be inhibited by phenolic compounds, and the effective concentration was dependent on the substituents of the phenol. A toxic amount of a phenolic compound was added to a fungal growth medium in the presence or absence of a naturally occurring phenol, and half of the replicates also received laccase. The medium was then inoculated with R. praticola, and the levels of phenols in the medium were monitored by high-performance liquid chromatography analysis. The addition of the laccase reversed the inhibitory effect of 2,6-xylenol, 4-chloro-2-methylphenol, and p-cresol. Other compounds, e.g., o-cresol and 2,4-dichlorophenol, were detoxified only when laccase was used in conjunction with a natural phenol such as syringic acid. The toxicity of p-chlorophenol and 2,4,5-trichlorophenol could not be overcome by any additions. The ability of the laccase to alter the toxicity of the phenols appeared to be related to the capacity of the enzyme to decrease the levels of the parent compound by transformation or cross-coupling with another phenol.     

Laccase-mediated detoxification of phenolic compounds

The ability of a polyphenoloxidase, the laccase of the fungus Rhizoctonia praticola, to detoxify phenolic pollutants was examined. The growth of the fungus could be inhibited by phenolic compounds, and the effective concentration was dependent on the substituents of the phenol. A toxic amount of a phenolic compound was added to a fungal growth medium in the presence or absence of a naturally occurring phenol, and half of the replicates also received laccase. The medium was then inoculated with R. praticola, and the levels of phenols in the medium were monitored by high-performance liquid chromatography analysis. The addition of the laccase reversed the inhibitory effect of 2,6-xylenol, 4-chloro-2-methylphenol, and p-cresol. Other compounds, e.g., o-cresol and 2,4-dichlorophenol, were detoxified only when laccase was used in conjunction with a natural phenol such as syringic acid. The toxicity of p-chlorophenol and 2,4,5-trichlorophenol could not be overcome by any additions. The ability of the laccase to alter the toxicity of the phenols appeared to be related to the capacity of the enzyme to decrease the levels of the parent compound by transformation or cross-coupling with another phenol.     

β-Glucan synthase induction in mushrooms grown on olive mill wastewaters

-1-3-Glucan synthase activity and its induction by olive mill wastewaters (OMW) was studied in ten fungal strains (Auricularia auricula-judae, Lentinula edodes, Pleurotus eryngii, Stropharia aeruginosa, Agrocybe aegerita, P. pulmonarius, Armillaria mellea, P. ferulae, P. ostreatus, P. nebrodensis). A microtiter-based enzymatic assay on -1-3-glucan synthase activity was carried out on all mycelia growth both on the control medium and on OMW. Among the fungi assayed, L. edodes -1-3-glucan synthase was highly enhanced in OMW. The main components of OMW, i.e. phenols and lipids, were added separately to the control medium, to highlight the mechanism of L. edodes -1-3-glucan synthase induction. A Southern blot analysis and PCR with degenerated primers were carried out to detect the presence of fks1-like genes in these Basidiomycetes. The sequences obtained from the ten Basidiomycota were remarkably similar to fks1 from Filobasidiella neoformans. Spectrofluorimetric and RT-PCR analyses of -1-3-glucan synthase were performed on the mycelia of L. edodes. In this fungus, a strong stimulation of -1-3-glucan synthase mRNA and protein was recorded in the presence of OMW and phenols.