Immobilization of pycnoporus coccineus laccase on Eupergit C: Stabilization and treatment of olive oil mill wastewaters

The use of olive oil mill wastewaters (OMW) as an organic fertilizer is limited by their phytotoxic effect, due to the high concentration of phenolic compounds. As an alternative to physico-chemical methods for OMW detoxification, the laccase from Pycnoporus coccineus, a white-rot fungus with the ability to decrease the chemical oxygen demand (COD) and color of the industrial effluent, is being studied. In this work, the P. coccineus laccase was immobilized on two acrylic epoxy-activated resins, Eupergit C and Eupergit C 250L. The highest activity was obtained with the macroporous Eupergit C 250L, reaching 110 U g^?1 biocatalyst. A substantial stabilization effect against pH and temperature was obtained upon immobilization. The soluble enzyme maintained ≥80% of its initial activity after 24 h at pH 7.0–10.0, whereas the immobilized laccase kept the activity in the pH range 3.0–10.0. The free enzyme was quickly inactivated at temperatures >50°C, whereas the immobilized enzyme was very stable up to 70°C. Gel filtration profiles of the OMW treated with the immobilized enzyme (for 8 h at room temperature) showed both degradation and polymerization of the phenolic compounds.     

Molecular microbial and chemical investigation of the bioremediation of two-phase olive mill waste using laboratory-scale bioreactors

Two-phase olive mill waste (TPOMW) is a semi-solid effluent that is rich in contaminating polyphenols and is produced in large amounts by the industry of olive oil production. Laboratory-scale bioreactors were used to investigate the biodegradation of TPOMW by its indigenous microbiota. The effect of nutrient addition (inorganic N and P) and aeration of the bioreactors was studied. Microbial changes were investigated by PCR-temperature time gradient electrophoresis (TTGE) and following the dynamics of polar lipid fatty acids (PLFA). The greatest decrease in the polyphenolic and organic matter contents of bioreactors was concomitant with an increase in the PLFA fungal/bacterial ratio. Amplicon sequences of nuclear ribosomal internal transcribed spacer region (ITS) and16S rDNA allowed identification of fungal and bacterial types, respectively, by comparative DNA sequence analyses. Predominant fungi identified included members of the genera Penicillium, Candida, Geotrichum, Pichia, Cladosporium, and Aschochyta. A total of 14 bacterial genera were detected, with a dominance of organisms that have previously been associated with plant material. Overall, this work highlights that indigenous microbiota within the bioreactors through stimulation of the fungal fraction, is able to degrade the polyphenolic content without the inoculation of specific microorganisms.     

Advantages in using non-isothermal bioreactors in bioremediation of water polluted by phenol by means of immobilized laccase from Rhus vernicifera

Laccase from Rhus vernicifera was immobilized on a polypropylene membrane chemically modified with chromic acid. Ethylenediamine and glutaraldehyde were used as spacer and bifunctional coupling agent, respectively. Phenol was used as substrate.To know how the immobilization procedures affected the enzyme reaction rate the catalytic behavior of soluble and insoluble laccase was studied under isothermal conditions as a function of pH, temperature and substrate concentration. From these studies, two main singularities emerged: (i) the narrower pH–activity profile of the soluble enzyme in comparison to that of the insoluble counterpart and (ii) the increase in pH and thermal stability of the insoluble enzyme.The laccase catalytic behavior was also studied in a non-isothermal bioreactor as a function of substrate concentration and size of the applied transmembrane temperature difference. It was found that, under non-isothermal conditions and keeping constant the average temperature of the bioreactor, the enzyme reaction rate linearly increased with the increase of the temperature difference.     

Isothermal and non-isothermal bioreactors in the detoxification of waste waters polluted by aromatic compounds by means of immobilised laccase from Rhus vernicifera

Laccase from Rhus vernicifera was immobilised on a nylon membrane chemically grafted with glycidyl methacrylate (GMA). Hexamethylenediamine (HMDA) and glutaraldehyde (GLU) were used as spacer and bifunctional coupling agent, respectively. Quinol was used as substrate.

To know how the immobilisation procedures affected the enzyme reaction rate the catalytic behaviour of soluble and insoluble laccase was studied under isothermal conditions as a function of pH, temperature and substrate concentration. From these studies, two main singularities emerged from the experimental data: (i) the narrower pH-activity profile of the insoluble enzyme in comparison to that of the soluble counterpart; (ii) the increase of the affinity of the immobilised enzyme for its substrate.

The behaviour of the catalytic membrane was also studied in a non-isothermal bioreactor as a function of substrate concentration and size of the applied transmembrane temperature difference. It was found that, under non-isothermal conditions and keeping constant the average temperature of the bioreactor, the enzyme reaction rate linearly increases with the increase of the temperature difference. These results have been discussed in the frame of reference of the process of thermodialysis driving thermodiffusive transmembrane substrate fluxes, which add to the diffusive ones.

The advantages of the catalytic process carried out under non-isothermal conditions have been thrown in relief through the evaluation of the reduction of the production times and of the percentage increases of the enzyme activity.     

Effect of extracts obtained from olive oil mill waste waters on Bacillus megaterium ATCC 33085

n -Propanol was the most effective solvent for extracting antibacterial substances from olive oil mill waste water ('alpechines'). Several phenolics were detected in propanol extracts that had bactericidal effects on Bacillus megaterium ATCC 33085, inhibiting sporulation and germination at 5.6 mmol/l total phenolics (expressed as syringic acid). The biological effect was increased in the presence of high glucose and NaCl concentrations and after β-glucosidase hydrolysis.     

Selection of non-conventional yeasts and their use in immobilized form for the bioremediation of olive oil mill wastewaters

The yeast population dynamics in olive wastewaters (OMW), sampled in five mills from Salento (Apulia, Southern Italy), were investigated. Three hundred yeasts were isolated in five industrial mills and identified by molecular analysis. Strains belonging to Geotrichum, Saccharomyces, Pichia, Rhodotorula and Candida were detected. Five G. candidum strains were able to grow in OMW as the sole carbon source and to reduce phenolics, chemical oxygen demand (COD) and antimicrobial compounds. One G. candidum isolate was selected for whole-cell immobilization in calcium alginate gel. The COD and phenolic reduction obtained with immobilized cells showed a 2.2- and 2-fold increase compared to the removal obtained with free cells, respectively. The immobilization system enhanced yeast oxidative activity by avoiding the presence of microbial protease in treated OMW. To our knowledge, this is the first report on G. candidum whole-cell immobilization for OMW bioremediation.     

Production of laccase from Trametes versicolor by solid-state fermentation using olive leaves as a phenolic substrate

The aim of the present study was to investigate whether olive leaves were feasible as a substrate for laccase production by the white-rot fungus Trametes versicolor FPRL 28A INI under solid-state fermentation conditions. Different experiments were conducted to select the variables that allow obtaining high levels of laccase activity. In particular, the effects of the initial moisture content, substrate particle size, supplementation with inorganic and organic nitrogen sources were evaluated. Highest laccase activity (276.62 ± 25.67 U/g dry substrate) was achieved with 80 % initial moisture content and 1.4–1.6 mm particle size of the substrate supplemented with yeast extract (1 % (w/w) nitrogen). Such a high activity was obtained without any addition of inducers.     

Degradation of waste waters from olive oil mills by Yarrowia lipolytica ATCC 20255 and Pseudomonas putida

Waste waters from olive oil processing may cause severe pollution in the Mediterranean area, since they have a high level of chemical oxygen demand (COD) (100–200 g/l) and contain other organic and inorganic compounds. In all olive oil producing countries, the reduction of pollution in olive oil mill waste waters at reasonable costs and using techniques suitable for most industrial applications is an unsolved problem. For this paper, the yeast Yarrowia lipolytica ATCC 20255 was grown on waste waters from an olive oil mill in a 3.5 1 fermenter under batch culture conditions. The results showed that the yeast was capable of reducing the COD value by 80% in 24 h. In this way, a useful biomass of 22.45 g/l as single cell protein (SCP) and enzyme lipase were produced. During this process, most of the organic and inorganic substances were consumed, only aromatic pollutants were still present in the fermentation effluents. Therefore, we used a phenol degrader, namely Pseudomonas putida, to reduce phenolic compounds in the fermentation effuents after removing Yarrowia lipolytica cells. P. putida was effective in reducing phenols in only 12 h.     

Use of coagulants in treatment of olive oil wastewater model solutions by induced air flotation

Natural polyelectrolytes are suitable coagulants for the treatment of industrial and minicipal wastewaters because they are safe and have environmental benefits. Chitosan, a natural cationic polyelectrolyte, and other similar coagulants were used in the treatment of an olive oil water suspension as a model for the processing wastewater. The effect of chitosan, starch, alum and ferric chloride on the coagulation of oil droplets were determined by the jar test apparatus and turbidometric measurements. Olive oil emulsion samples were prepared by the use of surface active agents and other agents that could form stable oil water emulsions. The effect of parameters such as pH, ionic strength and optimum dosage of the coagulants were determined in the jar test experiments. Following the jar experiments, with the optimum concentration of the suitable coagulant, the emulsions were placed in an induced air flotation (IAF) cell to separate the coagulated oil droplets from solution. In the air flotation experiments, the effect of temperature, surfactant concentration and air flowrate were determined on the decrease of turbidity and COD of the emulsion samples. In the jar experiments, chitosan and alum used together at concentrations of 15 and 25 ppm, respectively, at pH 6 produced the lowest turbidity values. In the air flotation experiments, a concentration of 100 ppm of chitosan, an air flowrate of 3 l/min, aeration time of 45 s, temperature of 20 degrees C and pH 6 produced optimum levels. At optimum conditions of coagulation and flotation stages, the COD of the olive oil emulsion could be reduced by 95%.     

Engineered tobacco and microalgae secreting the fungal laccase POXA1b reduce phenol content in olive oil mill wastewater

Olive oil mill wastewaters (OMWs) are characterised by low pH and a high content of mono- and polyaromatic compounds that exert microbial and phytotoxic activity. The laccase cDNA of the poxA1b gene from Pleurotus ostreatus, carrying a signal peptide sequence for enzyme secretion and driven by the CaMV 35S promoter, was cloned into a plant expression vector. Nuclear genetic transformation was carried out by co-cultivation of Agrobacterium tumefaciens with tobacco cv Samsun NN leaves and cells of five different microalgae accessions belonging to the genera Chlamydomonas, Chlorella and Ankistrodesmus. Transgenic plants and microalgae were able to express and secrete the recombinant laccase in the root exudates and the culture medium, respectively. In comparison to untransformed controls, the ability to reduce phenol content in OMW solution was enhanced up to 2.8-fold in transgenic tobacco lines and by up to about 40% in two microalgae accessions. The present work provides new evidence for metabolic improvement of green organisms through the transgenic approach to remediation.     

Engineered tobacco and microalgae secreting the fungal laccase POXA1b reduce phenol content in olive oil mill wastewater

Olive oil mill wastewaters (OMWs) are characterised by low pH and a high content of mono- and polyaromatic compounds that exert microbial and phytotoxic activity. The laccase cDNA of the poxA1b gene from Pleurotus ostreatus, carrying a signal peptide sequence for enzyme secretion and driven by the CaMV 35S promoter, was cloned into a plant expression vector. Nuclear genetic transformation was carried out by co-cultivation of Agrobacterium tumefaciens with tobacco cv Samsun NN leaves and cells of five different microalgae accessions belonging to the genera Chlamydomonas, Chlorella and Ankistrodesmus. Transgenic plants and microalgae were able to express and secrete the recombinant laccase in the root exudates and the culture medium, respectively. In comparison to untransformed controls, the ability to reduce phenol content in OMW solution was enhanced up to 2.8-fold in transgenic tobacco lines and by up to about 40% in two microalgae accessions. The present work provides new evidence for metabolic improvement of green organisms through the transgenic approach to remediation.     

Purification and characterization of a novel laccase from the ascomycete Trichoderma atroviride: Application on bioremediation of phenolic compounds

The extracellular laccase produced by the ascomycete Trichoderma atroviride was purified and characterized and its ability to transform phenolic compounds was determined. The purified laccase had activity towards typical substrates of laccases including 2,2′-azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS), dimethoxyphenol (2,6-DMP), syringaldazine and hydroquinone. The enzyme was a monomeric protein with an apparent molecular mass of 80 kDa and an isoelectric point of 3.5. The pH optima for the oxidation of ABTS and 2,6-DMP were 3 and 5, respectively, and the optimum temperature was 50 °C with 2,6-DMP. The laccase was stable at slightly acidic pH (4 and 5). It retained 80% of its activity after 4 h incubation at 40 °C. Under standard assay conditions, K_m values of the enzyme were 2.5 and 1.6 mM towards ABTS and 2,6-DMP, respectively. This enzyme was able to oxidize aromatic compounds present in industrial and agricultural wastewater, as catechol and o-cresol, although the transformation of chlorinated phenols required the presence of ABTS as mediator.     

Production of 10-hydroxystearic acid from oleic acid and olive oil hydrolyzate by an oleate hydratase from Lysinibacillus fusiformis

A recombinant enzyme from Lysinibacillus fusiformis was expressed, purified, and identified as an oleate hydratase because the hydration activity of the enzyme was the highest for oleic acid (with a k (cat) of 850?min(-1) and a K (m) of 540?μM), followed by palmitoleic acid, γ-linolenic acid, linoleic acid, myristoleic acid, and α-linolenic acid. The optimal reaction conditions for the enzymatic production of 10-hydroxystearic acid were pH 6.5, 35?°C, 4% (v/v) ethanol, 2,500?U ml(-1) (8.3?mg?ml(-1)) of enzyme, and 40?g l(-1) oleic acid. Under these conditions, 40?g l(-1) (142?mM) oleic acid was converted into 40?g l(-1) (133?mM) 10-hydroxystearic acid for 150?min, with a molar yield of 94% and a productivity of 16?g l(-1)?h(-1), and olive oil hydrolyzate containing 40?g l(-1) oleic acid was converted into 40?g l(-1) 10-hydroxystearic acid for 300?min, with a productivity of 8?g l(-1)?h(-1).     

Enhanced bioremediation of oil spills in the sea

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The dissolution of calcite in acid waters: mass transport versus surface control

SUMMARY. 1. A new experimental method for the study of kinetics and mechanism of reactions at the solid-liquid interface has shown that the dissolution of calcite in acidic waters is, under conditions of high mass transport, controlled by the first order heterogeneous reaction of H⁺ at the interface and not by diffusion as previously thought.
2. The implications of this for lake liming strategies (aimed at countering the effects of 'acid rain') are significant in that under typical liming conditions, the rate of calcite dissolution will be surface controlled and consequently appreciably slower than previously considered.     

Purification and biochemical characterization of a new alkali-stable laccase from Trametes sp. isolated in Tunisia: role of the enzyme in olive mill waste water treatment

A white-rot basidiomycete, isolated from decayed acacia wood (from Northwest of Tunisia) and identified as Trametes sp, was selected in a broad plate screening because of its ability to decolorize and dephenolize olive oil mill wastewater (OMW) efficiently. The major laccase was purified and characterized as a monomeric protein with apparent molecular mass of 61 kDa (SDS-PAGE). It exhibits high enzyme activity over broad pH and temperature ranges with optimum activity at pH 4.0 and a temperature of 60 °C. The purified laccase is stable at alkaline pH values. The enzyme retained 50 % of its activity after 90 min of incubation at 55 °C. Using ABTS, this laccase presented K _m and V _max values of 0.05 mM and 212.73 μmoL min^?1 mg^?1, respectively. It has shown a degrading activity towards a variety of phenolic compounds. The purified laccase was partially inhibited by Fe²⁺, Zn²⁺, Cd²⁺ and Mn²⁺, while Cu²⁺ acted as inducer. EDTA (10 mM) and NaN₃ (10 mM) were found to completely inhibit its activity. 73 % OMW was dephenolized after 315 min incubation at 30 °C with 2 U mL^?1 of laccase and 2 mM HBT.     

Respiration profiles in monitoring the composting of by-products from the olive oil agro-industry

The composting of olive press cake (OPC) repeatedly mixed either with olive mill wastewater (OPC+OMW) or with tap water (OPC+W) was studied using the thermogradient respirometer, an apparatus that determines the respiration rates from a substrate over a wide range of different temperatures (respiratory profile). The composting processes took place over a period of five months during which nine moistenings of the OPC were performed with the respective liquids. The composting resulted in detoxification of the materials used in both treatments, as indicated by seed germination tests. However, the repeated applications of OMW resulted in recurring thermophilic phases (following each application) and in greater pH and conductivity increases in the final product, as compared to water applications. Respiration measurements performed at 35 degrees C were good indicators of the mean metabolic potential in the compost piles (the mean respiration derived from the whole respiration profile over a wide range of environmental temperatures). However, respiration measurements at higher temperatures (48.5 degrees C) were better indicators of the respiration activity occurring in situ. Following the initial thermophilic phase, the respiration potential of the composts at high temperatures (42-63 degrees C) increased drastically compared to their respiration potential at lower temperatures (17-42 degrees C) indicating the establishment of a thermophilic microflora. Subsequently, only the periodic new substrate-C applications in the form of OMW resulted in increased ratios of low temperature-to-high temperature respiration potential. These ratios decreased again following the respective thermophilic phase that each new OMW application had induced.