Biodegradation of olive oil mill wastewaterby Coriolus versicolor and Funalia trogii:effects of agitation,initial COD concentration,inoculum size and immobilization

The biodegradation of olive oil mill wastewater (OOMW) by Coriolus versicolor and Funalia trogii was investigated. Initial COD concentration, agitation and inoculum size were all found to be significant for biodegradation. Adding glucose, sulphate or nitrogen had no effect on biodegradation. During growth in optimum conditions, C.versicolor removed approximately 63% COD, 90% phenol and 65% colour within 6 days and F. trogii removed approximately 70% COD, 93% phenol and 81% colour of the OOMW used. The fungi also excreted large amounts of extracellular laccase into the medium. High biodegradation yields were also obtained by fungi immobilized in calcium alginate gels.     

Plant growth hormone production from olive oil mill and alcohol factory wastewaters by white rot fungi

In this study, olive oil mill and alcohol factory wastewaters have been tested as growth media for the production of plant growth hormones. Funalia trogii ATCC 200800 and Trametes versicolor ATCC 200801 have been tested. Gibberellic acid (GA₃), abscisic acid (ABA), indole acetic acid (IAA), and cytokinin were determined in the culture media of these fungi. Both organisms produced enhanced levels of all three hormones in the presence of either of the wastewaters.     

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.     

Removal of low molecular weight phenols from olive oil mill wastewater using microalgae

The treatment of olive oil mill wastewater (OMW) with two phenol resistant algae, Ankistrodesmus braunii and Scenedesmus quadricauda, showed a limited reduction of phenol content after 5 d of treatment, irrespective of algal concentration. Otherwise, cultures of both algae, grown in the dark, degraded over 50% of the low molecular weight phenols contained in OMW, but they were not completely removed, but were biotransformed into other non-identified, aromatic compounds.     

Transformation of 2,4,6-trichlorophenol by the white rot fungi Panus tigrinus and Coriolus versicolor

The toxicity of thirteen isomers of mono-, di-, tri- and pentachlorophenols was tested in potato-dextrose agar cultures of the white rot fungi Panus tigrinus and Coriolus versicolor. 2,4,6-Trichlorophenol (2,4,6-TCP) was chosen for further study of its toxicity and transformation in liquid cultures of these fungi. Two schemes of 2,4,6-TCP addition were tested to minimize its toxic effect to fungal cultures: stepwise addition from the moment of inoculation and single addition after five days of growth. In both cases the ligninolytic enzyme systems of both fungi were found to be responsible for 2,4,6-TCP transformation. 2,6-Dichloro-1,4-hydroquinol and 2,6-dichloro-1,4-benzoquinone were found as products of primary oxidation of 2,4,6-TCP by intact fungal cultures and purified ligninolytic enzymes, Mn-peroxidases and laccases of both fungi. However, primary attack of 2,4,6-TCP in P. tigrinus culture was conducted mainly by Mn-peroxidase, while in C. versicolor it was catalyzed predominantly by laccase, suggesting a different mode of regulation of these enzymes in the two fungi.     

Removal of molecular weight fractions of COD and phenolic compounds in an integrated treatment of olive oil mill effluents

Previous works (Beccari et al. 1999b; Beccari et al. 2001a; Beccari et al. 2001b)on the anaerobic treatment of olive oil mill effluents (OME) have shown: (a) apre-treatment based on the addition of Ca(OH)₂ and bentonite was able toremove lipids (i.e. the most inhibiting substances present in OME) almostquantitatively; (b) the mixture OME – Ca(OH)₂ – bentonite, fed to amethanogenic reactor without providing an intermediate phase separation,gave way to high biogas production even at very low dilution ratios; (c) theeffluent from the methanogenic reactor still contained significant concentrationsof residual phenolic compounds (i.e. the most biorecalcitrant substances present inOME). Consequently, this paper was aimed at evaluating the fate of the phenolicfractions with different molecular weights during the sequence of operations(adsorption on bentonite, methanogenic digestion, activated sludge post-treatment).The results show that a very high percentage (above 80%) of the phenolic fractionbelow 500 D is removed by the methanogenic process whereas the phenolic fractionsabove 1,000 D are significantly adsorbed on bentonite; the 8-day activated sludgepost-treatment allows an additional removal of about 40% of total filtered phenoliccompounds. The complete sequence of treatments was able to remove more than the96% of the phenolic fraction below 500 D (i.e. the most toxic fraction towards plantgermination). Preliminary respirometric tests show low level of inhibition exerted bythe effluent from the methanogenic reactor on aerobic activated sludges taken fromfull-scale municipal wastewater plants.     

Phenolic removal in olive oil mill wastewater using loofah-immobilized Phanerochaete chrysosporium

Summary Olive oil mill wastewater (OMW) has a high organic load, and this is a serious concern of the olive industry. Conventional biological wastewater treatments, despite their simplicity and suitable performance are ineffective for OMW treatment since phenolics possess antimicrobial activity. In order to carry out a proper treatment of OMW, use of a microorganism able to degrade the phenolics is thus necessary. In this study the ability of Phanerochaete chrysosporium to degrade the phenolic compounds of OMW and to decrease the chemical oxygen demand (COD) using cells immobilized on loofah was examined. The basal mineral salt solution along with glucose, ammonium sulfate and yeast extract was used to dilute the OMW appropriately. The fungus did not grow on the concentrated OMW. The extent of removal in this bio-treatment, of total phenols (TP) and the COD were 90 and 50%, respectively, while the color and aromaticity decreased by 60 and 95%, respectively. The kinetic behavior of the loofah-immobilized fungus was found to follow the Monod equation. The maximum growth rate μ_max was 0.045 h⁻¹ while the Monod constant based on the consumed TP and COD were (mg/l) 370 and 6900, respectively.     

Bioremediation of olive oil mill wastewater and production of microbial biomass

Olive oil mill wastewater (OMWW) was used as a substrate for the culture of a mixture of edible fungi in order to obtain a potentially useful microbial biomass and to induce a partial bioremediation of this fastidious waste. Before fermentation, the OMWW underwent an alkaline-oxidative treatment with the aim of decreasing the polyphenolic content which is the main cause of its toxicity. The fungal mixture grew fairly well in the treated OMWW and reached a maximum of biomass production within about 14 days of fermentation at room temperature. Up to 150–160 g of wet biomass was obtained per liter of OMWW. Analysis of the partially dehydrated biomass revealed a protein content of about 13 g% and 6 g% of row fiber. A relevant presence of unsaturated fatty acids was found, as well as the presence of significant amounts of vitamins A and E, nicotinic acid, calcium, potassium and iron. The possibility of using the microbial biomass produced from OMWW as an additive to animal feed is discussed.     

Codigestion of olive oil mill wastewaters with manure, household waste or sewage sludge

Combined anaerobic digestion of oil mill effluent(OME) together with manure, household waste (HHW) orsewage sludge was investigated. In batch experimentsit was shown that OME could be degraded into biogaswhen codigested with manure. In codigestion with HHWor sewage sludge, OME dilution with water (1:5) wasrequired in order to degrade it. Using continuouslystirred lab-scale reactors it was shown thatcodigestion of OME with manure (50:50 and 75:25 OMEto manure ratios) was successful with a theoreticalOME utilization of 75% and with approx. 87%reduction of the lipids content in OME. An OMEutilization of approx. 55%, and lipid reduction of73% was reached in codigestion with HHW (50:50 and75:25 OME to HHW ratios). The results showed thatthe high buffering capacity contained in manure,together with the content of several essentialnutrients, make it possible to degrade OME withoutprevious dilution, without addition of externalalkalinity and without addition of external nitrogensource.     

DNA protecting and genotoxic effects of olive oil related components in cells exposed to hydrogen peroxide

In search for compounds, able to protect nuclear DNA in cells exposed to oxidative stress, extracts from olive leaves, olive fruits, olive oil and olive mill waste water were tested by using the “single cell gel electrophoresis” methodology (comet assay). Jurkat cells in culture were exposed to continuously generated hydrogen peroxide (11.8±1.5 μM per min) by direct addition into the growth medium of the appropriate amount of the enzyme “glucose oxidase” in the presence or absence of the tested total extracts. The protective effects of the tested extracts or isolated compounds were evaluated from their ability to decrease hydrogen peroxide-induced formation of single strand breaks in the nuclear DNA, while the toxic effects were estimated from the increase of DNA damage when the extracts or isolated compounds were incubated directly with the cells. Significant protection was observed in extracts from olive oil and olive mill waste water. However, above a concentration of 100 μg/ml olive oil extracts exerted DNA damaging effects by themselves in the absence of any H₂O₂. Extracts from olive leaves and olive fruits although protective, were also able to induce DNA damage by themselves. Main compounds isolated from the above described total extracts, like oleuropein glucoside, tyrosol, hydroxytyrosol and caffeic acid, were tested in the same experimental system and found to exert cytotoxic (oleuropein glucoside), no effect (tyrosol) or protective effects (hydroxytyrosol and caffeic acid). In conclusion, cytoprotective as well as cytotoxic compounds with potential pharmaceutical properties were detected in extracts from olive oil related sources by using the comet assay methodology.     

Effect of continuous olive mill wastewater applications, in the presence and absence of nitrogen fertilization, on the structure of rhizosphere–soil fungal communities

Olive mill wastewater (OMW) is rich in potentially toxic organics precluding its disposal into water receptors. However, land application of diluted OMW may result in safe disposal and fertilization. In order to investigate the effects of OMW on the structure of soil fungal groups, OMW was applied daily to pepper plants growing in a loamy sand and a sandy loam at two doses for a period of 3 months (total OMW equivalents 900 and 1800 m³ ha⁻¹). Nitrogen (N) fertilization alleviated N scarcity and considerably enhanced plant biomass production; however, when applied in combination with the high OMW dose, it induced plant stress. OMW applications resulted in marked changes in the denaturing gradient gel electrophoresis patterns of soil basidiomycete communities, while concurrent N fertilization reduced these effects. In contrast, the ascomycete communities required N fertilization to respond to OMW addition. Cloning libraries for the basidiomycete communities showed that Cryptococcus yeasts and Ceratobasidium spp. dominated in the samples treated with OMW. In contrast, certain plant pathogenic basidiomycetes such as Thanatephorus cucumeris and Athelia rolfsii were suppressed. The observed changes may be reasonably explained by the capacity of OMW to enrich soils in organic substrates, to induce N immobilization and to directly introduce OMW-derived basidiomycetous yeasts.     

Isolation of Enterobacteria able to degrade simple aromatic compounds from the wastewater from olive oil extraction

Summary Enterobacteria growing on wastewater from olive oil extraction were selected. Among this microflora, strains of Klebsiella oxytoca and Citrobacter diversus able to degrade simple monomeric aromatic compounds were isolated by enrichment culture of the effluent lacking simple sugars. In this preliminary investigation, the phenolic acids tested on solid and liquid media were gentisic, protocatechuic, p-hydroxybenzoic, benzoic, vanillic and ferulic. It was shown that the biodegradation of an aromatic acid is tightly dependent on both the type and the position of the radical substituted on the aromatic ring. Citrobacter was the most efficient strain in metabolizing ferulic acid in liquid medium at a concentration of 1.5 g/l. The substrate biodegradation yield achieved exceeded 86%.     

Reduction of phenolics content and COD in olive oil mill wastewaters by indigenous yeasts and fungi

Olive oil mill wastewaters (OOMW) cause a recurrent environmental pollution problem. The large concentration of phenolic compounds in the organic fraction of OOMW is principally responsible for the phytotoxicity and microbial growth inhibitory effects of the effluent. Candida boidinii, Geotrichum candidum, a Penicillium sp. and Aspergillus niger HA37 were isolated from OOMW. When cultivated directly on an undiluted OOMW-based medium containing 82 g l⁻¹ COD, these strains removed only 4–8% of chemical oxygen demand (COD) and phenolics. In contrast, reduction values attaining respectively 40–73% for phenolics and 45–78% for COD removal in the undiluted OOMW-based medium were obtained when using the strains gradually acclimated to high concentration of OOMW by successive stepwise transfer from media containing COD of 20.5 up to 82 g l⁻¹. Possibly, a sufficient production level of degradation and/or detoxification enzymes has to be attained to overcome the toxic effects of the phenolic fraction of concentrated OOMW. The present investigation calls attention to the necessity of acclimation for certain fungal and yeasts strains potentially useful for treating highly polluted effluents.     

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.     

Effects of agronomic application of olive mill wastewater in a field of olive trees on carbohydrate profiles, chlorophyll a fluorescence and mineral nutrient content

Olive mill wastewater (OMW) management is a serious environmental issue for the Mediterranean area where there is the most production of olive oil. OMW contains a high organic load, substantial amounts of plant nutrients but also several compounds with recognized toxicity towards living organisms. Moreover, OMW may represent a low cost source of water. We studied the influence of irrigation with OMW (amounts applied: 30, 60, 100 and 150 m³ h⁻¹) in a field of olive trees on root colonization, photosynthesis, chlorophyll fluorescence, leaf nutrient concentration and soluble carbohydrate. The soil fatty acid methyl ester (FAME) 16:1ω5 was used to quantify biomass of arbuscular mycorrhizal (AM) fungi and the root FAME 16:1ω5 analysis was used as index for the development of colonization in the roots. Agronomic application of OMW decreased significantly the abundance of the soil FAME 16:1ω5 and the root FAME 16:1ω5 in the soil amended with 60, 100 and 150 m³ ha⁻¹ OMW. Decreased root FAME 16:1ω5 due to OMW amendment was associated with a significant reduction of tissue nutrient concentrations in the olive trees. The highest application of OMW to the soil reduced significantly the olive trees uptake of N, P, K, Ca, Mg, Fe, Cu, Mn and Zn. Land spreading of OMW increased concentration of soluble carbohydrate in the olive leaves, mostly due to decreased sink demand for carbon by the root. In the olive trees amended with 150 m³ ha⁻¹ OMW, net CO₂ uptake rate (A), quantum yield of photosystem II electron transport (Φ_PSII), maximal photochemical efficiency of photosystem II (Fv/Fm), photochemical quenching (q_p) and the electron transport rate (ETR) were significantly depressed, whereas non-photochemical quenching (NPQ) was found to increase. Taken with data from experiments in field conditions, our results suggest that agronomic application of OMW alters the functioning of arbuscular mycorrhizas and can even disrupt the relationship between AM fungi and olive trees.