Biodegradation of different molecular-mass polyphenols derived from olive mill wastewaters by Geotrichum candidum

The decolourisation of fresh and stored olive mill wastewaters (OMW) and the biodegradation of three groups (F1, F2 and F3) of phenolic compounds by Geotrichum candidum were investigated. Separated phenolic compounds derived from natural OMW ultrafiltration using membranes with a cutoff 2and 100 kDa. G. candidum growth on fresh OMW decreased pH and reduced COD and colour of 75% and 65%, respectively. However, on the stored-black OMW a failure of COD and colour removal were observed. G. candidum activity on this later substrate was enhanced by the addition of a carbon source easily metabolised, misleading an improvement of the COD reduction and decolourization that reached 58% and 48%, respectively. Growth of G. candidum in the presence of F2 or F3 polyphenolic fractions induced high decolourisation and depolymerisation of phenolic compounds. Whereas, very week decolourisation and biodegradation were observed with F1 fraction. Moreover, the highest levels of lignin peroxidase (LiP) and manganese peroxidase (MnP) activities were obtained in the presence of F2 fraction. These results showed that increasing of molecular-mass of aromatics led to an increase in levels of depolymerisation, decolourisation and COD removal by G. candidum culture.     

Influence of salts and phenolic compounds on olive mill wastewater detoxification using superabsorbent polymers

For a selection of nine commercially available superabsorbent polymers, the absorption capacity was evaluated for the principal absorption-inhibition constituent of OMW, mineral salts and for phytotoxic-components, the phenolic compounds. A double exponential model was established for electrical conductivities ranging 4.2-25,000 microS cm(-1). For solutions of phenolic compounds ranging 0-0.5 g l(-1), a distribution coefficient near unit was achieved, while for OMW, the phenolic compounds were concentrated inside the gel as the distribution coefficient was 1.4. Correction of OMW pH towards neutrality was found to increase the absorption capacity by up to 35%. The phytotoxicity was assessed by the germination of Lepidium sativum. Inhibition in plant growth occurred for all OMW dilutions without superabsorbent polymers application. For 5% of OMW (COD 5 gl(-1) and 200 ppm of phenolic compounds) immobilised in PNa2 (1 gl(-1)), plant growth was promoted being observed a 120% growth germination, thus indicating that olive mill wastewater detoxification occurred.     

Scale down and optimization of olive mill wastewaters decolorization by Geotrichum candidum

Microbiol flora acclimated in continuous pilot scale bubble column fed with OMW was analysed. The most efficient isolated fungus was identified to white-rot fungus Geotrichum candidum. Decolorization of OMW by Geotrichum candidum was investigated by using Hadamard's matrix for screening the important parameters and optimize them in order to control the biological decolorization. Agitation favours the conversion of COD removed into Geotrichum candidum biomass especially with high arthoconodia and few mycelium. Dilution of OMW and aeration enhanced the mycelium growth and rammification which that allowed polyphenols hydrolysis and then a decolorization. The initial pH of OMW is suitable for its decolorization by Geotrichum candidum growth. Ammonium sulfate concentrations tested with different OMW dilutions showed that the COD:N:S ratio of 100:5:2 is suitable for higher black colour removal. With optimized conditions Geotrichum candidum growth on OMW in laboratory scale bubble column, the OD removal reached 70% and all fractions of polyphenolic compounds of OMW were oxidized.     

Involvement of lignin peroxidase in the decolourization of black olive mill wastewaters by Geotrichum candidum

AIM: Decolourization of black olive mill wastewaters (OMW) by depolymerization of phenolic compounds by Geotrichum candidum. METHODS AND RESULTS: Our results show that G. candidum is able to grow on black OMW supplemented with carbon source and nitrogen. The Geotrichum growth decreased the pH and induced a 49% of colour removal when the black OMW was supplemented with glycerol and diammonium tartrate (20 mm ammonium). An improvement of 10% of colour removal was observed when the culture was supplemented with veratryl alcohol. The decolourization was inhibited with glutamate as nitrogen source. CONCLUSION: Our results suggest the potential use of G. candidum in black OMW decolourization and support the concept that lignin peroxidase (LiP) of G. candidum is involved in the depolymerization of phenolic compounds. Significance and Impact of the Study: This is the first report of LiP production by G. candidum on OMW.     

Controversial role of fungal laccases in decreasing the antibacterial effect of olive mill waste-waters

Antibacterial diffusion tests (against Bacillus megaterium) detected both bacterial growth-promoting and growth-inhibiting components in olive mill waste-water (OMW). Mixtures of OMW aromatic components showed antibacterial effects that did not show antibacterial activity when tested as individual compounds. Strains of white rot fungi (WRF) producing different patterns of lignin modifying enzymes (LMEs) have been evaluated for OMW remediation under nutritional conditions affecting the LMEs produced. The decrease of both the content in OMW phenols and in the OMW antibacterial activity was compared with fungal growth and LMEs production. OMW addition to the cultures increased fungal growth and laccase activity irrespectively of the nutritional conditions of the cultures. Laccase was the sole LME activity that increased after OMW addition to the cultures. Neither the increased growth of WRF in OMW-containing cultures, their content in laccase nor the amount of OMW phenols were direct indications of a greater decrease in OMW antibacterial effect. The higher decrease in OMW antibacterial activity was obtained in cultures of Phanerochaete flavido-alba in an N-rich media.     

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.     

Integrated biological process for olive mill wastewater treatment

The biological process for OMW treatment is based on an aerobic detoxification step followed by methanization step and aerobic post-treatment.The first aerobic detoxification step of OMW supplemented with sulfate and ammonium was carried out by the growth of Aspergillus niger in a bubble column. This step decreased OMW toxicity and increased its biodegradability because of phenolic compounds degradation. Growth of A. niger resulted in 58% COD removal, with production of biomass containing 30% proteins (w/w). Filtration of OMW was enhanced by this fermentation because the suspended solids were trapped in the mycelium. The filtrate liquid was then methanized using an anaerobic filter packed with flocoor. This reactor showed a short start up and a good stability. COD removal was around 60% and the methane yield (1 CH₄/g COD removed) was close to the theoretical yield.The anaerobic filter effluent was treated in an activated sludge fluidized reactor containing olive husk as a packing material. Husks were maintained in fluidization state by the aeration. This step induces COD removal at 45% and sludge (up to 2 g/dm³).The entire process allowed a global COD reduction up to 90%; however, the black colour due to polyphenolic compounds with high molecular weight persisted.     

Ligninolytic enzymes activities of Oyster mushrooms cultivated on OMW (olive mill waste) supplemented media, spawn and substrates

Ligninolytic enzymes activities (laccases, peroxidases (total, MnP and MiP) and aryl-alcohol oxidase (AAO)) were measured during the cultivation of six commercial Pleurotus sp. strains on MMP media, on cereal grains (spawn) and on straw substrates (the three commonly utilized cultivation steps to obtain fruiting bodies) supplemented with several concentrations of autoclaved (OMW) or gamma-irradiated (iOMW) olive mill waste. Results indicated that all the strains were able to grow on MMP media and spawn containing up to 30% OMW and iOMW and on straw substrates mixed with 50% OMW. None of the strains showed AAO activity and there was not a single strain which showed the highest laccases and peroxidases activities, independently of the utilized substrate. Pleurotus mycelia adjusted their enzymatic mechanisms depending on their variety, type of substrate, concentration of OMW or iOMW added. OMW was a better supplement to use than iOMW because OMW induced higher exo-enzymes activities.     

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.     

Improvement of fermentative decolorization of olive mill wastewater by Lactobacillus paracasei by cheese whey's addition

The effect of the cheese whey's (CW) addition on the fermentative decolorization of olive mill wastewater (OMW) by Lactobacillus paracasei, with and without pH adjustment by lime, was investigated. Mixtures OMW/CW at different proportions were fermented. The highest colour removal (47%) and total phenolic reduction (22.7%) of OMW were obtained after cofermentation of OMW/CW at proportions of 10/90, respectively. The decrease of pH after cofermentation of the two wastewaters, induced the precipitation of whey proteins with phenolic compounds and, so, improves decolorization. These removal yields reached 64% and 34%, respectively after precipitation by adjustment of pH at 7 with lime at the end of cofermentation. These improvements were correlated to a clarification of wastewaters by precipitation of whey proteins with phenolic compounds. An enhanced decolorization (up to 93%) and a total phenolic reduction (50%) of the mixture were obtained when cofermentation sequentially pH corrected by lime addition was investigated.     

Lipase production by Aspergillus ibericus using olive mill wastewater

Olive mill wastewater (OMW) characteristics make it a suitable resource to be used as a microbial culture media to produce value-added compounds, such as enzymes. In this work, the ability of the novel species Aspergillus ibericus to discolor OMW and produce lipase was studied. An initial screening on plates containing an OMW-based agar medium and an emulsified olive oil/rhodamine-B agar medium was employed to select the strain A. ibericus MUM 03.49. Then, experiments in conical flasks with liquid OMW-based media showed that the fungus could growth on undiluted OMW, with a chemical oxygen demand (COD) of 97 ± 2 g/L, and to produce up to 2,927 ± 54 U/L of lipase. When pure OMW was used in the media, the maximum COD and color reduction achieved were 45 and 97 %, respectively. When OMW diluted to 10 % was used, A. ibericus was able to reduce phenolic and aromatic compounds by 37 and 39 %, respectively. Additionally, lipase production was found to be promoted by the addition of mineral nutrients. When the fermentations were scaled up to a 2-L bioreactor, A. ibericus produced up to 8,319 ± 33 U/L of lipase, and the maximum COD and color reduction were 57 and 24 %, respectively.     

Decolourisation and dephenolisation potential of selected <Emphasis Type="Italic">Aspergillus</Emphasis> section <Emphasis Type="Italic">Nigri</Emphasis> strains – <Emphasis Type="Italic">Aspergillus tubingensis</Emphasis> in olive mill wastewater

Aspergillus section Nigri strains Aspergillus aculeatus Ege-K 258, A. foeditus var. pallidus Ege-K156, A. niger Ege-K 4 and A. tubingensis Ege-K 265 were used to treat olive mill wastewater (OMW) in an investigation aimed at exploring their dephenolisation and decolourisation ability and, consequently, the economic feasibility of using any or all of these strains in a pre-treatment step in the processing of OMW. Of these strains A. tubingensis Ege-K 265 resulted in an 80% decolourisation of twofold-diluted OMW and a 30% decolourisation of undiluted OMW; in addition, it was able to remove approximately 30% of all phenolic compounds in both twofold-diluted and undiluted OMW. We conclude that A. tubingensis Ege-K 265 could be effectively used in the pre-treatment step of a combined aerobic-anaerobic process to solve the environmental problems caused by OMW in Mediterranean countries.     

Decolourization of olive mill waste-waters by the white-rot fungus Phanerochaete chrysosporium: involvement of the lignin-degrading system

Summary The decolourization of olive mill waste-waters (OMW) by Phanerochaete chrysosporium was investigated. OMW decolourization occurred during the primary phase of growth when glycerol was used as the carbon source, and during secondary metabolism in nitrogen-limited cultures. The decolourization was found to be extensive (74% of colour removal, 80% of chemical oxygen demand removal) when the cultures were supplement d with veratryl alcohol and flushed with O₂. The biodegradation system was repressed with glutamate as a nitrogen source. These results suggest that all or part of the lignin-degrading system of P. chrysosporium played a role in biodegradation of OMW. The decolourization of OMW corresponds to depolymerization of high-molecular-mass aromatics combined with mineralization of a wide range of monoaromatic compounds. Correspondence to: S. Sayadi     

Olive mill wastewater stabilization in open-air ponds: impact on clay-sandy soil

The aim of this work was to study the natural biodegradation of the stored olive mill wastewater (OMW) in ponds and the infiltration as well as the impact on soil of the effluent in the evaporation pond used for the storage over the past eight years. For this, two approaches were considered. First, a laboratory-scale column was used for the infiltration of OMW through soil (clay and sand) to predict the effect of the clayey soil in reducing OMW pollution. Second, the ponds including the effluent annually stored and having this clayey structure were investigated. At the laboratory-scale, a modification of OMW contents was noticed, with the elimination of 95% of total suspended solids (TSS), 60% of chemical oxygen demand (COD), 40% of total organic carbon (TOC), 50% of total P, 50% of phenols and 40% of minerals (K+, Mg++ and Na+). The experimented soil was able to restrain the considerable effects of OMW pollution. In the ponds, the granulometric characteristics, the physico-chemical and the biological parameters of the soil profile from the contaminated pond were compared to those of a control soil, located near the contaminated pond. Property modifications of the contaminated soil were noted, especially pH, electrical conductivity, COD and microflora. These changes can be explained by the infiltration of OMW constituents, which were noticed in the soil layers, especially phenolic compounds that have a negative effect on the ground water.     

Biodegradation of polyphenols with immobilized Candida tropicalis under metabolic induction

During olive oil production, large quantities of olive mill wastewater (OMW) are produced. This wastewater material, containing a high level of phenolic compounds, poses a serious environmental problem in almost all Mediterranean countries. Candida tropicalis YMEC14 was used as an extremophile strain to design an aerobic biotreatment process to detoxify OMW and reduce its polluting organic load. The process was enhanced by directing yeast metabolism towards biodegradation pathways using hexadecane as co-metabolite and by immobilizing yeast cells in calcium alginate beads. Under immobilization conditions, C. tropicalis YMEC14 grown at 40 degrees C in OMW supplemented with hexadecane resulted in 69.7%, 69.2% and 55.3% reduction of chemical oxygen demand, monophenols and polyphenols, respectively, after a 24-h fermentation cycle.     

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.     

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.     

The effect of pretreatment on anaerobic activity of olive mill wastewater using batch and continuous systems

This study was focused on several physico-chemical and biological treatment methods that may affect the reduction of the organic load in olive mill wastewater (OMW). In this study, removal of 95% of the phenolic compounds present in OMW was achieved using sand filtration and subsequent treatment with powdered activated carbon in a batch system. This pretreatment for OMW was found to enhance the anaerobic activity of the sludge in the batch system significantly. The efficiency of organic load removal achieved by the anaerobic treatment of untreated OMW in batch reactors with tap water dilution factors below 1:10, reached approximately 65% chemical oxygen demand (COD) removal. However, in the up-flow sludge anaerobic blanket (UASB) reactor, COD removal efficiency of 80–85% was reached at a hydraulic retention time (HRT) of 5 days with an influent COD concentration of 40 g l⁻¹ and organic loading rate (OLR)=8 g⁻¹ COD l⁻¹ per day.     

Olive oil mill waste waters decoloration and detoxification in a bioreactor by the white rot fungus Phanerochaete flavido-alba

Olive oil mill wastewater (OMW) is produced as waste in olive oil extraction. With the purpose of treating this highly polluting waste, a number of experiments were conducted in a laboratory-scale bioreactor with the white rot fungus Phanerochaete flavido-alba (P. flavido-alba). It is known that this fungus is capable of decolorizing OMW in static or semistatic cultures at Erlenmeyer scale and at 30 degrees C. The objective of this work was to prove that P. flavido-alba could decolorize OMW in submerged cultures and that it is capable of reducing OMW toxicity at room temperature (25 degrees C) and in a laboratory-scale bioreactor. In the experiments conducted, manganese peroxidase (MnP) and laccase enzymes were detected; however, unlike other studies, lignin peroxidase was not found to be present. Decoloration obtained after treatment was 70%. The reduction of aromatic compounds obtained was 51%, and the toxicity of the culture medium was reduced by up to 70%. We can therefore state that P. flavido-alba is capable of reducing important environmental parameters of industrial effluents and that prospects are positive for the use of this process at a larger scale, even when working at room temperature.     

Use of <Emphasis Type="Italic">Pichia fermentans</Emphasis> and <Emphasis Type="Italic">Candida</Emphasis> sp. strains for the biological treatment of stored olive mill wastewater

Of 105 isolates screened for growth on plates containing olive mill wastewater (OMW), five were selected and identified as Pichia fermentans (Y1, Y4) and Candida sp. (Y2, Y11, and Y18). On the basis of their ability to use phenol at 716 mg l⁻¹, strains Y2 (15% reduction) and Y4 (18% reduction) were then used to detoxify stored OMW under various operational conditions. Yeast treatment of OMW increased the pH and, in the best conditions (aeration and no glucose addition), the COD decreased (47%) and phytotoxicity was also decreased (56%) probably due to the changes in the composition of phenolic compounds.