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.     

Detoxification of olive mill wastewaters by Moroccan yeast isolates

A total of 105 yeast strains were isolated from Moroccan olive oil production plants and evaluated for their ability to grow in olive oil mill wastewaters (OMW). The 9 isolates that grew best on OMW were selected for further study to evaluate their effect on removal of organic pollutants and OMW phytotoxicity (barley seed germination test). The results showed that at least four yeast isolates effectively lowered the toxicity of this effluent in addition to providing very useful materials in terms of both yeast biomass (6 g/l DW) and an irrigation fluid. This group of yeast isolates significantly reduced the concentration of total phenols (44% removal) and Chemical Oxygen Demand, COD (63% removal). The best germination rate of 80% for undiluted OMW was obtained for strain Candida holstii that also increased the pH from 4.76 to 6.75. Principal component analysis of the results obtained for the best yeast strains confirmed the importance of COD and total phenol reduction along with increase of organic nitrogen and final pH for the improvement of germination rates and phytotoxic reduction. This study has highlighted the potential of indigenous yeasts in detoxification of olive mill wastewaters.     

Characterisation of Paenibacillus jamilae strains that produce exopolysaccharide during growth on and detoxification of olive mill wastewaters

A total of 10 bacterial strains were isolated from a compost of corn treated with olive mill wastewaters (OMW) and selected by their capacity to synthesize exopolysaccharides (EPS). Morphological, physiological, biochemical and nutritional tests were used for a phenotypic study. A numerical analysis showed that all strains were 90% similar to each other. A DNA–DNA hybridization assay confirmed that all the strains belonged to Paenibacillus jamilae species. All the characterized strains were able to produce EPS growing on OMW batch cultures. The strain which was able to produce the highest EPS yield was chosen to perform an assay for testing its putative detoxifying activity, and it showed to reduce more than half the toxic capacity of the OMW. The results presented in this study, indicated the possible perspectives for using these bacterial strains to produce EPS and contribute to the bioremediation of the waste waters that are produced in the olive oil elaboration process.     

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 mill wastes: Biochemical characterizations and valorization strategies

The olive mill waste generated from olive oil extraction is a major environmental issue, particularly in Mediterranean areas. The extraction of olive oil is achieved through discontinuous or continuous processes. The two processes yield three fractions: a solid residue and two liquid phases (oil and olive mill wastewater). The characterization of these two by-products showed that they are mainly composed of phenolic compounds, carbohydrates, organic acids and mineral nutrients variably distributed depending on the process employed and the agronomic practices. Untreated olive by-products discharged between November and March into the environment are a major ecological issue for olive oil-producing countries due to their high toxic organic loads, low pH, and high chemical and biological demands. In this context, recent research studies highlight on the treatment approaches and valorization options for dealing with olive mill waste residues, predominantly those allowing for the recovery of valuable natural components such as phenolic compounds, dietary fibers, animal feed, biofuel, biogaz, enzymes, polymers and other. The impact of the chemical heterogeneity and water content of olive mill by-products about these processes of valorization and bioconversion is discussed.     

Determination of aerobic biodegradation kinetics of olive oil mill wastewater

Aerobic biological treatment was conducted for the treatment of high strength olive oil mill wastewater (OMW). Two different approaches were used for kinetic modeling of OMW biodegradation. TOC removal and CO₂–C evolution were monitored in an open and a closed bioreactor systems, respectively. Gompertz, Refractory organics plus first-order (RFO) and Chen–Hashimoto equations were applied to estimate the kinetic parameters by using the data from bioreactors. Furthermore, change in oxidation stage of carbon was monitored and temperature dependency of OMW biodegradation was investigated based on activation energy. At room temperature, 64% of TOC was removed in the open bioreactor while cumulative CO₂–C evolution was 6.32 g L⁻¹ in closed the bioreactor. Higher biodegradation efficiency and kinetic parameters were obtained at 25 °C rather than 10 °C. Gompertz and RFO equations provided better fitting with CO₂–C and TOC data, respectively. Experimental and kinetic estimations indicated that OMW constituted of approximately 30% refractory organics. The comparison of two different modeling approaches showed that kinetic modeling based on CO₂–C provided better correlation with the experimental data. Temperature coefficient indicated that biological degradation of OMW is slightly dependent on temperature.     

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.     

The effect of olive mill wastewaters variability on xanthan production

AIMS: Xanthan production by Xanthomonas campestris from several olive mill wastewaters (OMW) was investigated. METHODS AND RESULTS: Maximum xanthan production of 4 g l(-1) was obtained in media with 50% OMW as sole source of nutrients. OMW storage decreased effluent quality for xanthan production. The range of effluent concentration for X. campestris growth and xanthan production varied depending on OMW extraction METHOD: Wastewaters from press and two-phase extraction methods required higher dilution rates (< 10%) than those from the three-phase extraction method (50%). Nitrogen supplementation improved xanthan production in press and two-phase OMW. CONCLUSION: Factors affecting wastewaters composition, namely, waste storage, time of olive harvesting, and method for oil extraction, were found to influence xanthan production in shake-flask cultures. SIGNIFICANCE AND IMPACT OF THE STUDY: Conditions for xanthan production from OMW should be optimized in accordance with the nature of the waste material.     

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.     

Biological treatment of olive mill wastewater by non-conventional yeasts

The ability of lipolytic yeasts to grow on olive mill wastewater (OMW)-based medium and to produce high-value compounds while degrading this waste, was tested. OMW collected from three-phase olive mills from the North region of Portugal were characterized and used. OMW with COD ranging from 100 g L⁻¹ to 200 g L⁻¹ were supplemented with yeast extract and ammonium chloride. Studies of OMW consumption were carried out in batch cultures of Candida rugosa, Candida cylindracea and Yarrowia lipolytica. All strains were able to grow in the OMW-based media, without dilution, to consume reducing sugars and to reduce COD. C. cylindracea was the best strain concerning the lipase production and the reduction of phenolic compounds and COD. For all strains, the phenols degradation was quite difficult, mostly when more easily degradable carbon source is still present in the medium. Among the phenolic compounds tested catechol is the most inhibitory to the cells.     

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.     

Olive-mill wastewaters: a promising substrate for microbial lipase production

The present study investigated the valorization of olive-mill wastewater (OMW) by its use as a possible growth medium for the microbial production of extra-cellular lipase. To this end, strains of Geotrichum candidum (NRRL Y-552 and Y-553), Rhizopus arrhizus (NRRL 2286 and ISRIM 383), Rhizopus oryzae (NRRL 6431), Aspergillus oryzae (NRRL 1988 and 495), Aspergillus niger (NRRL 334), Candida cylindracea (NRRL Y-17506) and Penicillium citrinum (NRRL 1841 and 3754, ISRIM 118) were screened. All strains were able to grow on the undiluted OMW, producing extra-cellular lipase activity. C. cylindracea NRRL Y-17506 showed the highest lipase activity on all the typologies of OMW used. Its lipase production on OMW was markedly affected by the type of nitrogen source and was induced by the addition of olive oil. The highest activity (9.23 IU ml(-1)) of the yeast was obtained on OMW supplemented with NH(4)Cl (2.4 g l(-1)) and olive oil (3.0 g l(-1)).     

Xanthan production from olive-mill wastewaters

Olive mill wastewaters (OMW) are a by-product from olive oil manufacture that cause environmental pollution. These wastes have been used as substrate for the production of the extracellular polysaccharide xanthan by Xanthomonas campestris NRRL B1459-S4L41. Growth and xanthan production on dilute OMW as a sole source of nutrients were obtained at OMW concentrations below 60%, yielding a maximal xanthan production of 4.4gl⁻¹ at 30–40% OMW concentration. Addition of nitrogen and/or salts led to significantly increased xanthan yields with a maximum of 7.7gl⁻¹. The N/salts supplements also allowed an increase in the optimal OMW concentration. Inocula pre-grown on OMW can be used. Results suggest that an improved xanthan yield could be obtained with adequate balance between waste concentration and nitrogen or salt supplementation. OMW is proposed as a low-cost substrate for xanthan production with the additional environmental benefit of this use.     

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.     

Microbiological and physicochemical characterization of olive mill wastewaters from a continuous olive mill in Northeastern Portugal

The microbiological and physicochemical characterization of samples from the different wastewaters generated during oil extraction in a continuous olive mill was performed. The main aim was to determine which of the physicochemical parameters were the best fitted to correctly characterize these residual waters. High correlations were obtained for COD, DOC, K, P and N contents with the sampling points, allowing the distinction of olive washing waters (OWW) from olive centrifuge waters (OCW) and olive mill wastewaters (OMW). These parameters were sufficient for a rapid and less costly chemical characterization of these waters. Phenols and oil and grease contents, together with low pH and dissolved oxygen contents, and high organic loads, were the most toxic for microbial populations. Microbial characterization showed that fungi were well adapted to these stressing environmental characteristics and the reuse of OMW after aerobic treatment with microbial species isolated from the effluent is considered.     

Olive-derived biomass as a renewable source of value-added products

Olive oil is a characteristic product of Mediterranean countries, and its production is rapidly expanding to many other regions due to the health benefits attributed to the consumption of high-quality olive oil. Taking into account that the oil content is, on average, only 20% by weight of olives, a great amount of biomass, including leaves, pits, and pomace, is produced in the mill along with the main product. Moreover, the pruning of olive trees and the production of the olive pomace also generates a great amount of biomass. Currently, the usual practices for disposal of these different biomasses do not take advantage of the wide range of products that can be produced from them. This work summarizes the most relevant practices considering both the bioprocesses studied for the exploitation of the olive-derived biomass and the potential products obtained. The integration of processes in a single facility, i.e., the concept of a biorefinery based on olive-derived biomass, is also reviewed, including technoeconomic and environmental assessment.     

Novel static composting method for bioremediation of olive mill waste

This paper presents results obtained on the evaluation of static composting process aimed at bioremediation of the hazardous solid olive mill waste (OMW). The static composting process carried out in gas-permeable polyethylene bags followed the fluctuating temperature and oxygen profiles similar to those seen in aerated composting systems. Static composting resulted in apparent increases and decreases in values for total nitrogen and C:N ratios respectively during the process. The amount of nitrogen (>3%) in the composting end product was in agreement with the Italian legislation (Decreto Legislativo 29 aprile 2010, n. 75) specification for nitrogen fertilizer. A gradual decrease in polyphenols during the storage of compost resulted in a non-phytotoxic composted organic matter high in humic substances. Different respirometric tests also stated high biological stability of the end compost product.     

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.     

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.     

Two-stage biological treatment of olive mill wastewater with whey as co-substrate

Olive mill wastewater (OMW) is a highly polluting wastewater, caused by a high organic load and phenol content. These characteristics suggest that it may be suitable for aerobic treatment and anaerobic bacterial digestion. Aerobic treatment coupled with anaerobic bacterial digestion may be economically feasible as the methane produced is a valuable energy source while simultaneously purifying the OMW. In an attempt to improve the overall performance of the process, the addition of a co-substrate such as whey to the aerobic treatment pre-treatment of OMW by the yeast Candida tropicalis was studied.The two-stage system operated satisfactorily up to an organic loading rate (OLR) of 3.0 kg COD L⁻¹ day⁻¹ with a biogas production rate of 1.25 L_biogas L_reactor⁻¹ day⁻¹ and a total COD reduction in excess of 93% (62% COD reduction in aerobic pretreatment and 83% COD reduction in anaerobic digestion). Fifty-four percent of the phenol was biodegraded during the aerobic treatment stage, and biogas with between 68% and 75% methane was produced during anaerobic digestion.