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.     

Transgenic tobacco plants expressing a fungal laccase are able to reduce phenol content from olive mill wastewaters

A biotechnological approach was applied to reduce phenol content in olive mill wastewaters by transgenic tobacco plants. The cDNA laccase of poxC gene from Pleurotus ostreatus, carrying its own signal peptide for extracellular secretion, was transferred into the Nicotiana tabacum genome. Transgenic tobacco plants were obtained and the recombinant enzyme was secreted into the rhizosphere by the plant root apparatus, confirming the ability of the plant machinery to recognize the fungal POXC peptide signal leader appropriately as secretory tag. Total laccase activity assayed by ABTS in transgenic lines increased sharply compared to control plants. Moreover, plants cultivated in a hydroponic solution with the addition of olive mill wastewaters were able to reduce the total phenol content up to 70%.     

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.     

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.     

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.     

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.     

Production of added‐value metabolites by Yarrowia lipolytica growing in olive mill wastewater‐based media under aseptic and non‐aseptic conditions

Yarrowia lipolytica ACA‐YC 5033 was grown on glucose‐based media in which high amounts of olive mill wastewaters (OMWs) had been added. Besides shake‐flask aseptic cultures, trials were also performed in previously pasteurized media while batch bioreactor experiments were also done. Significant decolorization (～58%) and remarkable removal of phenolic compounds (～51% w/w) occurred, with the latter being amongst the highest ones reported in the international literature, as far as yeasts were concerned during their growth on phenol‐containing media. In nitrogen‐limited flask fermentations the microorganism produced maximum citric acid quantity ≈19.0 g/L [simultaneous yield of citric acid produced per unit of glucose consumed (Y_Cit/Glc)≈0.74 g/g]. Dry cell weight (DCW) values decreased at high phenol‐containing media, but, on the other hand, the addition of OMWs induced reserve lipid accumulation. Maximum citric acid concentration achieved (≈52.0 g/L; Y_Cit/Glc≈0.64 g/g) occurred in OMW‐based high sugar content media (initial glucose added at ≈80.0 g/L). The bioprocess was successfully simulated by a modified logistic growth equation. A satisfactory fitting on the experimental data occurred while the optimized parameter values were found to be similar to those experimentally measured. Finally, a non‐aseptic (previously pasteurized) trial was performed and its comparison with the equivalent aseptic experiment revealed no significant differences. Yarrowia lipolytica hence can be considered as a satisfactory candidate for simultaneous OMWs bioremediation and the production of added‐value compounds useful for the food industry.     

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.     

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.     

Treatment of olive mill wastewater in pilot-scale vertical flow constructed wetlands

Pilot-scale constructed wetlands (CW) were constructed and operated to treat pre-treated olive mill wastewater. Pilot-scale units comprising three identical series with four pilot-scale vertical flow CWs were operated for one harvest season in a Greek olive mill plant. The pilot-scale CWs were filled with various porous media (i.e., cobble, gravel, and sand) of different gradations. Two series of pilot-scale units were planted with common reeds and the third (control) was unplanted. Mean influent concentrations were 14,120 mg/L, 2841 mg/L, 95 mg/L, 123 mg/L and 506 mg/L for COD, phenols, ortho-phosphate, ammonia and TKN, respectively. Despite the rather high influent concentrations, the performance of the CW units was very effective since it achieved removals of about 70%, 70%, 75% and 87% for COD, phenols, TKN and ortho-phosphate, respectively. COD, phenol and TKN removal seems to be significantly higher in the planted series, while ortho-phosphate removal shows no significant differences among the three series. Temperature and pollutant surface load seem to affect the removal efficiency of all pollutants. Compared to previous studies, pollutant surface loads applied here were higher (by one or two orders of magnitude). Even though high removal efficiencies were achieved, effluent pollutant concentrations remained high, thus preventing their use for irrigation or immediate disposal into the environment.     

Biodegradation performance of an anaerobic hybrid reactor treating olive mill effluent under various organic loading rates

The primary objective of this study was to evaluate the performance of a 20 l lab scale anaerobic hybrid reactor (AHR) combining sludge blanket in the lower part and filter in the upper part under varying organic loading rates (OLRs) in order to study biodegradation of olive mill effluent (OME). For this purpose, some parameters, such as total phenols, effluent chemical oxygen demand (COD), suspended solids (SS), volatile fatty acids (VFAs), and pH in the influent and effluent, and removal efficiencies for those parameters (except pH) were continuously monitored throughout the experimental period of 477 days. Eleven different organic loadings between 0.45 and 32 kg COD m⁻³ day⁻¹ were imposed by either varying influent COD or hydraulic retention time (HRT). The results demonstrated that the AHR reactor could tolerate high influent COD concentrations. Removal efficiencies for the studied pollution parameters were found to be as follows: COD, 50–94%; total phenol, 39–80%; color, 0–54%; and suspended solids, 19–87%. The levels of VFAs in the effluent, which was principally acetate, butyrate, iso-butyrate, and propionate, varied between 10 and 2005 mg l⁻¹ depending upon OLRs. A COD removal efficiency of 90% could be achieved as long as OLR is kept at a level of less than 10 kg COD m⁻³ day⁻¹. However, a secondary treatment unit for polishing purposes is necessary to comply with receiving media discharge standards.     

Composting of olive leaves and pomace from a three-phase olive mill plant

The composting of olive leaves and olive pomace from a three-phase olive mill was tested as a method for solid waste reuse. The process was carried out using a compost windrow and mixing olive leaves and pomace at a ratio of 1:2. Compost was retained in the windrow for 60 days during which thermophilic temperatures developed for the first 40 days. The compost was then placed into a closed area to mature for another 60 days. The final product proved to be high quality amendment with C/N 27.1 and high nutrient concentrations (N, 1.79%; P, 0.17%; K, 4.97%; Na, 2.8%). Mature compost presented the highest germination index (198%) reported to date, as the germination index in the majority of previous studies is under 80%. Furthermore, tests revealed that addition of 31.5 tons of compost per ha, could increase lettuce yield by 145%.     

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.     

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.     

Two-phase olive mill waste composting: community dynamics and functional role of the resident microbiota

In this study, physico-chemical modifications and community dynamics and functional role of the resident microbiota during composting of humid husk from a two-phase extraction system (TPOMW) were investigated. High mineralization and humification of carbon, low loss of nitrogen and complete degradation of polyphenols led to the waste biotransformation into a high-quality compost. Viable cell counts and denaturing gradient gel electrophoresis (DGGE) profiling of the 16S rRNA genes showed that the thermophilic phase was characterized by the strongest variations of cell number, the highest biodiversity and the most variable community profiles. The isolation of tannin-degrading bacteria (e.g. Lysinibacillus fusiformis, Kocuria palustris, Tetrathiobacter kashmirensis and Rhodococcus rhodochrous) suggested a role of this enzymatic activity during the process. Taken together, the results indicated that the composting process, particularly the thermophilic phase, was characterized by a rapid succession of specialized bacterial populations with key roles in the organic matter biotransformation.     

Low-molecular-weight components of olive oil mill waste-waters

A new lignan 1-hydroxy-2-(4-hydroxy-3-methoxyphenyl)-6-(3-acetyl-4-hydroxy-5-methoxyphenyl)-3,7-dioxabicyclo[3.3.0]octane, the secoiridoid 2H-pyran-4-acetic acid,3-hydroxymethyl-2,3-dihydro-5-(methoxycarbonyl)-2-methyl-, methyl ester, the phenylglycoside 4-[beta-D-xylopyranosyl-(1-->6)]-beta-D-glucopyranosyl-1,4-dihydroxy-2-methoxybenzene and the lactone 3-[1-(hydroxymethyl)-1-propenyl] delta-glutarolactone were isolated and identified on the basis of spectroscopic data including two-dimensional NMR, as components of olive oil mill waste-waters. The known aromatic compounds catechol, 4-hydroxybenzoic acid, protocatechuic acid, vanillic acid, 4-hydroxy-3,5-dimethoxybenzoic acid, 4-hydroxyphenylacetic acid, 3,4-dihydroxyphenylacetic acid, tyrosol, hydroxytyrosol, 2-(4-hydroxy-3-methoxy)phenylethanol, 2-(3,4-dihydroxy)phenyl-1,2-ethandiol, p-coumaric acid, caffeic acid, ferulic acid, sinapic acid, 1-O-[2-(3,4-dihydroxy)phenylethyl]-(3,4-dihydroxy)phenyl-1,2-ethandiol, 1-O-[2-(4-hydroxy)phenylethyl]-(3,4-dihydroxy)phenyl-1,2-ethandiol, D(+)-erythro-1-(4-hydroxy-3-methoxy)-phenyl-1,2,3-propantriol, p-hydroxyphenethyl-beta-D-glucopyranoside,2(3,4-dihydroxyphenyl)ethanol 3beta-D-glucopyranoside, and 2(3,4-dihydroxyphenyl)ethanol 4beta-D-glucopyranoside were also confirmed as constituents of the waste-waters.     

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.     

Optimisation of biodegradation conditions for the treatment of olive mill wastewater

The aim of the present paper was to optimise the conditions of aerobic treatment of olive mill wastewater. To do so, the waste was treated following the experimental optimal design methodology studying the set of factors susceptible to influence the treatment (pH, C/N ratio, aeration and temperature). The results of a first series of experiments showed a strong correlation between the reduction in the levels of polyphenols and three of the parameters studied, i.e. the C/N ratio, aeration and temperature. Optimised conditions led to a 94% drop in polyphenols.

Then, for a finer study of the conditions, just two parameters were varied, the pH and the C/N ratio. The results showed that the conditions of pH modification (addition of lime or sodium hydroxide) and the C/N ratio (urea or ammonium nitrate) allowed the microbiological activity to be very significantly improved. This led to polyphenol reductions of 51% and 76%.     

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.     

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.