Dry Olive Mill Residue Research Articles

Enzymatic mechanisms and detoxification of dry olive-mill residue by Cyclocybe aegerita, Mycetinis alliaceus and Chondrostereum purpureum

The detoxification of dry olive-mill residue (DOR), a lignocellulosic by-product, was investigated using three agaric fungi: Cyclocybe aegerita, Mycetinis alliaceus and Chondrostereum purpureum. The lignin-modifying enzymes (LME) secretion pattern of the above-mentioned fungi such as DyP-type peroxidase (DyP), laccase (Lac), unspecific peroxygenase (UPO), and manganese peroxidase (MnP) was determined in presence and absence of DOR. In C. aegerita, a laccase (Lac) and unspecific peroxygenase (UPO) induction was found when DOR was present in the medium. Thus, these enzymes appeared to be responsible for DOR detoxification and, indirectly, its plant-growth-promoting effect. In the experiment performed with M. alliaceus, no differences were found in DyP-type peroxidase (DyP) secretion when the basal barley medium was supplemented with DOR. However, MnP and Lac activities in DOR-barley reached a maximum after 5 weeks of incubation with a concomitant decline in DOR phytotoxicity. C. purpureum completely eliminated DOR phytotoxicity but no significant production of LME was detected in soy or barley fungal media. Other enzymatic mechanisms were also investigated, in relation to hydrolytic enzymes and the intracellular system cytochrome P450 monooxygenase (CYPs). Our results suggest the participation of a complex enzymatic system (intra and extracellular) in C. purpureum for the biotransformation of DOR.

Integrated biovalorization of wine and olive mill by-products to produce enzymes of industrial interest and soil amendments

An integral and affordable strategy for the simultaneous production of lignin-modifying and carbohydrate active enzymes and organic amendment, with the aid of a saprobe fungus was developed by using olive oil and wine extraction by-products. The polyporal fungus Trametes versicolor was cultivated in soy or barley media supplemented with dry olive mill residue (DOR) as well as with grape pomace and stalks (GPS) in solid state fermentation (SSF). This strategy led to a 4-fold increase in the activity of laccase, the principal enzyme produced by SFF, in DOR-soy media as compared to controls. T. versicolor managed to secrete lignin-modifying enzymes in GPS, although no stimulative effect was observed. GPS-barley media turned out to be the appropriate medium to elicit most of the carbohydrate active enzymes. The reuse of exhausted solid by-products as amendments after fermentation was also investigated. The water soluble compound polymerization profile of fermented residues was found to correlate with the effect of phytotoxic depletion. The incubation of DOR and GPS with T. versicolor not only reduced its phytotoxicity but also stimulated the plant growth. This study provides a basis for understanding the stimulation and repression of two groups of enzymes of industrial interest in the presence of different carbon and nitrogen sources from by-products, possible enzyme recovery and the final reuse as soil amendments.

Solid state fermentation of olive mill residues by wood- and dung-dwelling Agaricomycetes: Effects on peroxidase production, biomass development and phenol phytotoxicity

The in vivo conversion of dry olive mill residue (DOR) by wood- and dung-dwelling fungi − Auricularia auricula-judae, Bjerkandera adusta and Coprinellus radians − increases peroxidase secretion up to 3.2–3.5-fold (∼1.3, 3.5 and 7.0Ug−1 DOR for dye-decolorizing peroxidase, manganese peroxidase and aromatic peroxygenases, respectively). The incubation of DOR with these fungi produced a sharp decrease in total phenolic content (100% within 4wk), a reduction in phytotoxicity as well as a certain degree of plant growth caused by the stimulating effect of fungal-treated DOR. These findings correlate with a characteristic shift in the fragmentation pattern of water-soluble aromatics (detected at 280nm) from low (0.2, 1.5 and 2.2kDa, respectively) to high molecular mass (35 to >200kDa), which demonstrates the presence of a polymerization process. Phenol-rich agricultural residues are a useful tool for enzyme expression and production studies of peroxidase-producing Agaricomycetes which could make DOR a valuable organic fertilizer.

Production of bacterial cellulose by Gluconacetobacter sacchari using dry olive mill residue

Bacterial Cellulose (BC), produced by many bacteria specially those belonging to the Gluconacetobacter genus, is a very peculiar cellulose form that bears unique mechanical and structural properties that can be exploited in numerous applications. However, the production costs of BC are very high because of the use of quite expensive culture media. In this sense, the purpose of this work was to evaluate the possibility of use residues from the olive oil production industry as nutrient and carbon source for the production of BC by Gluconacetobacter sacchari. The dry olive mill residue (DOR) was submitted to water extraction at 40 and 100 °C (DOR40 and DOR100) and to hydrolysis with H2SO4 1M (DOR100H) in order to obtain sugar rich aqueous extracts to be used for BC production. The BC production obtained without addiction of any type of nutrients was 0.81 g L−1 for DOR40 and 0.85 g L−1 for DOR100 after 96 h incubation, which corresponded respectively to 32 and 34% of the production achieved with conventional HS culture medium (around 2.5 g L−1). In order to enhance the production of BC, the residues were supplemented with nitrogen (N) and phosphate (P) sources to overcome possible nutritional limitations. It was verified an increase on the BC production between 21.5% (N8 P4,5) and 43.2% (N1 P8) when compared with no supplementation. These are promising results to overcome high BC production costs.

Valorization of olive mill residues: Antioxidant and breast cancer antiproliferative activities of hydroxytyrosol-rich extracts derived from olive oil by-products

Phenolic composition, antioxidant and breast cancer antiproliferative activities of water and methanol/water derived extracts from olive pomace (OP) and dry olive mill residue (DOR), from Portuguese industries, were studied. DOR water (DORW) extracts showed the highest extraction yield; as well as the highest total phenolic content (TPC) and hydroxytyrosol (HT) (∼25mg/g extract). HPLC–ESI-MS analysis identified HT in both OP and DOR, whereas HT-1-glucoside, tyrosol, oleuropein aglycone isomers, verbascoside and oleuropein were only detected in DOR. Additionally, a de(carboxymethyl)oleuropein aglycone isomer, in aldehyde form, was reported for the first time in DOR. DOR water extract also presented the most effective DPPH scavenging capacity and antiproliferative activity, comparatively to OP water (OPW) extract. Moreover, antioxidant potential of phenolic compounds present in DORW extract was comparable to HT, and to butylated hydroxyanisole (BHA), a widely used food industry antioxidant. Phenolic compounds present in DORW extract also showed comparable tumor antiproliferative activity on MDA-MB-231, relatively to HT and 5-fluorouracil (5-FU), a well-known cytostatic agent. MDA-MB-231 cell growth inhibition, upon 5-FU incubation was even incremented in the presence of DORW extract.These results demonstrate DOR extracts potential as source of phenolic compounds for nutraceutical applications, as food supplements, opening new strategies for its olive mill residues valorization.

Bioremediation of dry olive-mill residue removes inhibition of growth induced by this waste in tomato plants

The disposal of dry olive-mill residue, the waste product from olive oil production, is a serious environmental issue. Dry olive-mill residue, being rich in organic and inorganic nutrients, could be used as fertilizer; however, it contains phenolic compounds that can inhibit plant growth. In order to clarify whether bioremediation of this waste could be a valuable strategy for its reuse, the effect of aqueous extract of dry olive-mill residue, untreated or bioremediated by the saprobe fungi Coriolopsis rigida and Penicillium chrysogenum-10, has been analyzed in relation to some physiological parameters of tomato plants. The data show that aqueous dry olive-mill residue significantly reduces the biomass of roots and shoots. In particular, it causes a dramatic reduction in root length, area, and volume as well as in the number of root tips. At an early stage, aqueous dry olive-mill residue also reduces the content of chlorophyll a and b and the efficiency of PS II. The inhibition of growth seems to be due to the increase in phenolic compounds that induce oxidative stress. Interestingly, when plants are treated with aqueous dry olive-mill residue bioremediated by saprobe fungi a decrease in phenolic content and an alleviation of oxidative stress occur. In conclusion, the results show that bioremediation of aqueous dry olive-mill residue is a useful tool to remove most of the inhibiting effects of this waste on plant growth.

Oxidative stress induced in sunflower seedling roots by aqueous dry olive-mill residues.

The contamination of soils with dry olive-mill residue can represent a serious problem as being an environmental stressor in plants. It has been demonstrated that inoculation of aqueous extract of olive oil-mill residue (ADOR) with saprobe fungi removes some phenolic compounds. In this paper we studied the effect of ADOR uninoculated or inoculated with saprobe fungi in sunflower seedling roots. The germination and root growth, O2·- generation, superoxide dismutase (SOD) and extracellular peroxidases (EC-POXs) activities, and the content of some metabolites involved in the tolerance of stress were tested. The roots germinated in ADOR uninoculated show a decrease in meristem size, resulting in a reduction of the root length and fresh weight, and in the number of layers forming the cortex, but did not alter the dry weight, protein and soluble amino acid content. ADOR caused the decreases in O2·- generation and EC-POX′s activities and protein oxidation, but enhanced SOD activity, lipid peroxidation and proline content. Fluorescence imaging showed that ADOR induced O2·- and H2O2 accumulation in the roots. The increase in SOD and the decrease in EC-POX′s activities might be involved in the enhancement of H2O2 content and lipid peroxidation. Control roots treated with ADOR for 10 min show an oxidative burst. Roots germinated in ADOR inoculated with saprobe fungi partially recovered normal levels of ROS, morphological characteristics and antioxidant activities. These results suggested that treatment with ADOR caused a phytotoxic effect during germination inducing an oxidative stress. The inoculation of ADOR with saprobe fungi limited the stress.

Defence response of tomato seedlings to oxidative stress induced by phenolic compounds from dry olive mill residue

ADOR is an aqueous extract obtained from the dry olive mill residue (DOR) which contains the majority of its soluble phenolic compounds, which are responsible for its phytotoxic properties. Some studies have shown that ADOR negatively affects seed germination. However, to date, few studies have been carried out on the effect of ADOR on the oxidative stress of the plant. It is well known that saprobe fungi can detoxify these phenolic compounds and reduce the potential negative effects of ADOR on plants. To gain a better understanding of the phytotoxic effects and oxidative stress caused by this residue, tomato seeds were germinated in the presence of ADOR, treated and untreated with Coriolopsis rigida, Trametes versicolor, Pycnoporus cinnabarinus and Penicillium chrysogenum-10 saprobe fungi. ADOR sharply reduced tomato seed germination and also generated high levels of malondialdehyde (MDA), O2- and H2O2. However, bioremediated ADOR did not negatively affect germination and reduced MDA, O2- and H2O2 content in different ways depending on the fungus used. In addition, the induced defense response was studied by analyzing the activity of both antioxidant enzymes (superoxide dismutase (SOD), catalase, ascorbate peroxidasa, glutathione reductase (GR), peroxidases and coniferil alcohol peroxidasa) and detoxification enzymes (glutathione-S-transferase (GST)). Our findings suggest that, because ADOR is capable of inducing oxidative stress, tomato seedlings trigger a defense response through SOD, GR, and GST activity and through antioxidant and lignification processes. On the other hand, the bioremediation of ADOR plays an important role in counteracting the oxidative stress induced by the untreated residue.

Non-supplemented aqueous extract from dry olive mill residue: A possible medium for fungal manganese peroxidase production

The low moisture content of the dry olive mill residue (DOR), solid waste from the two-phase olive oil extraction process, enables its long-term storage and its ready reconstitution with water thus leading to a candidate liquid growth medium for the microbial production of enzymes of commercial interest. To test this hypothesis, two white-rot fungi (WRF), namely Lentinus tigrinus CBS 577.79 and Phlebia sp. DABAC 9, were grown in shaken cultures on non-supplemented aqueous extracts of the dry olive mill residue (ADOR) and compared for their ability to grow therein and produce Mn-dependent peroxidase (MnP). Then, in order to assess the feasibility of scale transfer to the reactor level, the two fungi were grown in a 3-L bubble-column reactor on 25% (w/v) ADOR at 28°C and an aeration rate of 0.3vvm. Under these conditions, L. tigrinus and Phlebia sp. produced 42.60±1.74 and 38.88±1.68 nkatal mL−1 MnP with respective mean volumetric productivities of 222.9 and 324.1nkatalL−1h−1. These results clearly show that the use of non-supplemented ADOR as a production medium enables a significant MnP production thus representing an excellent basis for process optimization.

Dry matter and root colonization of plants by indigenous arbuscular mycorrhizal fungi with physical fractions of dry olive mill residue inoculated with saprophytic fungi

We studied the influence of indigenous arbuscular mycorrhizal (AM) and saprobe fungi on the phytotoxicity of the physical fractions of dry olive mill residue (DOR). The physical extractions of DOR gave an aqueous (ADOR) and an exhausted (SDOR) fraction with less phytotoxicity for tomato than the original samples. The indigenous AM were able to decrease the phytotoxicity of SDOR inoculated with Trametes versicolor and Pycnoporus cinnabarinus on tomato. However, incubation of ADOR with both saprophytic fungi did not decrease its phytotoxicity in presence of the indigenous AM fungi. The percentage of root length colonized by indigenous AM strongly decreased in presence of DOR, around 80% of decrease at dose of 25 g kg-1of DOR, but the level of mycorrhization was higher in presence of ADOR or SDOR (38% and 44% of decrease respectively at the same dose). There were no relationships between the effects of the physical fractions of DOR incubated with the saprobe fungi on AM colonization and on plant dry weight of tomato. Our results suggest that the phytotoxicity of the olive residues can be eliminated by the combination of physical extraction and by saprobe fungal inoculation and the use of this agrowaste as organic amendment in agricultural soil may be possible.

Short-term impact of dry olive mill residue addition to soil on the resident microbiota

The short-term response of the resident soil bacterial and fungal communities to the addition of 5% (w/w) of either dry olive mill residue (DOR), DOR treated with Phlebia sp. (PTDOR) or DOR previously extracted with water (WEDOR) was investigated. As opposed to bacteria, the diversity of fungi increased upon the amendments as assessed by denaturing gradient gel electrophoresis of 18S rDNA. Over the first 30 days, phospholipid fatty acids analyses indicated a gradual decrease in the relative abundances of Gram + bacteria (from 44.8% to 37.9%) and a concomitant increase of Gram − bacteria (from 37.3% to 51.2%) in DOR-amended soil. A considerable increase in the fungal/bacterial ratio was observed after 7 days in DOR, WEDOR and PTDOR-amended soils with respect to the control (0.316, 0.165 and 0.265, respectively, vs. 0.011). The overall microbial activity was stimulated by the amendments as indicated by the higher activity levels of both dehydrogenase and fluorescein diacetate hydrolase. These results indicate that DOR at the application level examined is not toxic on soil microorganisms.

Immobilized Inocula of White-Rot Fungi Accelerate both Detoxification and Organic Matter Transformation in Two-Phase Dry Olive-Mill Residue

The potential use for agronomic purposes of dry olive-mill residue (DOR), solid waste from the olive oil two-phase extraction process, might be impaired by its phytotoxicity. Although fungal treatments can detoxify DOR, long times are required for these processes. The objective of this study was to assess whether the addition of immobilized fungal inocula to DOR might improve colonization rates, thus reducing the time necessary for its detoxification and bioconversion. Inocula of Panus tigrinus CBS 577.79 and Phlebia sp. DABAC 9 immobilized on either chopped maize stalks or polyurethane sponge (PS) led to higher removals of both phenols and phytotoxicity from DOR than free inocula after 4 weeks of incubation. Best dephenolization (85%) was with PS-immobilized Phlebia sp., the use of which reduced germinability inhibition of Lepidium sativum and Lactuca sativa by 80 and 71.4%, respectively. Regardless of the type of inoculant, a low degree of humification was obtained.

Organic matter transformation and detoxification in dry olive mill residue by the saprophytic fungus Paecilomyces farinosus

Dry olive mill residue (DOR), the by-product of the two-phase extraction process, is very rich in organic matter and nutritionally relevant cations. For this reason, the agronomic use of this waste has been suggested although DOR exhibits significant phytotoxicity. The objective of this study was to investigate the impact of Paecilomyces farinosus on both organic matter modification and detoxification of this waste. Humification ratio in DOR colonized by the fungus for 20 weeks was increased by about 65% with respect to the abiotic control and humification index reached 0.38, a value that characterizes well-humified materials. High performance size-exclusion chromatography of humic acids from fungal cultures showed a marked increase in both weight-averaged and number-averaged molecular weights with respect to abiotic controls. Water-soluble phenols were reduced by 45% in 20-week-old P. farinosus cultures on DOR and mass-balance ultra-filtration showed that the relative abundance of the molecular weight fraction of phenols above 30 kDa increased from 31 to 72% suggesting the occurrence of polymerization. Experiments performed with alfalfa grown on soils containing 2.5% (w/w) of abiotic controls and fungal-treated DOR showed that phytotoxicity was totally suppressed in the waste that underwent fungal treatment.

Saprobe fungi decreased the sensitivity to the toxic effect of dry olive mill residue on arbuscular mycorrhizal plants

We studied the influence of olive mill dry residue (DOR) treated with saprobe fungi on growth of tomato and alfalfa colonized by Glomus deserticola. The application of 25 g kg −1 of dry DOR to soil decreased the shoot and root dry weight of tomato and alfalfa. Plants were more sensitive to the toxicity of DOR when colonized with the arbuscular mycorrhizal (AM) fungi. The sensitivity of both plants to the toxicity of DOR differed according to whether they were colonized by G. deserticola or by indigenous AM fungi. The phytotoxicity of DOR towards tomato and alfalfa was decreased by incubation the residue before planting with saprobe fungi for 20 wk. The beneficial effects of AM fungi on plant growth added with DOR incubated with saprobe fungi depend of the type of the plant and AM fungi. The contribution of AM fungi to the beneficial effect of DOR incubated with saprobe fungi varied according to the type of the plant and AM fungi. G. deserticola increased the shoot and root dry weight of plants when they were grown in the presence of DOR incubated with saprobe fungi for 20 wk. The beneficial effect of saprobe fungi on the dry weight and the level of AM mycorrhization of plants seem to be related to the decrease caused by these fungi in the phenol concentration in DOR. However, the toxicity of DOR due to substances other than phenols can not be ignored. The use of certain saprobe and AM fungi allows the possibility of using DOR as an organic fertilizer.

Chemical characterization and effects on Lepidium sativum of the native and bioremediated components of dry olive mill residue

Dry olive mill residue (DOR) from the olive oil production by two phase centrifugation system was fractionated by a consecutive continuous solid–liquid extraction obtaining the EAF, PF, MF and WF fractions with ethyl acetate, n-propanol, methanol and water, respectively. The chemical, chromatographic and mass spectrometric analyses showed EAF, PF and MF to be mainly composed of simple phenols, phenolic acids, flavonoids and glycosilated phenols (glycosides of phenols, secoiridoids and flavonoids), whereas WF was mainly consisting of polymerin, the metal organic polymeric mixture previously identified in olive oil mill waste waters and composed of carbohydrates, melanin, proteins and metals (K, Na, Ca, Mg and Fe). The identification in DOR of oleoside, 6′-β-glucopyranosyl-oleoside and 6′-β-rhamnopyranosyl-oleoside, and of its organic polymeric component, known as polymerin, are reported for the first time in this paper. The inoculation of the previously mentioned fractions with saprobe fungi Coriolopsis rigida, Pycnoporus cynnabarinus or Trametes versicolor indicated these fungi to be able to metabolize both the phenols and glycosilated phenols, but not polymerin. In correspondence, EAF, PF, MF and WF, which proved to be toxic on Lepidium sativum, decreased their toxicity after incubation with the selected fungi, WF showing to be also able to stimulate the growth of the selected seeds. The phytotoxicity appeared mainly correlated to the monomeric phenols and, to a lesser extent, to the glycosilated phenols, whereas polymerin proved to be non toxic. However, the laccase activity was not associated with the decrease of phytotoxicity. The valorization of DOR as a producer of high added value substances of industrial and agricultural interest in native form and after their bioremediation for a final objective of the total DOR recycling is also discussed.

Organic matter evolution and partial detoxification in two-phase olive mill waste colonized by white-rot fungi

Dry olive mill residue (DOR) is a solid waste arising from the olive oil two-phase extraction system. The objective of the present study was to investigate the impact of an aerobic treatment of DOR with selected lignin-degrading fungi on both organic matter evolution and residual phytotoxicity of the waste. To this aim, several white-rot fungi, including Phlebia radiata, Coriolopsis rigida, Phanerochaete chrysosporium, Pycnoporus cinnabarinus, Poria subvermispora and Pleurotus pulmonarius were inoculated under axenic conditions for 2 and 20 weeks. The chemical composition and phytotoxicity of DOR were scarcely affected by fungi after 2 weeks incubation. By contrast, both a significant depletion of phenolic compounds and a partial removal of phytoxicity towards Lycopersicum esculentum plants were generally obtained after 20 weeks. The most effective fungus in degrading lignin, total phenols and in removing phytotoxicity was C. rigida. A high correlation was observed between phytotoxicity and phenols added to soil with the waste.

Reusing ethyl acetate and aqueous exhausted fractions of dry olive mill residue by saprobe fungi

Some saprobe fungi ( Phlebia radiata, Trametes versicolor, Coriolopsis rigida, Pycnoporus cinnabarinus, Fomes sclerodermus or Pleurotus pulmonarius) were able to bioconvert the ethyl acetate fraction (DEAF) and the corresponding aqueous exhausted fraction (EAF) of dry olive mill residue (DOR), reducing their phytotoxicity on Lepidium sativum seeds. Large amount of hydroxytyrosol together with other eight monomeric phenols were found in the native DEAF fraction, which represents a good source of antioxidants. P. radiata, T. versicolor and F. sclerodermus caused an effective phytotoxicity reduction of EAF in the concentration range of 25–3 g l −1. In particular, in the range between 12.5 and 3 g l −1, the EAF samples inoculated with P. radiata and F. sclerodermus surprisingly stimulated the germinability of L. sativum, suggesting their use as a potential biofertilizer. This is the first report which showed the bioconversion of the above fractions in shorter time with respect to the previous findings concerning DOR. The possible implications of laccase in the decrease of DEAF and EAF phytotoxicity was also discussed.

Saprobic fungi decrease plant toxicity caused by olive mill residues

We studied the influence of dry olive mill residue (DOR), as affected by saprobic fungi on the growth of soybean ( Glycine max) and tomato ( Lycopersicum esculentum). The application of 60 g kg −1 of DOR decreased the dry weight of tomato and soybean plants mainly because of its phenolic content. The phytotoxicity of DOR decreased when it was incubated in the presence of almost all of the saprobic fungi tested. All saprobic fungi tested in our experiments were able to decrease the phenol content of DOR, and a correlation between the decrease in the phenol content of DOR by saprobic fungi and the increase in the dry weight of plants was found. However, the toxicity of these residues to either tomato or soybean plants did not decrease in the presence of all saprobic fungi in spite of a decrease in phenolic compounds. Some of the saprobic fungi tested eliminate completely the phytotoxicity of DOR. F. oxysporum 738 and F. lateritum were capable of transforming the DOR, after incubation for 20 weeks, so that the tomato plants to which this transformed DOR was applied increased significantly in shoot dry weight compared with the tomato plants which did not receive DOR. These results open the possibility of the use of DOR as organic fertilizer.