Evolution of Hydroxytyrosol, Hydroxytyrosol 4-ß-d-Glucoside, 3,4-Dihydroxyphenylglycol and Tyrosol in Olive Oil Solid Waste or "Alperujo".

The main by-product generated from the olive oil two-phase extraction system, or alperujo, is undoubtedly a rich source of bioactive components, among which phenolics are one of the most important. The evolution of four of its main phenolics: hydroxytyrosol (HT), hydroxytyrosol 4-ß-d-glucoside (Glu-HT), 3,4-dihydroxyphenylglycol (DHPG) and tyrosol (Ty) was studied over two seasons and in ten oil mills under similar climatological and agronomic conditions, for the first time using organic extraction and high-performance liquid chromatography (HPLC-DAD) determination. The results show that HT (200-1600 mg/kg of fresh alperujo) and Ty (10-570 mg/kg) increase, while DHPG (10-370 mg/kg) decreases only in the last month of the season and Glu-HT (1400-0 mg/kg) decreases drastically from the beginning. This evolution is similar between different seasons, with a high correlation between Glu-HT, HT, and Ty. On the other hand, it has been verified that a mixture of alperujos from all the oil mills, which is what the pomace extractor receives, is a viable source of a liquid fraction which is rich in the phenolics studied through organic extractions and especially after the application of a thermal treatment, obtaining values of 4.2 g/L of HT, 0.36 g/L of DHPG, and 0.49 g/L of Ty in the final concentrated liquid fraction.

Exploitation of virgin olive oil by-products (Olea europaea L.): phenolic and volatile compounds transformations phenomena in fresh two-phase olive pomace ('alperujo') under different storage conditions.

BACKGROUND: Much effort has recently been spent for re-using virgin olive oil by-products as nutraceutical ingredients for human diet thanks to their richness in bioactive phenols, but their management is not easy for producers. We aimed to provide useful information for a better management of fresh olive pomace before drying, by studying the phenolic and volatile compounds transformations phenomena of fresh olive pomace stored under different conditions planned to simulate controlled and uncontrolled temperature conditions in olive oil mills. RESULTS: The evolution of the phenolic and volatile compounds was studied by high-performance liquid chromatography-diode array detector mass spectrometry (HPLC-DAD-MS) and headspace solid-phase microextraction gas chromatography-mass spectrometry (HS-SPME-GC-MS). The phenolic profile varied rapidly during storage: the verbascoside content decreased about 70% after 17 days even at 4 °C, while the content of simple phenols such as hydroxytyrosol and caffeic acid increased over time. The low temperature was able to slow down these phenomena. A total of 94 volatile organic compounds (VOCs) were detected in the fresh olive pomace, with a prevalence of lipoxygenase (LOX) VOCs (78%), mainly aldehydes (19 490.9 µg kg-1 ) despite the higher number of alcohols. A decrease in LOX volatiles and a quick development of the ones linked to off-flavors (carboxylic acids, alcohols, acetates) were observed, in particular after 4 days of storage at room temperature. Only storage at 4 °C allowed these phenomena to be slowed down. CONCLUSION: To preserve the natural phenolic phytocomplex of fresh olive pomace before drying and to avoid off-flavors development, storage in open containers must be avoided and a short storage in cold rooms (7-10 days) is to be preferred. © 2021 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Bio-hydrolysis of used soybean oil: environmental-friendly technology using microbial consortium.

In this work, strains of Bacillus subtilis were inoculated in consortium with Rhodotorula mucilaginosa into spent soy oil as aiming to biological treatment and low-cost reuse. The microorganisms were previously isolated and selected for the lipolytic capacity of the alperujo residue generated during the processing of olive oil. For fermentation, bioassays containing Rhodotorula mucilaginosa isolated from alperujo and Candida rugosa CCMA 00371, both co-inoculated with Bacillus subtilis CCMA 0085 in medium containing (% w/v) 0.075 glucose and 0.375 (NH4)3 PO4 in 75 mL of water and 75 mL of spent soy oil. Despite the low biomass productivity, it has favorable characteristics to be used in animal feed supplementation. Spent soy oil was used as a carbon source proven by Bartha respirometer. The strains of R. mucilaginosa UFLA RAS 144 and B. subtilis CCMA 0085 are promising inoculants for oil degradation and can be applied in a waste treatment system.

Biogas Potential of the Side Streams Obtained in a Novel Phenolic Extraction System from Olive Mill Solid Waste.

The olive oil production is an important industrial sector in many Mediterranean areas, but it is currently struggled by the necessity of a proper valorisation of the olive mill solid waste or alperujo. The alperujo is the main by-product generated during the two-phase olive oil extraction, accounting for up to 80% of the initial olive mass. The alperujo is a source of valuable compounds, such as the pomace olive oil or highly interesting phenolic compounds. In the present research, a novel biorefinery approach has been used for phenolic compounds recovery. However, the extraction of these valuables compounds generates different exhausted phases with high organic matter content that are required to be managed. This study consists of the evaluation of the anaerobic biodegradability of the different fractions obtained in a novel biorefinery approach for the integral valorisation of alperujo. The results show that the different phases obtained during the biorefinery of the alperujo can be effectively subjected to anaerobic digestion and no inhibition processes were detected. The highest methane yield coefficients were obtained for the phases obtained after a two-months storages, i.e., suspended solids and liquid phase free of suspended solids, which generated 366 ± 7 mL CH4/g VS and 358 ± 6 mL CH4/g VS, respectively. The phenol extraction process reduced the methane yield coefficient around 25% due to the retention of biodegradable compounds during the extraction process. Regardless of this drop, the anaerobic digestion is a suitable technology for the stabilization of the different generated residual phases, whereas the high market price of the extracted phenols can largely compensate the slight decrease in the methane generation.

Rhodococcus bacteria as a promising source of oils from olive mill wastes.

The accumulation of triacylglycerols (TAG) is a common feature among actinobacteria belonging to Rhodococcus genus. Some rhodococcal species are able to produce significant amounts of those lipids from different single substrates, such as glucose, gluconate or hexadecane. In this study we analyzed the ability of different species to produce lipids from olive oil mill wastes (OMW), and the possibility to enhance lipid production by genetic engineering. OMW base medium prepared from alperujo, which exhibited high values of chemical oxygen demand (127,000 mg/l) and C/N ratio (508), supported good growth and TAG production by some rhodococci. R. opacus, R. wratislaviensis and R. jostii were more efficient at producing cell biomass (2.2-2.7 g/l) and lipids (77-83% of CDW, 1.8-2.2 g/l) from OMW than R. fascians, R. erythropolis and R. equi (1.1-1.6 g/l of cell biomass and 7.1-14.0% of CDW, 0.1-0.2 g/l of lipids). Overexpression of a gene coding for a fatty acid importer in R. jostii RHA1 promoted an increase of 2.2 fold of cellular biomass value with a concomitant increase in lipids production during cultivation of cells in OMW. This study demonstrates that the bioconversion of OMW to microbial lipids is feasible using more robust rhodococal strains. The efficiency of this bioconversion can be significantly enhanced by engineering strategies.

Three-year study of fast-growing trees in degraded soils amended with composts: Effects on soil fertility and productivity.

Currently, worries about the effects of intensive plantations on long-term nutrient supply and a loss of productivity have risen. In this study two composts were added to degraded soils where this type of intensive crops were growing, to avoid the soil fertility decrease and try to increase biomass production. For the experiment, two degraded soils in terms of low organic carbon content and low pH were selected in South-West Spain: La Rábida (RA) and Villablanca (VI) sites. Both study sites were divided into 24 plots. In RA, half of the plots were planted with Populus x canadensis "I-214"; the other half was planted with Eucalyptus globulus. At the VI site, half of the plots were planted with Paulownia fortunei, and the other plots were planted with Eucalyptus globulus. For each tree and site, three treatments were established (two organic composts and a control without compost), with four replications per treatment. The organic amendments were "alperujo" compost, AC, a solid by-product from the extraction of olive oil, and BC, biosolid compost. During the three years of experimentation, samples of soils and plants were analyzed for studying chemical and biochemical properties of soil, plant growth and plant nutritional status and biomass production. The composts increased total organic carbon, water-soluble carbon, nutrients and pH of soil only in the most acidic soil. Soil biochemical quality was calculated with the geometric mean of the enzymatic activities (Dehydrogenase, ß-glucosidase, Phosphatase and Urease activities) determined in soils. The results showed a beneficial improvement in comparison with soils without compost. However, the best results were found in the growth and biomass production of the studied trees, especially in Eucalyptus. Nutritional levels of leaves of the trees were, in general, in the normal established range for each species, although no clear effect of the composts was observed. The results of this study justify the addition of compost to guarantee good biomass production and maintain or improve soil management in degraded soils, especially in acid soils.

Inhibitory and synergistic effects of natural olive phenols on human platelet aggregation and lipid peroxidation of microsomes from vitamin E-deficient rats.

PURPOSE: This study explored the in vitro antioxidant and anti-platelet activities of hydroxytyrosol, hydroxytyrosol acetate, 3,4-dihydroxyphenylglycol and two phenolic olive extracts. These compounds and extracts were obtained from a new industrial process to hydrothermally treat the alperujo (160 °C/60 min), a by-product of olive oil extraction. METHODS: The extracts and the purified compounds were obtained chromatographically using both ionic and adsorbent resins. The antioxidant activity was determined by measuring inhibition of human platelet aggregation and inhibition of lipid peroxidation in liver microsomes of vitamin E-deficient rats. RESULTS: The positive effect of the extracts on the inhibition of platelet aggregation is showed, being higher in the case of hydroxytyrosol acetate up to 38%, and for the first time, its synergist effect with hydroxytyrosol has been proved, obtaining more than double of inhibition. The phenolic extracts and the isolated phenols showed good results for inhibiting the lipid oxidation, up to 62 and 25%, respectively. A synergistic effect occurred when the hydroxytyrosol acetate and the 3,4-dihydroxyphenylglycol were supplemented by hydroxytyrosol. CONCLUSION: These results suggest the extract and these compounds obtained from a novel industrial process could be natural alternatives for the prevention of diseases related to cardiovascular disorder or oxidative damage.

Production of 4-ethylphenol in alperujo by Lactobacillus pentosus.

BACKGROUND: Alperujo is the paste generated from the two-phase extraction system of olive oil. This wet pomace must be stored for several months and, during this period, the formation of 4-ethyphenol provokes a strong off-odour. The aim of this work was to identify the microorganisms able to produce this volatile phenol. RESULTS: Yeast and bacterial strains were isolated from stored alperujo and tested for their ability to metabolize p-coumaric acid and form 4-ethylphenol. Among them, Lactobacillus pentosus was the microorganism that both in synthetic medium and alperujo gave rise to 4-ethylphenol formation. This microorganism did not grow in alperujo acidified to pH 2, thereby confirming that acidification as the best method to control odour emissions during alperujo storage. CONCLUSION: Lactic acid bacteria, particularly Lactobacillus pentosus, can be responsible for the formation of the off-odour caused by 4-ethylphenol during the storage of alperujo. This odour can be prevented by acidifying the alperujo.

Quality of trace element contaminated soils amended with compost under fast growing tree Paulownia fortunei plantation.

The use of fast growing trees could be an alternative in trace element contaminated soils to stabilize these elements and improve soil quality. In this study we investigate the effect of Paulownia fortunei growth on trace element contaminated soils amended with two organic composts under semi-field conditions for a period of 18 months. The experiment was carried out in containers filled with tree different soils, two contaminated soils (neutral AZ and acid V) and a non contaminated soil, NC. Three treatments per soil were established: two organic amendments (alperujo compost, AC, and biosolid compost, BC) and a control without amendment addition. We study parameters related with fertility and contamination in soils and plants. Paulownia growth and amendments increased pH in acid soils whereas no effect of these factors was observed in neutral soils. The plant and the amendments also increased organic matter and consequently, soil fertility. Positive results were also found in soils that were only affected by plant growth (without amendment). A general improvement of "soil biochemical quality" was detected over time and treatments, confirming the positive effect of amendments plus paulownia. Even in contaminated soils, except for Cu and Zn, trace element concentrations in leaves were in the normal range for plants. Results of this mid-term study showed that Paulownia fortunei is a promising species for phytoremediation of trace element polluted soils.

Composted organic wastes from the pharmaceutical and agro-food industries induce soil bioactivity and nodulation in alfalfa.

BACKGROUND: Environmentally friendly agriculture needs to reduce the use of synthetic fertilizers and to reclaim nutrients from organic wastes. In this study the effect of five doses (0, 12, 24, 48 and 96 t ha(-1) ) of two two-phase olive mill waste (TPOMW)-based composts on the bioactivity and chemical characteristics of an agricultural soil and their potential to fertilize alfalfa (Medicago sativa) and stimulate nodulation were assessed during a two-year incubation experiment. The two composts were prepared either with the olive mill waste alone (compost A), which served as control, or mixed with a liquid fatty-proteinaceous hydrolyzate waste (FPH) from the pharmaceutical industry (compost AH). RESULTS: Compost AH resulted in greater N immobilization than compost A because the former supplied the soil with easily degradable C and N, which increased microbial biomass and activity. Both compost mineralizations during the first year of incubation supplied the soil with more nutrients (mainly N), more so with A than with AH. Nevertheless, plant growth was similar in soils amended with either A or AH. Both composts induced nodulation similarly and the highest dose (96 t ha(-1) ) increased the formation of nodules by a factor of 11 compared with the four lower doses. CONCLUSION: TPOMW serves as an effective ground material for co-composting with liquid wastes such as FPH. TPOMW supplies key nutrients and stimulates nodulation in alfalfa.

Assessing the effect of organic amendments on the degradation of profoxydim in paddy soils: Kinetic modeling and identification of degradation products.

The fate and behavior of herbicides can be altered in an unpredictable way when organic amendments are added to soil as a beneficial management tool. The objective of this work was to investigate the effect exerted by the addition of two different organic amendments (alperujo compost and biochar) to soil in the degradation of one of the most relevant new generation rice herbicides, profoxydim. In unamended soils, the degradation of profoxydim was quite fast and was governed by both chemical (DT50steril soil = from 1.52 to 9.21 days) and microbial (DT50nonsterile soil = from 0.47 to 0.53 days) processes. Alperujo- and biochar-amended soils significantly increased the persistence of the herbicide in both soils, especially in the presence of biochar, due to the high capacity absorption of this amendment, increasing DT90 from 1.92 to 3.54 days for DT90unamended to 41.02-48.41 days for DT90biochar amended. Different kinetics models applied to fit the observed dissipation datasets showed that a HS biphasic model fits well with the dissipation of profoxydim in amended and unamended soils. For the first time, five degradation products (DPs) were identified by HPLC-QTOF-MS/MS in soil and a degradation pathway was described. Main DP was generated via oxidation of the sulfur atom to give rise to the corresponding sulfoxide derivative, with this DP being more persistent than the active substance. These outcomes can be very useful for the assessment of the environmental risk associated with the use of profoxydim in rice crops and the application of organic amendments as potential measures for minimizing the risk of contamination of natural water resources.

Effects of massive desiccation of olive waste residues on air quality.

New industries are proliferating in the recovery of agri-food wastes, such as those involved in the revaluation of alperujo, generated in the production of olive oil. Despite the potential environmental benefits, their activity is not exempt from new forms of emissions, aggravated by the massification of waste treatments. This work reports a six-month field campaign carried out in an alperujo desiccation plant which can serve as a proxy for these emerging industries in the Mediterranean countries. The study focused on air quality parameters, covering criteria pollutants, metals and microbiological load of particulate matter and the characterization of volatile organic compounds (VOCs). The results show a slight contribution of the factory to the NOx levels in the surroundings (3.0-12.5 µg/m3). Statistically significant effects were not observed for ozone, CO, SO2, or PM10. Concerning the levels of metals, concentrations were low and calculated health risk indexes indicated safe conditions in the area. The most abundant elements were Na (6.5 × 102 ng/m3), K (4.0 × 102 ng/m3), Al (2.7 × 102 ng/m3), Zn (2.1 × 102 ng/m3), Ca (2.16 × 102 ng/m3), Fe (3.6 × 101 ng/m3) and Mg (3.2 × 101 ng/m3). Bacterial counts, with a mean value of 15.9 CFU/m3, showed a seasonal shift, mainly explained by weather (air moisture and temperature) and PM2.5 concentration. The genomic analysis showed Cutibacterium as the dominant genus during the cold months while Bacillus predominated in the warm season. The VOCs with higher average concentrations were acetic acid (130 µg/m3), nonanoic acid (124 µg/m3), benzoic acid (29.7 µg/m3), octanoic acid (19.9 µg/m3) and nonanal (4.70 µg/m3), with the rest of compounds in concentrations below 4 µg/m3. Odorant pollutants with the greatest contribution to olfactory nuisance were aldehydes (from pentanaldehyde to decanaldehyde), acetic acid and phenol. Although the observable effects of the waste treatments were low, several parameters showed an influence on the environment which should be assessed to foresee and prevent long-term consequences.

Ant Community Is Not Influenced by the Addition of Olive Mill Pomace Compost in Two Different Olive Crop Managements.

Industrial production of olive oil generates large amounts of solid waste called 'alperujo'. Its compost can fertilize many crops, especially olives. Furthermore, superintensive orchards are increasing their surface globally due to higher production and savings in different costs. Ants are considered an important part of the arthropod community in olive orchards and could even play a significant role in pest control. The tree canopy and ground were sampled to compare the ant assemblage in plots fertilized with compost and mineral products in two groves with different types of crop management (superintensive and traditional) over two years. The numbers of ants in both types of fertilization in each grove were not statistically different (p > 0.05), indicating that the type of fertilization did not have a significant impact on its populations in the ground or in the canopy, but the number of individuals was significantly higher in the superintensive grove than in the traditional grove (both in the ground and in the canopy, p < 0.01). The most frequent species in the ground were Pheidole pallidula, Plagiolepis smitzii and Aphaenogaster senilis (superintensive grove) and Pheidole pallidula, Tetramorium gr semilaeve, Plagiolepis pygmaea, and Tapinoma nigerrimum (traditional grove). In the canopy, the most frequent species were Plagiolepis spp. in both groves. Differences in ant densities and species between the groves could be due to the different management, especially of the soil, but it must be confirmed using more replicas and longer periods of study.

Application of Thermo-Malaxation Followed by Three-Phase Centrifugation to Enable the Biorefinery of Alperujo, the Main By-Product of Olive Oil.

The pomace olive oil sector needs to improve its use of the main olive oil by-product, called alperujo, which is currently used mainly for combustion after extraction of pomace oil, with all the problems this process entails due to the high degree of humidity, organic load and phytotoxic substances. In this work, a solution at an industrial level that uses thermo-malaxation at a temperature close to 65 °C for one or two hours followed by centrifugation in three phases is proposed. In this way, over 40% of the pomace oil that is rich in minor compounds, a solid with a lower degree of humidity (55%), and a liquid aqueous fraction that is rich in bioactive compounds such as phenolics and sugars are obtained. This aqueous fraction can be treated through subsequent storage stages to increase its content of the main phenolic, hydroxytyrosol, to up to 1.77 g/L, decreasing its percentage of insoluble solids by up to 1.9%, making it possible to obtain extracts that are rich in hydroxytyrosol using systems that are commonly in place at the industrial level. The aqueous fraction, without phenolics, could be used for energy production. A solid with a slightly higher fat content than the initial alperujo remains, thus the rest of the oil content can be extracted from it using solvent, making it, once defatted, suitable for application in subsequent bioprocesses.

An Acid-Free Alternative to Pectin Production from the Cell Walls of Olive Oil Waste and Different Fruits Using Choline Chloride.

The pectin from the cell walls of olive waste (alperujo) and apple, orange and strawberry fruits was extracted using choline chloride (ChCl) and the yield and chemical and structural compositions were compared to pectin extracted using citric acid (CA) and ammonium oxalate/oxalic acid (AOOA). According to the results, the alperujo pectin extracted using ChCl from alcohol-insoluble residue (AIR) showed a higher yield (2.20-2.88% on the basis of dry weight of AIR) than using CA (0.65-1.22%) but lower than using AOOA (3.92-5.42%). For fruit pectin, the highest yield was obtained using CA (8.81-16%), followed by AOOA (5.4-6.63%), although for apple pectin, ChCl gave a similar yield (5.36%) to AOOA. The uronic acid contents in all ChCl pectins (45.9-70.6% dry basis AIR) were higher or similar to that of the other extracting agents (30.6-65.2%), although a lower level of neutral sugar side chains was detected, with a lower degree of branching and degree of methylation. The NMR and FT-IR spectroscopy of the pectin isolated using ChCl confirmed its slightly different structural composition with respect to CA and AOOA pectin. Therefore, depending on the source material and functionality, pectin isolated using ChCl could be an acid-free alternative to pectin production.

Bioconversion of alperujo into an alternative feed for ruminants by pretreatment with live yeasts and/or exogenous fibrolytic enzymes.

Extraction of olive oil through a two-stage centrifugation process produces a large amount of phytotoxic waste known as alperujo. This research was performed to bioconvert alperujo into enriched ruminant feed by pretreatment with exogenous fibrolytic enzymes (EFE) or/and live yeasts (LY). These additives were used in a completely randomized design with 3 EFE doses (0, 4, and 8 µl/g dry matter) and 3 LY doses (0, 4, and 8 mg/g dry matter) in a 3 × 3 factorial arrangement. Fermented alperujo with both EFE doses converted some of their hemicellulose and cellulose to simple sugars and increased bacterial abundance in the rumen. As a result, it shortens the lag time of rumen fermentation, increases the rate and amount of rumen fermentation, and improves digestibility. This improvement provides additional energy that can be used by ruminants to produce milk and by rumen microbiota to produce short-chain fatty acids. Fermented alperujo with a high dose of LY decreased their antinutritional compounds and reduced their high content of lipid. In the rumen, this waste became rapidly fermentable, and rumen bacteria became more abundance. Fermented alperujo with a high dose of LY + EFE accelerated rumen fermentation and improved rumen digestibility, energy available for milk production, and short-chain fatty acids compared to the use of LY or EFE alone. This synergistic interaction between these two additives increased protozoa abundance in rumen and the ability of rumen microbiota to bioconvert ammonia-nitrogen to microbial protein. Ultimately, fermentation alperujo with EFE + LY is a good strategy with minimum investment for a social sustainable economy and environment.

Replacement of Vitamin E by an Extract from an Olive Oil By-Product, Rich in Hydroxytyrosol, in Broiler Diets: Effects on Growth Performance and Breast Meat Quality.

The hypothesis of this experiment was that a liquid rich in hydroxytyrosol (HT) obtained from "alperujo", an olive oil by-product, could replace part of the added vitamin E (VE) as an antioxidant in poultry diets. There were five diets that differed exclusively in the substitution of supplemental VE (0 to 40 mg/kg, with differences of 10 mg/kg) by HT (30 to 0 mg/kg, with differences of 7.5 mg/kg). The basal diet was based on corn and soybean meal and provided 10 mg VE/kg. From 0 to 39 d of age, the growth performance of the birds was not affected by diet. The birds were slaughtered at 39 d of age to evaluate the quality of the breast, and malonaldehyde concentration, pH, color, and drip loss were measured. In terms of meat lipid oxidation, the combination of 22.5 mg HT/kg and 10 mg of added VE/kg equalized to a diet supplemented with 40 mg VE/kg. Meat color improved in broilers fed 7.5 mg HT/kg and 30 mg VE/kg. It is concluded that once the nutritional requirements of the birds in VE are satisfied, the dietary supplementation with the olive oil by-product rich in HT can be used as a strategy to spare VE in broiler diets.

Modeling the antimicrobial effects of olive mill waste extract, rich in hydroxytyrosol, on the growth of lactic acid bacteria using response surface methodology.

The objective of this study is to assess the inhibitory effects of an aqueous extract from olive oil mill waste (alperujo) on the growth of a lactic acid bacteria (LAB) cocktail consisting of various strains of Lactiplantibacillus pentosus and Lactiplantibacillus plantarum species. For this purpose, response surface methodology was employed using two independent variables (pH levels 3.5-5.55; hydroxytyrosol concentration ranging from 0.93-2990 ppm). The response variable was the average inhibition per treatment on the LAB cocktail (expressed as a percentage). The developed model identified significant terms, including the linear effect of hydroxytyrosol and pH, their interaction, and the quadratic effect of pH. Maximum inhibition of the LAB cocktail was observed at progressively higher concentrations of hydroxytyrosol and lower pH values. Therefore, complete inhibition of LAB in the synthetic culture medium could only be achieved for concentrations of 2984 ppm hydroxytyrosol at a pH of 3.95. These findings suggest that extracts derived from "alperujo" could be utilized as a natural preservative in acidified foods with a bitter flavor and antioxidant requirements.

Facile one-step synthesis of a versatile nitrogen-doped hydrochar from olive oil production waste, "alperujo", for removing pharmaceuticals from wastewater.

In line with the principles of zero waste and recycling, alperujo (AL) was used in this study to produce a value-added product: hydrochar (HC) with high adsorption capacity. An optimization of the hydrothermal carbonization (HTC) conditions, such as temperature, residence time, and water/solid ratio, was carried out to maximize the adsorption capacity. Eight HCs were obtained, and an in-depth comparative characterization, as well as adsorption tests of two pharmaceuticals with very different physicochemical properties (fluoxetine (FLX) and cefazolin (CFZ)), were performed. This first step allowed for elucidation of the best candidates to carry out nitrogen grafting on their surface, resulting in the HC obtained at a higher water/solid ratio and temperature, and longer residence time: 3-220°C-2.5 h with a maximum uptake of 4.6 and 0.4 mg/g for FLX and CFZ, respectively. After that, a facile one-step, one-pot synthesis of nitrogen-doped hydrochars (N-HC) was developed to prepare a versatile bio-adsorbent with enhanced adsorption capacity. Two N-HCs were prepared using urea (U-HC) and polyethyleneimine (PEI-HC) and were intensively characterized to shed light on the adsorption mechanism. In both cases, amide groups were formed, which favored the adsorption process. PEI-HC acquired an outstanding maximum adsorption capacity of 983.84 mg/g for CFZ, and 29.31 mg/g for FLX, and the process was well described by the Freundlich isotherm and pseudo-second-order kinetic model. A co-adsorption test was performed using PEI-HC for both pharmaceuticals, finding that the adsorption process occurs in different active sites because there was no interference between the pollutants. This fact corroborates the versatility of the new bio-adsorbent synthesized.

Extraction of polyphenols associated with pectin from olive waste (alperujo) with choline chloride.

The main by-product from olive oil extraction (alperujo) was extracted with hot water, citric acid, natural deep eutectic solvent (choline chloride: citric acid), and only choline chloride. The purified extracts were composed of macromolecular complexes constituting polyphenols associated with pectin. The extracts were structurally characterized by FT-IR and solid-NMR spectroscopy and an in vitro test revealed distinct antioxidant and antiproliferative activity, depending on the extracting agents. The choline chloride-extracted complex contained the highest amount of polyphenols among the examined agents, which exhibited a strong antioxidant activity and significant antiproliferative capacity. However, the complex extracted by hot water showed the highest antiproliferative capacity in vitro against the colon carcinoma Caco-2 cell line. In this finding, choline chloride could be used as a novel, green and promising alternative to the conventional extracting agent for the production of complexes that combine the antioxidant activity of phenolic compounds and the physiological effects of pectic polysaccharides.