Olive Vegetation Water Research Articles

Effect of phenols extracted from a by-product of the oil mill on the shelf-life of raw and cooked fresh pork sausages in the absence of chemical additives

Replacing chemical additives in meat preparations with natural compounds is a matter of great interest today, both to consumers and to the food sector. The effect of an extract rich in phenols obtained from olive vegetation water (an agricultural by-product) on the pH, weight loss after cooking, diacylglycerols (DAGs), peroxide value (POV), thiobarbituric acid reactive species (TBARS), and cholesterol oxidation products (COPs) of raw and cooked fresh pork sausages prepared without chemical additives was evaluated before and after aerobic storage at 2 ± 2 °C for 14 d. Adding the extract at concentrations of 0.075 and 0.15 g/100 g resulted in a decrease in pH, DAGs, POV, TBARS and COPs; notably, the COPs levels were 4- and 17-fold lower in raw and cooked sausages, respectively. Sensory analysis revealed significant differences between control samples and those enriched with the extract, but the enriched samples were never considered unpleasant by the panellists. Storing the raw sausages for 14 d and subsequently cooking them led to 58% and 49% decreases in phenols, respectively. The purified phenols from olive oil wastewater proved to be an effective antioxidant, thus demonstrating themselves to be a potential ingredient to ensure the quality and safety of meat preparations.

Effect of an olive phenolic extract added to the oily phase of a tomato sauce, on the preservation of phenols and carotenoids during domestic cooking

The protective effect of a phenolic extract from olive vegetation water on carotenoids and other phytonutrients has been evaluated during a home-cooking procedure to prepare tomato sauce. To specifically investigate on the complex mechanisms involved in the thermal degradation and solubilization of carotenoids, the olive phenols were added to a refined olive oil, chosen as control fat, in order to distinguish the effect of the simple oily matrix from that of the phenols occurring in virgin olive oils.Three sauces were prepared using a commercial tomato “passata”, carrots, celery and onion. Two sauces contained refined olive oil to which was added a phenolic extract at 40 or 60 mg/100 g of polyphenols, the third was the control with refined olive oil. The results revealed positive effect of the phenolic extract on the phytonutrient content of the tomato sauce. In the experimental sauces for the hydrophilic phenols, an improvement of approximately 100% was observed at both levels of phenolic extract addition in comparison to the control. There was a greater than 50% increase in a-tocopherol and a 43% increase in carotenoids with the lower level of phenols addition, and greater than 58% with the higher level of enrichment.

Effect of a Phenolic Extract from Olive Vegetation Water on Fresh Salmon Steak Quality during Storage.

This study aimed to evaluate the antioxidant and antimicrobial effects of a phenolic extract from olive vegetation water on fresh salmon steaks stored at 4°C under modified atmosphere. Twenty-four salmon steaks were respectively immersed in solutions of the diluted phenolic extract at 1.5 g/L (A), 3 g/L (B), and water only as a control (CTR), packaged within a protective atmosphere (70% carbon dioxide, 25% nitrogen and 5% oxygen) and then stored at 4°C. After 2 h, and 3 and 6 days of storage, the fish samples were analysed for the total viable count, Enterobacteriaceae count, pH, colour (CIE L*a*b* colour system), phenolic composition, atocopherol content, antioxidant activity by 2,2- diphenyl- 1-picrylhydrazyl (DPPH˙) assay, and thiobarbituric reactive substances (TBARS). A 3 g/L phenolic extract contributed positively to the hygienic quality of the salmon by reducing the microbial growth during storage. The treated samples were slightly yellower than the CTR but only at the beginning of storage. The flesh contained 6.2% of the total polyphenols present in the initial solutions, with various percentages of the single fractions. After 6 days storage, the α- tocopherol content in the CTR and A samples was statistically lower than the B group that also showed the lowest DPPH˙ and TBARS values. In conclusion, the phenolic extract increased the microbiological quality and antioxidant concentration and decreased the lipid oxidation of salmon steaks during storage at 4°C under modified atmosphere.

Use Efficiency of Cyanobacteria and Olive Vegetation Water (Cyano/Ovw) Biofertilizer for Olive Trees under Different Mineral Npk Levels

Use Efficiency of Cyanobacteria and Olive Vegetation Water (Cyano/Ovw) Biofertilizer for Olive Trees under Different Mineral Npk Levels

Using a Concentrate of Phenols Obtained from Olive Vegetation Water to Preserve Chilled Food: Two Case Studies.

Phenols are plant metabolites characterised by several interesting bioactive properties such as antioxidant and bactericidal activities. In this study the application of a phenols concentrate (PC) from olive vegetation water to two different fresh products – gilt-head seabream (Sparus aurata) and chicken breast – was described. Products were treated in a bath of PC (22 g/L; chicken breast) or sprayed with two different solutions (L1:0.75 and L2:1.5 mg/mL; seabream) and then stored under refrigeration conditions. The shelf life was monitored through microbiological analyses – quality index method for seabream and a specific sensory index for raw breast. The secondary products of lipid-peroxidation of the chicken breast were determined using the thiobarbituric acid reactive substances (TBARs) test on cooked samples. Multivariate statistical techniques were adopted to investigate the impact of phenols and microbiological data were fitted by DMfit software. In seabream, the levels of PC did not highlight any significant difference on microbiological and sensory features. DMfit models suggested an effect only on H2S producing bacteria with an increased lag phase compared to the control samples (C: 87 h vs L2: 136 h). The results on chicken breast showed that the PC bath clearly modified the growth of Pseudomonas and Enterobacteriaceae. The phenol dipping was effective in limiting lipid-peroxidation (TBARs) after cooking. Treated samples disclosed an increase of shelf life of 2 days. These could be considered as preliminary findings suggesting the use of this concentrate as preservative in some fresh products.

Agricultural by-products with bioactive effects: A multivariate approach to evaluate microbial and physicochemical changes in a fresh pork sausage enriched with phenolic compounds from olive vegetation water

The use of phenolic compounds derived from agricultural by-products could be considered as an eco-friendly strategy for food preservation. In this study a purified phenol extract from olive vegetation water (PEOVW) was explored as a potential bioactive ingredient for meat products using Italian fresh sausage as food model. The research was developed in two steps: first, an in vitro delineation of the extract antimicrobial activities was performed, then, the PEOVW was tested in the food model to investigate the possible application in food manufacturing. The in vitro tests showed that PEOVW clearly inhibits the growth of food-borne pathogens such as Listeria monocytogenes and Staphylococcus aureus. The major part of Gram-positive strains was inhibited at the low concentrations (0.375–3mg/mL). In the production of raw sausages, two concentrates of PEOVW (L1: 0.075% and L2: 0.15%) were used taking into account both organoleptic traits and the bactericidal effects. A multivariate statistical approach allowed the definition of the microbial and physicochemical changes of sausages during the shelf life (14days). In general, the inclusion of the L2 concentration reduced the growth of several microbial targets, especially Staphylococcus spp. and LABs (2log10CFU/g reduction), while the increasing the growth of yeasts was observed. The reduction of microbial growth could be involved in the reduced lipolysis of raw sausages supplemented with PEOVW as highlighted by the lower amount of diacylglycerols. Moisture and aw had a significant effect on the variability of microbiological features, while food matrix (the sausages' environment) can mask the effects of PEOVW on other targets (e.g. Pseudomonas). Moreover, the molecular identification of the main representative taxa collected during the experimentation allowed the evaluation of the effects of phenols on the selection of bacteria. Genetic data suggested a possible strain selection based on storage time and the addition of phenol compounds especially on LABs and Staphylococcus spp. The modulation effects on lipolysis and the reduction of several microbial targets in a naturally contaminated product indicates that PEOVW may be useful as an ingredient in fresh sausages for improving food safety and quality.

New approaches to virgin olive oil quality, technology, and by‐products valorization

Extra virgin olive oil (EVOO) merchandize quality is based on analytical parameters describing alteration status and assuring genuineness, but qualitative/quantitative composition of those quality markers related to health and sensory aspects such as monounsaturated fatty acids, phenolic compounds as secoiridoid derivatives, and C5 and C6 volatile compounds are not mentioned. This paper focuses on the new approaches to the definition of EVOO quality that might include also healthy and sensory parameters and on the agronomic and technological factors that most influence their variability. Crushing and malaxation are discussed as critical points of the technological process since their direct involvement on the phenolic release and on the aroma generation. Variables such as time, temperature, and oxygen level, during malaxation, were analyzed to design new implant systems for the improvement of EVOO quality. Flash thermal conditioning (FTC) of the crushed olive pastes was briefly discussed as a new approach to the technological process. The rapid reaching of the optimal malaxing temperature seems to improve polyphenols and volatile concentration. The paper shows also recent studies on the valorization of the mechanical extraction co‐products as stoned pomaces for the animal feeding and vegetation waters phenolic concentrates and extracts for functional foods and ingredients production. This article provides an overview of the Author keynote lecture delivered at the Euro Fed Lipid Congress and focuses on the group's works.The GA report, shows the new approach to the EVOO technological process through the use of the flash thermal conditioning (FTC) of the crushed olive pastes to improve EVOO quality and the results obtained in terms of by‐products valorization showing the new applications of pomaces as animal feed supplement and the recovery of the crude phenolic concentrate (CPC) from olive vegetation water that could be used for functional food and ingredients production.

Effect of an olive phenolic extract on the quality of vegetable oils during frying

The potential of a phenolic extract (PE) from olive vegetation water (OVW) to limit the negative effects of frying was tested after adding it at different concentrations to a refined olive oil (RO). Its efficacy was also compared to ROs containing butylated hydroxytoluene (BHT) and an extra virgin olive oil (EVOO) with a high polyphenol content. Analyses of the oils collected after 30min, 1, 2, 4, 6, 8, 10 and 12h of frying at 180°C, demonstrated that degradation of the polyphenols was proportional to the original content; at a concentration of at least 400mg/kg of polyphenols, PE was able to reduce oxidation of the tocopherols and the emission of low-molecular-weight aldehydes better than BHT and with similar results to the EVOO. In addition, secoiridoid oxidative compounds were examined by high-performance liquid chromatography/triple quadrupole tandem mass spectrometry with electrospray ionisation.

CO-COMPOSTING ALPERUJO WITH OTHER AGRO-INDUSTRIAL RESIDUES AS SUSTAINABLE PRACTICE FOR ITS RECYCLING

The main by-product generated by Argentinean olive oil industry is a semisolid cake made of olive pulp, stone and vegetation water locally called alperujo. It is reported that composting represents an interesting option to recycle it; however, this practice is rarely implemented by olive industries in Argentina. The aim of this work was to evaluate two low-technology methods, open and covered piles with mechanical turnovers, for their effect on composting a mixture of alperujo, olive leaves and grape stalk (56:22:22) with or without urea addition. Temperature, organic matter content (OM) and phenolic compounds (PC) were measured to follow up the composting process. While electrical conductivity (EC), pH, macro- and micro-nutrients, as well as germination index (GI) of Medicago sativa seeds were determined at the end of the experiment in order to assess compost quality. Additionally, to infer the soil and water conditions required to apply the obtained compost, the agricultural index (AgIndex) was calculated. Results showed that all piles presented similar evolution patterns, achieving maturity within 150 days. Temperature rose upwards in the thermophilic phase and next it decreased through the maturation phase. pH increased along the experiment and reached slightly alkaline level at maturity. OM content and C/N ratio decreased, this decrease being higher in covered piles than in open ones. Interestingly, PC was highly reduced in all treatments and was inversely correlated with the increase in GI. Furthermore AgIndex indicated that the obtained compost may be appropriate for amending soils with excellent drainage characteristics, low salt content and good water quality. The results suggest that evaluated methods are suitable for composting alperujo, decrease its toxicity and obtain a product appropriate for amending soils where olive groves are usually implanted. Undoubtedly the long time required to reach maturity may represent a negative feature. In this sense further works, focused on conditions to accelerate the process should be carried out.

Polyphenols from olive mill waste affect biofilm formation and motility in Escherichia coli K-12.

Olive mill wastes are sources of phenolic compounds with a wide array of biological activities, including antimicrobial effects. A potential option for bioremediation to overcome ecological problems is the reutilization of these natural compounds in food production. The aim of this work was to gain a better understanding of the antimicrobial mode of action of a phenols extract from olive vegetation water (PEOVW) at molecular level by studying Escherichia coli as a model microorganism. Genome-wide transcriptional analysis was performed on E. coli K-12 exposed to PEOVW. The repression of genes for flagellar synthesis and the involvement of genes linked to biofilm formation and stress response were observed. Sub-inhibitory concentrations of PEOVW significantly decreased biofilm formation, swarming and swimming motility, thus confirming the gene expression data. This study provides interesting insights on the molecular action of PEOVW on E. coli K-12. Given these anti-biofilm properties and considering that biofilm formation is a serious problem for the food industry and human health, PEOVW has proved to be a high-value natural product.Olive mill wastes are sources of phenolic compounds with a wide array of biological activities, including antimicrobial effects. Genome-wide transcriptional analysis was performed on E. coli K-12 exposed to phenols extract from olive vegetation water (PEOVW). Sub-inhibitory concentrations of PEOVW significantly decreased biofilm formation, swarming and swimming motility. Given these anti-biofilm properties PEOVW has proved to be a high-value natural product.

Hydroxytyrosol Is the Major Anti-Inflammatory Compound in Aqueous Olive Extracts and Impairs Cytokine and Chemokine Production in Macrophages

Substances in olive products contribute to improved health as suggested by epidemiological data. In this study we assessed the effects of hydroxytyrosol (HT) on inflammatory mediators, cytokines and chemokines, and identified anti-inflammatory constituents of aqueous olive extracts, I.E., olive vegetation water (OVW). Murine macrophages (RAW264.7 cells) were stimulated with lipopolysaccharide (LPS) in the absence or presence of substances; inflammatory mediators [nitric oxide (NO), prostaglandin E₂ (PGE₂), cytokines, interleukins, chemokines] were determined by the Griess reaction, EIA, or multiplex ELISA (Luminex technology). Expression of inflammatory genes was determined by RT-PCR. Aqueous olive extracts were fractionated by preparative HPLC and the fractions investigated for their effects on NO and PGE₂ production. Results were further analyzed by principal component analysis. HT inhibited production of NO and PGE₂ with an IC₅₀ of 11.4 and 19.5 µM, respectively, reflecting strong anti-inflammatory activity. HT and OVW diminished secretion of cytokines (IL-1 α, IL-1 β, IL-6, IL-12, TNF- α), and chemokines (CXCL10/IP-10, CCL2/MCP-1). HT and OVW concentration-dependently reduced the expression of genes of inducible nitric oxide synthase (iNOS), IL-1 α, CXCL10/IP-10, MIP-1 β, matrix metalloproteinase-9, and prostaglandin E₂ synthase (PGES). The effects of HT were partly mediated VIA the NF- κB pathway, as shown by RT-PCR analysis. HT was identified as the main bioactive compound of OVW. The data provide a molecular basis for elucidating the effects of HT on inflammatory processes. The effects of HT on NO and chemokine production point to their impact on chronic inflammatory processes in endothelium or arthritis.

Effects of olive oil solid waste on growth and nutrient uptake of faba bean, onion, and radish plants

Although, olive oil industry is crucial for Turkey, olive oil solid waste or olive vegetation water, a by-product of the industry, leads to environmental pollution during olive oil production. This study investigated the fields where olive oil solid waste was used as an organic substance. Olive oil solid waste in different concentrations (0, 10, 20, 30 and 40 t ha-1) were applied to faba bean, onion and radish plants grown (in 1 m2 plots) in the field conditions. Germination rates and growth of the plants were monitored. The application at the rates of 10 to 30 t ha-1were determined as the most appropriate rates for both faba bean and onion. The higher rates of application negatively affected germination and growth of plants. The application at 20 t ha-1 concentration of olive oil solid waste provided the best results for faba bean and onion plants. However, none of the application rates were found suitable for radish. Keywords: Olive oil solid waste; germination; plant growth; plant analysis

Functional milk beverage fortified with phenolic compounds extracted from olive vegetation water, and fermented with functional lactic acid bacteria

Functional milk beverages (FMB100 and FMB200) fortified with phenolic compounds (100 and 200mg/l) extracted from olive vegetable water, and fermented with γ-amino butyric acid (GABA)-producing (Lactobacillus plantarum C48) and autochthonous human gastro-intestinal (Lactobacillus paracasei 15N) lactic acid bacteria were manufactured. A milk beverage (MB), without addition of phenolic compounds, was used as the control. Except for a longer latency phase of FMB200, the three beverages showed an almost similar kinetic of acidification, consumption of lactose and synthesis of lactic acid. Apart from the beverage, Lb. plantarum C48 showed a decrease of ca. Log 2.52-2.24 cfu/ml during storage. The cell density of functional Lb. paracasei 15N remained always above the value of Log 8.0 cfu/ml. During fermentation, the total concentration of free amino acids markedly increased without significant (P > 0.05) differences between beverages. The concentration of GABA increased during fermentation and further storage (63.0 ± 0.6-67.0 ± 2.1mg/l) without significant (P > 0.05) differences between beverages. After fermentation, FMB100 and FMB200 showed the same phenolic composition of the phenol extract from olive vegetable water but a different ratio between 3,4-DHPEA and 3,4-DHPEA-EDA. During storage, the concentrations of 3,4-DHPEA-EDA, p-HPEA and verbascoside of both FMB100 and FMB200 decreased. Only the concentration of 3,4-DHPEA increased. As shown by SPME-GC-MS analysis, diactetyl, acetoin and, especially, acetaldehyde were the main volatile compounds found. The concentration of phenolic compounds does not interfere with the volatile composition. Sensory analyses based on triangle and paired comparison tests showed that phenolic compounds at the concentrations of 100 or 200mg/l were suitable for addition to functional milk beverages.

Improvement of bioactive phenol content in virgin olive oil with an olive-vegetation water concentrate produced by membrane treatment

Olive-vegetation water (OVW) is very rich in phenols, which can cause pollution problems, due to their high hydrophilicity and antimicrobial activity. In this study, a three-phase membrane system was applied for the recovery of hydrophilic phenols from fresh OVW in an industrial plant, through a prior enzymatic treatment. This innovation yielded both a crude phenolic concentrate (CPC) and a very high reduction in OVW pollution. Furthermore, the CPC was utilised in a virgin olive oil (VOO) extraction process with the aim of improving VOO phenolic content. The results obtained with four different olive cultivars showed that the CPC extraction increased the phenolic content of the VOOs without any alteration of their aroma profiles.

Olive extract supplement decreases pain and improves daily activities in adults with osteoarthritis and decreases plasma homocysteine in those with rheumatoid arthritis

The aim of this study was to determine the effects of a polyphenolic-rich olive extract on pain, inflammation, loss of mobility, and patients' general quality of life associated with osteoarthritis (OA) and rheumatoid arthritis (RA). In this double-blind, randomized, placebo-controlled study, the efficacy of freeze-dried olive vegetation water (OVW) on subjects with a diagnosis of OA or RA was evaluated. The treatment group received 400 mg of freeze-dried OVW per day for 8 weeks. Subjects were assessed using the Health Assessment Questionnaire–Disability Index. In addition, the Disease Activity Score With 28-Joint Count was used to evaluate joint pain and inflammation, and the Profile of Moods State questionnaire for overall well-being. During the same time intervals, serum samples were taken for clinical and biochemical tests. The RA subjects in the OVW treatment group showed significant decreases in serum homocysteine levels after 8 weeks of treatment. There was no significant change in any other clinical marker, including markers of kidney and liver function, at any time during the study, suggesting that the supplement was safe. There was significant improvement by treatment group for the Health Assessment Questionnaire–Disability Index. Subjects with OA in the OVW treatment group showed significant improvement in the Disease Activity Score With 28-Joint Count index. Thus, after a relatively short treatment with OVW, subjects with OA and RA reported decreased pain and improvement in activities of daily living. In addition, subjects with RA had a statistically significant decrease in serum homocysteine. High homocysteine levels in patients with RA have been associated with higher rates of mortality from cardiovascular events.

Matter transfer during virgin olive oil elaboration

In the course of the process of elaboration of virgin olive oil (whose main stages are crushing, malaxation and centrifugation) the transfer of several minor components to the triacylglycerol constituent of the oily globules originally present in the cells of the fruit mesocarp is produced. Such minor components are chemical species present in the olive fruits, as well as those resulting from chemical or enzymatic processes which take place in the olive paste upon crushing. In this paper several types of transferable minor components, as well as those parameters affecting the rates of transfer are studied.Geometric and physical variations of interfacial regions between the oil and other systems in contact with it are discussed. These systems are olive vegetation water, olive pulp components, and fragments of the woody endocarp, as well as the atmospheric air and the tools and equipment of the oil mill. This article concludes with some considerations about the improvements of virgin olive oils achieved by controlling the transfer of minor components.

Production of Natural Fruity Aroma by Geotrichum candidum

Based on its aromatic potential, Geotrichum candidum isolated from olive vegetation water was tested for the production of volatile compounds. When G. candidum was cultivated on media with glucose as the carbon source, flavor volatile compounds were produced and accumulated in the broth. Fruity flavoring compounds (pineapple-like) such as esters and alcohols were analyzed by gas chromatography coupled to mass spectrometry, including ethyl esters of acetic acid and butyric acid, methyl-3-butan-1-ol, and methyl-2-propan-1-ol. Their synthesis corresponded to the stationary growth phase of the strain. Production of the volatile compounds reached 9.5 g/L of 2-hexanoic acid ethyl ester and 1.6 g/L of benzaldehyde as the main concentrated molecules. Ethyl alcohol seems to be an intermediate metabolite in this pathway.

Hydrolyzed Olive Vegetation Water in Mice Has Anti-Inflammatory Activity

Fruit and vegetable simple and polyphenols are potent antioxidants. One of the most effective in terms of free radical scavenging is 3,4-dihydroxyphenyl ethanol or hydroxytyrosol (HT), a simple phenol found predominantly in Olea europea, or the olive plant. HT is most abundant in the aqueous fraction of olive pulp with trace amounts in the olive oil fraction and in the leaves. For these experiments, we evaluated the anti-inflammatory activity of olive vegetation water (OVW), which we showed previously to have potent antioxidant activity. Because some simple phenols and polyphenols with antioxidant activity have shown varying anti-inflammatory activities, we tested OVW and HT for their ability to inhibit the production of tumor necrosis factor-alpha (TNF-alpha), a pivotal cytokine in inflammation. In lipopolysaccharide (LPS)-treated BALB/c mice, a model system of inflammation, OVW at a dose of 125 mg/mouse (500 mg/kg) reduced serum TNF-alpha levels by 95%. In the human monocyte cell line, THP-1, OVW reduced LPS-induced TNF-alpha production by 50% at a concentration of 0.5 g/L (equivalent to approximately 0.03 g/L simple and polyphenols). OVW had no toxic effects in vitro or in vivo. When OVW was combined with glucosamine, a component of proteoglycans and glycoproteins that was shown to decrease inducible nitric oxide synthase production in cultured macrophage cells, the 2 compounds acted synergistically to reduce serum TNF-alpha levels in LPS-treated mice. These findings suggest that a combination of OVW and glucosamine may be an effective therapy for a variety of inflammatory processes, including rheumatoid and osteoarthritis.

Ultrafiltration plant for olive vegetation waters by polymeric membrane batteries

Within the reconversion activities of the industrial complex of S. Eufemia Lamezia (Cz, Italy) into an environmental service plant system, a vegetation water treatment plant was started in November 1995 suitable to treat a nominal capacity of 300 m 3/d flowing from olive-oil mills. The treatment plant is based on the following steps: 1) Dirty water storage, 2) oil removal system, 3) Settling of suspended solids, 4) Tangential filtration on polymeric membrane, 5) Eluate treatment by means of polymeric membranes in a double-step biological process complying with Italian standards. The final reconversion of the industrial complex into an environmental service plant system will be composed of a sewage treatment line, an oil vegetation treatment line, a solid waste incineration line, and a compost solid waste line.

Biomethanization

Vegetation water in the olive constitutes 45–50% of the fruit, with a total content of 170kgm −3, of which 150kgm −3 are organic compounds (80% of these can be directly converted to methane). Anaerobic digestion of this waste yields methane with an average content of 37m 3m −3 of vegetation water and an energetic yield of 325KwH, of which 30% can be converted into electrical energy and 63% into calorific energy by the use of cogeneration processes. This is sufficient to cover the energy needs of both an olive oil production plant and an anaerobic reactor for the integral purification of wastewater. From an economic point of view, and taking into account the costs of electric (15ptsKwH −1) and calorific energy (3.2ptstherm −1), the intrinsic value of olive vegetation water would be 2148ptsm −3. For the optimization of this process, the following lines of research were investigated: (i) development of a biological process for the biodegradation of the phenolic compounds responsible for the inhibition of the biomethanization process; (ii) selection of the nature and the characteristics of an appropriate support for the immobilization of anaerobic bacteria; and (iii) development of a system and high density cellular reactor to obtain optimum conditions—hydraulic residence time, methane yield and efficiency for the purification process.