MEDUSA WATER, EU Programme INCO-MED ICA3-CT-1999-00010; OLEICO, EU Programme LIFE04/ENV/IT/000409; MOTIVE, Portuguese FCT Programme POCI/AMB/56616/2004; LIFEN-Energia, Portuguese FCT Programme PTDC/ENR/69755/2006; MEDOLICO, EU Programme ENPI-CBCMED I-B/2.1/090

Olive oil production is a traditional agricultural industry in Mediterranean countries and Portugal is one of the ten major producers. This industry generates an effluent, olive mill wastewater (OMW), which does not undergo any treatment and, usually, is stored in evaporation lagoons or spread on the land. Disposal of olive oil mill wastewaters is a serious environmental problem due to its high organic loading, presence of polyphenols and tannins, high content in suspended solids and acidity, which contributes to its ecotoxicity. In this work it was intended to study the biodegradation of OMW by microrganisms naturally present in these wastewaters. Thus, an aerobic biological treatment system: a packed-bed batch reactor was applied to a OMW from a mill on northern of Portugal, exploring its autochthon microbial population as inoculum. The biodegradation ability of OMW by microrganisms naturally present in these wastewaters was assessed, by following the evolution of the process and monitoring several of its physico-chemical parameters. Furthermore, an ecotoxicological evaluation, using chronic toxicity tests (Pseudomonas putida growth inhibition test and Vibrio fischeri growth inhibition test), was performed to follow the detoxification capacity of the system as well as its potential to be used in the treatment of this type of agroindustrial effluent.

Remediation of olive mill wastewater (OMW) is an important issue associated with olive-oil manufacturing, a widespread activity in the Mediterranean area. This high organic loading effluent contains water, organic acids, high-molecular-weight polyphenols such as tannins, antocyanins and catechins, which are considered to be responsible for its brownish black colour and toxic properties. The composition of OMWs is highly variable with respect to each individual component, depending on the process conditions and on the agricultural specificities. In this work, the ability of a “white-rot” fungus, Bjerkandera paranensis, to use undiluted OMW from a two phase process mill (COD = 11.1 gl -1 ; Phenol Content = 3.9 gl -1 ; ColourAbs395nm = 7.8) as a substrate was studied. The biodegradation potential of B. paranensis was assessed monitoring several physico-chemical parameters. A chronic ecotoxicity test (Vibrio fisheri growth inhibition test) was carried out to follow the detoxification ability of this fungus. In work, the results demonstrate that OMW was a suitable medium for cultivation of B. paranensis, with corresponding changes in the physico-chemical properties of the OMW. The results showed that B. paranensis removed 93% phenols and 54% COD from the culture medium within 21 days of treatment. In addition, the IC50s values obtained for the different treated samples showed a significant decrease in the effluent chronic toxicity to V. fischeri when the OMW pH was adjusted to 6.0 prior to the treatment (71.8 %), highlighting the OMW detoxification capacity of B. paranensis.

Lignocellulosic biomass can be utilized to produce ethanol, a promising alternative energy source for the limited crude oil. Wheat straw is an abundant agricultural residue which can be used as lignocellulosic raw material for bioconversion. There are mainly two processes involved in the bioconversion: hydrolysis of cellulose in the lignocellulosic biomass to produce reducing sugars, and fermentation of the sugars to ethanol. The current study involved the optimization of enzymatic hydrolysis of a wheat straw pretreated by acid hydrolysis, using a mixture of commercial cellulases: celluclast 1.5L + Novozym 188, with further fermentation of the hydrolisate’ sugar content by three ethanologenic strains, namely two yeast of Saccharomyces cerevisiae (strains F and K) and a bacterial strain, Zymomonas mobilis (strain CP4). The fermentation assays, using undiluted hydrolisate with or without nutrient supplements, were monitored by the evaluation of glucose and ethanol yields. In the assays using no supplemented hydrolisate the results obtained for the two yeasts strains F and K, and Zymomonas mobilis were 74%, 79% and 58% of ethanol yield, respectively. However, when the hydrolisate was supplemented the fermentation results showed a better bioconversion process by the Z. mobilis, reaching 98% ethanol yield while the two strains of S. cerevisiae used maintained their behaviour. So, the fermentation results showed the necessity of the addition of nutrients for a good bioconversion process by the Z. mobilis, resulting in better ethanol yield than S. cerevisiae strains (F and K) from WSP hydrolisate.

In this study, different strains of Saccharomyces cerevisiae have been screened for the ability of bioethanol production. Yeasts were grown in synthetic liquid medium containing two different substrates: sucrose at different concentrations (10 to 400g/l) and cane molasses (120g/l of sucrose). The screening was made in batch regime and the growth rates, ethanol and biomass productions were determined. The results indicate a flocculent yeast strain – F as the more suitable microorganism to produce ethanol, presenting the highest value of growth rate (0.49h -1 ) and ethanol yield (0.40g/g) with 120g/l of sucrose concentration. In addition, ethanol production was also studied in a continuous process with the selected yeast strain (F strain), with sucrose and cane molasses (120g/l) at different dilution rates (0.05-0.5 h -1 ). Data showed that when dilution rate raised to 0.4h -1 the highest sugar conversion, 85% and 90%, were achieved with an ethanol production of 40.5g/l and 50.8g/l for sucrose and molasses, respectively.

The optimization of metabolic pathways is of fundamental importance for strategies aimed at improving the economics and yield of the lignocellulose-to-ethanol processes. Although Escherichia coli is capable of metabolizing a wide variety of substrates including hexoses and pentoses, its hexose metabolism is inferior to that of Zymomonas mobilis, an obligate, ethanologenic bacterium. We therefore inserted and expressed Z. mobilis genes encoding essential enzymes involved in the fermentation pathway, alcohol dehydrogenase II (adh II) and pyruvate decarboxylase (pdc), into E. coli, resulting in increased cell growth and ethanol production. Ethanol concentrations of > 30 g/L were obtained on 10% glucose. Additionally, since pyruvate is mainly assimilated through pyruvate formate lyase (pfl) and forms formic acid and acetyl coenzyme A, metabolic redirection was attempted through gene knockout by Red-mediated recombination to decrease the byproducts of pyruvate metabolism. Under microaerobic conditions, pflA- and pflB-mutants produced more ethanol (163% and 207%, respectively) relative to the parent strain, using glucose as a carbon source.

Olive oil production is a traditional agricultural industry in Mediterranean countries and Portugal is one of the ten major producers. This industry generates an effluent, olive mill wastewater (OMW), which does not undergo any treatment and, usually, is stored in evaporation lagoons or spread on the land. Disposal of olive oil mill wastewaters is a serious environmental problem due to its high organic loading, presence of polyphenols and tannins, high content in suspended solids and acidity, which contributes to its ecotoxicity.

Platinum group metals (PGMs) are highly demanding metals in current high-techs. However, their supply is limited due to their scarcity in natural resources and expensive mining. Solvent extraction in combination with microbial communities were employed for the recovery of palladium from aqueous media. Accordingly, extraction of Pd(II) by N,N′-dimethyl-N,N′-dicyclohexylthiodiglycolamide (DMDCHTDGA) in toluene and subsequent stripping by acidic thiourea were carried out, followed by bio-recovery employing biogenic sulphide generated from sulphate-reducing bacteria. The results revealed an excellent extraction performance of DMDCHTDGA towards Pd(II) from metal complex mixtures in 2, 4 and 6 M HCl. However, Fe(III) was co-extracted from 4 and 6 M HCl, being completely removed with deionized water during scrubbing. 94–99% of extracted Pd(II) were stripped using acidic thiourea. Over 99% of stripped Pd(II) were recovered using two solutions containing biogenic sulphide: an effluent from a bioremediation process of acid mine drainage treatment, and a supernatant solution from Postgate B nutrient medium. 78–99% of scrubbed Fe(III) were bio-recovered. Analysis of all the precipitates collected during palladium bio-recovery showed that they were composed by Pd and S, which is consistent with the synthesis of nanosized PdS. The elemental analysis of the iron precipitate also suggests the presence of iron sulphide. Henceforth, the method engaged is environmentally sustainable, safe and cheap, thus attractive to be employed, to separate and recover both Pd and Fe from waste materials.

Olive oil production is a traditional agricultural industry in Mediterranean countries and Portugal is one of the ten major producers. This industry generates an effluent, olive mill wastewater. This effluent does not undergo any treatment and is usually stored in evaporation lagoons or spread on the land. This can have a negative impact in the environment since this effluent has a high level of organic matter leading to a high chemical oxygen demand. In addition it has also a high content of polyphenols that contributes to the ecotoxicity of this effluent.Different techniques for the treatment of these wastewaters have been studied. In this work a 60 litre vessel was filled with a packaging of plastic material consisting of a cubic geometry (Biological Carrier Media from Rauschert). The non-inoculated reactor was filled with effluent from an olive mill farm (from Alfândega da Fé, Trás-os-Montes) and the effluent was re-circulated daily for homogeneity. COD, colour, nitrogen, solids and phosphorous were measured to follow the evolution of the system. Microbial composition and polyphenols were also evaluated. As an indicator of the microbial activity in the reactor, lipase activities were measured. Ecotoxicity tests were carried out to follow the detoxification capacity of the system as well as its potential for using in the treatment of this type of agroindustrial effluent.

The olive oil industry is one of the most typical and economically important Portuguese agro-industries, 29,900 tons of olive oil having been produced in 2002/2003. This industry generates large amounts of olive oil wastewaters (OOWW), which are difficult to degrade and thus cause a negative environmental impact. Jet-loop reactors (JACTO) developed and scaled-up by our group have been successfully used for biological treatment of winery and OOWW. This study aimed to determine the interactions of reactor hydrodynamics with microflora profiles during bio-treatment of OOWW. Bio-treatment was performed using a 20-dm3 JACTO bioreactor achieving a chemical oxygen demand (COD) and phenolic compounds removal rate of 70% at a hydraulic retention time of 12 days. Bio-treatment was scaled-up to 200-dm3 JACTO bioreactor, reaching 87% COD removal and 80% phenolic compounds removal. Microflora present on OOWW were identified on samples taken before, during, and at the end of bio-treatment. Identification of isolates was carried out at genus and/or species level. Samples from the bio-treatments did not show any fungi; most of the isolates belonged to the Bacillus genus (with predominance of Bacillus megaterium, Bacillus sphaericus, and Brevibacillus brevis). The good COD and phenolic compounds removal rate indicates that the microbial community selected during the treatment is well adapted to the stress conditions imposed by this special type of bioreactor.

To search for reliable testing inocula alternatives to activated sludge cultures, several model microbial consortia were compared with activated sludge populations for their functional diversity. The evaluation of the metabolic potential of these mixed inocula was performed using the Biolog EcoPlates and GN and GP MicroPlates (Biolog, Inc., Hayward, California). The community‐level physiological profiles (CLPPs) obtained for model communities and activated sludge samples were analyzed by principal component analysis and hierarchic clustering methods, to evaluate the ability of Biolog plates to distinguish among the different microbial communities. The effect of different inocula preparation methodologies on the community structure was also studied. The CLPPs obtained with EcoPlates and GN MicroPlates showed that EcoPlates are suitable to screen communities with a metabolic profile similar to activated sludge. New, well‐defined, standardized, and safe inocula presenting the same metabolic community profile as activated sludge were selected and can be tested as surrogate cultures in activated‐sludge‐based bioassays.

Reference materials are important tools for maintaining high‐quality assurance standards, including for biological materials. A significant number of environmental international standards, including biodegradability and toxicity, involve utilization of activated sludge (AS) inocula. The absence of inoculum standardization in these tests is a potential source of error influencing the results. In this study three defined microbial consortia, two commercial inocula and a designed bacterial inoculum, were evaluated as an AS alternative seed for the Zahn–Wellens test, using diethylene glycol as the reference chemical. The results showed that to achieve diethylene glycol biodegradation with these inocula, a number of 10 5 colony‐forming units per milliliter of effective degrader microorganisms had to be present. Moreover, the addition of AS supernatant to the test mixtures improved inocula performance (the biodegradability curves), bringing them closer to those obtained with AS inocula. Among the three defined consortia, the designed inoculum replicates bested the AS behavior in the range of concentrations tested, with degradation completed in 12–14 days. Comparisons of the community substrate utilization profiles corroborated these results, showing that the designed inoculum profile was the most similar to the AS profile. The biodegradability and metabolism results provided support for the assertion that the designed inoculum can be used in the Zahn–Wellens test and as a base to develop reference inocula for other biodegradability and toxicity tests. © 2006 Wiley Periodicals, Inc. Environ Toxicol 21: 131–140, 2006.

In this study, the composting performance of two olive oil husk piles was compared using two different aeration processes: aeration by mechanical turning or by forced air-injection. The results showed that after the maturation phase both piles had a similar chemical composition and the same level of organic matter degradation (around 55%). However, the time necessary to reach the thermophilic phase was shorter for the turned pile (2 months in comparison to 3 months for the air-injected pile) and the humification degree achieved was slightly higher (94% versus 83%). Both piles showed the presence of an active microbial community, with an increase by 2-3 orders of magnitude in the number of cultured microbial colonies during the composting process. No significant difference in quantitative or qualitative terms was found in the microbial populations of both piles, nor was a clear succession between a mesophilic and a thermophilic population observed. In terms of industrial application, the mechanical turning process seems to be better since it is a lower energy and time consuming process.