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The use of alternative and renewable resources, such as biomass, for the production of energy and chemicals is motivated by decreasing oil reserves and environmental concerns. Acidogenic fermentation of biomass for the production of ethanol and acetic, propionic, butyric and lactic acids offers major advantages in that it allows: (i) non germ free conditions; (ii) using very diversified, less pretreated and cheap biomass; (iii) producing molecules and corresponding esters consumed on a large scale by the industries. The product recovery, separation and concentration steps downstream of the biomass transformation remain a major challenge for the application of acidogenic fermentation which produces very dilute (concentrations of only a few percent) and complex aqueous solutions of acids and ethanol. A number of separation processes can be potentially used, but a process which is technically, energetically and environmentally promising has not been developed yet. In this work, a novel extraction - re-extraction process is presented which is based on the transfer of the acids and ethanol to a glycerol phase via an intermediate solvent phase. In the first, extraction step, tri-n-octylamine based solvents are chosen for their preferential extraction of acids. In the second, re-extraction step, glycerol is used to extract the acids from the intermediate solvent. Glycerol being a by-product of the growing biodiesel production, its use opens perspectives for the transformation of glycerol into short chain esters, directly using the acids recovered. The mass based distribution coefficients were experimentally measured. Both model solutions and real fermentation broths were used. Solutions were stirred to reach equilibrium and then centrifuged. The concentrations of butyric acid in the aqueous phase before and after extraction were determined by gas chromatography. The influence of the pH, the temperature and the composition of the aqueous phase on the recovery of the acids w

Les substances perturbatrices du système endocrinien sont des substances qui, de par leur capacité à induire des changements hormonaux chez les organismes vivants, génèrent des préoccupations dans le domaine de la qualité des eaux et, par extension, dans le domaine du traitement des effluents aqueux. Particulièrement, ce projet de recherche s’est attardé sur l’élimination des perturbateurs endocriniens phénoliques nonylphénol (NP), bisphénol A (BPA) et triclosan (TCS) en solution aqueuse à l’aide de la laccase (E.C. 1.10.3.2) sécrétée par la souche fongique Coriolopsis polyzona. Cette oxydase est une métalloprotéine pouvant catalyser l’oxydation d’une vaste gamme de substances phénoliques. En premier lieu, l’impact du pH et de la température sur l’élimination de ces composés à l’aide de la laccase libre en utilisant un design factoriel. L’oxydation de ces composés produit des oligomères (dimère à pentamère) via le couplage des radicaux phénoxy produits par l’action de la laccase. Il s’avère que les substances produites suite à l’oxydation du NP et du BPA par la laccase ont perdu leurs similitudes structurales avec l’estrogène. Ainsi, l’élimination de l’activité estrogénique de ces substances est directement liée à la transformation des composés. Finalement, l’utilisation d’ABTS comme médiateur a permis d’augmenter le taux d’oxydation enzymatique de ces composés chimiques. Puis, de façon à augmenter la possibilité d’utilisation de la laccase dans des biotechnologies environnementales, cette enzyme a été immobilisée sur un support siliceux et via la réticulation d’agrégats. L’impact des conditions d’immobilisation sur l’activité enzymatique, la stabilité du catalyseur et les propriété biocatalytiques apparentes a été déterminé pour différentes stratégies d’immobilisation. Globalement, l’immobilisation génère un biocatalyseur stable vis-à-vis les dénaturations chimique, physique et biologique. Particulièrement, l’immobilisation sur un support solide produit un biocatal, (AGRO 3) -- UCL, 2008

Energy crops can be used to feed anaerobic digesters and produce renewable energy. However, sustainability of this option requires that it contributes to a net production of renewable energy and a net reduction of fossil CO2 emission. In this paper, the net balance of CO2 emission and renewable energy production is assessed for maize and grass energy crops produced in several agricultural systems relevant for Southern Belgium and surrounding areas. The calculated net energy yields are 8-25 (maize) and 7.4-15.5 (grass) MWh of renewable CH4 per MWh of fossil energy invested, depending on the agricultural option considered. After conversion to electricity, the specific CO2 emissions range from 31 to 104 kg(CO2)MWhelectricity(-1), depending on the case considered. This corresponds to a significant reduction in CO2 emissions compared to the current reference gas-steam turbine technology which produces 456 kg(CO2)MWhelectricity(-1).

Laboratory data on the behaviour of the pathogenic form of the prion protein (PrP(Sc)) in environmental matrices such as sewage sludge is scarce. Direct experiments with this misfolded protein require strict safety measures, pathogen class-3 facilities and costly reagents. However, preliminary data can be generated by non-pathogenic model systems that involve lower costs and simpler manipulation. We chose amyloid-like fibrils formed from the well-studied protein lysozyme as a model because, in the case of an accidental contamination of sewage sludge, PrP(Sc) would most likely be in the form of amyloid fibrils. All amyloid fibrils have similar structural features and tend to bind to thioflavin-T, thereby enhancing the fluorescence yield of the dye. We used this fluorescence enhancement to monitor amyloid fibrils introduced into activated sludge. We observed that, in the presence of sludge flocs, the concentration of amyloid fibrils that are detectable through the enhancement of thioflavin-T fluorescence decreased as a function of time, most likely due to hydrolysis of the fibrils by sludge proteases. Some of the fluorescence loss seems also due to the binding of sludge exopolymers to amyloid fibrils.

Understanding the behavior of enzymes at solid surfaces requires coping with the complexity of the interfaces. Examples are given to illustrate key considerations: (i) influence of the adventitious contamination; the amount of glucose oxidase adsorbed on stainless steel in conditions producing an electrochemical effect is very low compared to the amount of organic contaminants; (ii) structure (multilayered, random) of an adsorbed phase containing an enzyme and other organic constituents (self-assembled monolayer, adventitious contaminants); (iii) competition between proteins for adsorption and influence of the adsorption procedure (simultaneous or sequential) and (iv) reality vs. expectation in enzyme (glucose oxidase) immobilization by tentative covalent grafting. The study of enzyme adsorption in several model systems (catalase on surface-modified carbon blacks, beta-glucosidase on clay at different pHs, beta-glucosidase on sand and surface-modified sand) showed that a strong adsorption, whether by electrostatic or hydrophobic interactions, provokes an extensive deactivation. After adsorption, which is a quick process, the enzyme activity may continue to decrease both in the adsorbed phase and in the solution owing to dynamic processes which involve the adsorbed enzyme and exchanges between the adsorbed phase and the liquid phase. (c) 2008 Elsevier Ltd. All rights reserved.

The Insect Cell - Baculovirus Expression Vector System (IC-BEVS) is widely used for the production of complex recombinant (glyco)proteins. The simplicity of insect cell cultivation in suspension serum-free media and the easy construction of recombinant baculovirus vectors have made the BEVS quite an effective expression system. On the other hand, the BEVS is a transient lytic system that may present some drawbacks in purification and potential degradation of the products. Among the various insect cell lines, the High-Five cell line has a great potential for the production of recombinant proteins using the BEVS in stirred bioreactors, reaching high cell densities and high protein production levels. Moreover, these cells can tolerate environmental stresses and can be cultivated on a large scale (Chapter 1). Unfortunately, up to now, there have been limited data available regarding suitable culture conditions and the metabolism of High-Five cells, a key requirement for the rational development of new processes. The overall goal of the present work was the study of these High-Five cells, in order to develop sophisticated new processes as alternatives to batch cultivation. The original contributions have been developed along two axes. The first axis concerns the study of the physiology and metabolism of High-Five cells. At first, we undertook a study aiming to prevent cell ring formation on suspension culture recipient walls (Chapter 2). Next, we analyzed environmental factors affecting insect cell growth and death, by comparing and developing methods able to distinguish between apoptosis and necrosis of cells (Chapter 3). The comprehensive study of the extended metabolism of High-Five cells was done using a metabolic flux network that takes account of the catabolism but also the anabolism of uninfected and baculovirus-infected cells (Chapter 4). The second axis was the application of the previously gained knowledge on High-Five cells to develop high-density systems spec, (AGRO 3)--UCL, 2007

2,4,6-trinitrotoluene (TNT) is a toxic and recalcitrant pollutant contaminating soils and groundwater. Therefore, characterization of microbial populations of TNT-contaminated soils and isolation of bacteria degrading this pollutant are of primordial importance. Comparison of hybridizations of 16S rRNA derived from uncontaminated and TNT-contaminated soil samples required the development of a functional ANOVA model. Specifically, a statistical tool was necessary to compare dissociation curves obtained from thermal dissociation analysis of RNA hybridizations to DNA microarrays, and to determine if the dissociation curves significantly differed. To test and validate the model, we used dissociation curves from in vitro transcribed 16S rRNA amplified from two environmental samples hybridized to a phylogenetic microarray. Detection and rejection of outlier curves was important for appropriate discrimination between curves. The identification of significantly different curves was more efficient with the model than approaches relying on measurements at a single temperature. This functional ANOVA analysis was used to improve discrimination between hybridizations of two soil microbial communities. Following hybridization of in vitro transcribed 16S rRNA derived from an uncontaminated and a TNT-contaminated soil sample to an oligonucleotide microarray containing group- and species-specific perfect match (PM) probes and mismatch (MM) variants, thermal dissociation was used to analyze the nucleic acid bound to each PM-MM probe set. Functional ANOVA of the dissociation curves generally discriminated PM-MM probe sets when values of Td (temperature at 50% probe-target dissociation) could not. Maximum discrimination for many PM and MM probes often occurred at temperatures greater than Td. Comparison of signal intensities measured prior to dissociation analysis from hybridizations of the two soil samples revealed significant differences in domain-, group-, and species-specific probe, The amount of DNA extracted decreased in the highly contaminated soil samples (1.4 and 28.5 g TNT/kg soil). After 7 days of incubation of these soil samples, there was a clear shift of their original flora to a population dominated by Pseudomonadaceae, Xanthomonadaceae, Comamonadaceae and Caulobacteraceae. When the TNT concentration was lower (140 mg TNT/kg soil), a moderate shift in the bacterial population was observed. These results indicate that TNT affects soil bacterial diversity and richness by selecting for a narrow range of bacterial species that belong mostly to Pseudomonadaceae and Xanthomonadaceae. TNT-contaminated soil samples probably contained TNT-degrading bacteria. In order to isolate bacteria that can denitrate TNT, enrichment cultures were carried out with TNT as sole nitrogen source and in the absence of oxygen. These cultures were established starting with an uncontaminated or a TNT-contaminated soil inoculum, in the presence or absence of ferrihydrite. A significant release of nitrite was observed in the liquid culture containing TNT, ferrihydrite and inoculum from a TNT-contaminated soil. Under these conditions, Pseudomonas aeruginosa was the predominant bacterium in the enrichment, leading to the isolation of P. aeruginosa ESA-5 as a pure strain. The isolate had TNT denitration capabilities as confirmed by nitrite release in oxygen-depleted cultures containing TNT and ferrihydrite. Concomitantly, TNT-reduced compounds were detected as well as unidentified polar metabolites. The concentration of nitrite released from TNT was proportional to the concentration of ferrihydrite in the medium. The release of nitrite was lower when the concentration of initially spiked TNT was reduced by one order of magnitude. Under these conditions, the concentration of nitrite peaked and then its concentration slowly decreased and production of ferrous ions was detected. A decrease of nitrite concentration and production of ferrous ion were observed when TNT was, (AGRO 3)--UCL, 2007

Green fluorescent protein (GFP) was used as a reporter protein to investigate the interactions and fate of the active conformation of proteins in wastewater sludge. GFP was chosen because its fluorescence is dependent on the integrity of its native conformation. We identified factors that cause the loss of GFP fluorescence when this protein is introduced in aerobic or anaerobic sludge. In both systems, soluble polymers present in the liquid fraction caused an initial loss of fluorescence, but stabilized the remaining fluorescent GFP molecules. In aerobic sludge, interaction of GFP with the sludge solids initially caused an additional loss of fluorescence but the remaining fluorescence kept fairly constant over the following hours. In anaerobic sludge, on the contrary, interaction of GFP with the sludge solids caused a temperature dependent loss of fluorescence, probably related to the presence of precipitated ferrous sulfide. No direct relationship was found between sludge protease activity and loss of GFP fluorescence, suggesting that proteases are not the primary factor controlling the fate of the active form of proteins in wastewater sludges.

The reactions occurring between gases/aerosols and silicate ash particles in volcanic eruption plumes remain poorly understood, despite the fact that they are at the origin of a range of volcanic, environmental, atmospheric and health effects. In this study, we apply X-ray photoelectron spectroscopy (XPS), a surface-sensitive technique, to determine the chemical composition of the nearsurface region (2-10 nm) of nine ash samples collected from eight volcanoes. In addition, atomic force microscopy (AFM) is used to image the nanometer-scale surface structure of individual ash particles isolated from three samples. We demonstrate that rapid acid dissolution of ash occurs within eruption plumes. This process is favoured by the presence of fluoride and is believed to supply the cations involved in the deposition of sulphate and halide salts onto ash. AFM imaging also has permitted the detection of extremely thin (< 10 nm) coatings on the surface of ash. This material is probably composed of soluble sulphate and halide salts mixed with sparingly soluble fluoride compounds. The surface approach developed here offers promising aspects for better appraising the role of gas/aerosol-ash interaction in dictating the ability of ash to act as sinks for various volcanic and atmospheric chemical species as well as sources for others. (c) 2007 Elsevier B.V. All rights reserved.

The fate of active proteins in environmental matrices such as wastewater sludge, soil, and organic amendments is not well understood . In the present paper, we report the use of green fluorescent protein (GFP) as a probe protein to investigate the behaviour of proteins in wastewater sludge. We developed a procedure to quantitatively detect the active form of this protein in such a matrix. The procedure is based on the fluorimetric analysis of GFP in the separated liquid and solid fractions of sludge. We then tested the suitability of the approach by monitoring GFP added to aerobic and anaerobic sludge. Under aerobic conditions at 20 degrees C, most GFP immediately associated with the sludge solid fraction. About 20% of the fluorescence due to solid fraction associated GFP was still present after 72 h, which suggests a relative persistence of proteins in this system. Under anaerobic conditions at 35 degrees C, fluorescence signal due to GFP was reduced by 90% after only 6 h.

The surface composition of particles present in the fine earth (< 2 mm) of 50 soil horizons differing in composition and pedogenetic origin (13 soil profiles) was analysed by X-ray photoelectron spectroscopy (XPS) to assess the capability and limitations of this technique and to gain better knowledge of the soil samples. The surfaces were systematically enriched in carbon, sometimes up to 1000 times, indicating that the soil particle surfaces are coated with organic substances, even in horizons where the bulk organic content is less than 0.1 g kg(-1). The distribution of carbon in the various oxidation states was 0.569 +/- 0.008 C-[0], 0.275 +/- 0.004 C[+1], 0.089 +/- 0.003 C[+2] and 0.066 +/- 0.002 C[+3] for most horizons (mean +/- standard error, 69 data). Only Andosol surface horizons systematically had surface organic matter in a more oxidized state. After correcting the results for the presence of organic coatings, we found that Si was generally depleted and Al enriched at the surface of soil particles, while Fe was either depleted or enriched depending on the sample considered. However, the coating of the coarser soil particles by the finer ones and their differential composition explained this observation and limits the interest of XPS for characterizing the surface enrichment of inorganic elements in crude soil samples. These limitations should be considered when interpreting XPS results in future work. Nevertheless, XPS can analyse the adsorbed organic matter and its functional composition of carbon without the need for any chemical or physical extraction that might alter the structure and composition of the organic molecules.

A procedure was developed for handling natural soil particles and probing their native surface structure by atomic force microscopy (AFM) under water. This procedure was used to investigate the nanometer scale organisation of organic matter at the surface of sand particles taken from three soil horizons. The latter were selected for the contrasted properties of their organic matter, namely Podzol E and Bh horizons and a Cambisol A-B horizon. The presence of an adsorbed layer was visualised at the surface of Podzol Bit and Cambisol particles in the form of aggregated structures that interacted with the AFM probe. Surface analysis by X-ray photoelectron spectrometry (XPS) confirmed the carbonaceous nature of this adsorbed layer. Displacement of organic matter by the scanning probe was directly evidenced for Podzol Bh sand particles. Such displacement was not observed for Cambisol particles. A dramatic effect of drying on the concentration, nanometer scale distribution and properties of the adsorbed organic matter was clearly demonstrated by combining AFM imaging and XPS analysis. The procedure developed here gives access to direct, nanoscale information of the surface structure of sand particles and offers promising prospects for the characterisation of other environmentally-relevant particles in native conditions. (C) 2002 Elsevier Science B.V. All rights reserved.

Laboratory- and pilot-scale studies were conducted in order to adjust and optimize the in-situ conditions for bioremediation of a soil contaminated with trichlororethene (TCE) and nickel. Results from laboratory studies showed that the indigenous microorganisms of the soil were limited by the type of electron donor. A better TCE dechlorination was obtained when the electron donor was composed of a mixture of methanol and lactate, as compared to that with methanol alone. Addition of up to 10 mM of sulphate as external electron acceptor (in combination with TCE) and with a mixture of methanol and lactate as electron donor had no significant effect on the TCE reducing activity of indigenous microorganisms of the soil, while higher concentrations (15 and 20 mM) yielded a lower dechlorination. Long term operation of a large pilot-scale soil reactor demonstrated the feasibility of a single-process in situ soil remediation. Results showed that, on one hand, TCE was progressively and stepwise reduced to cis-dichloroethene (DCE), vinyl chloride (VC) and finally to ethene, using only the indigenous microorganisms of the soil. On the other hand, stimulating the activity of sulphate-reducing bacteria of the soil with the addition of sulphate as electron acceptor was efficient in precipitating nickel as nickel sulphide.