For over 50 years, synthetic petrochemical-based plastics have been produced in ever growing volumes globally and since their first commercial introduction; they have been continually developed with regards to quality, colour, durability, and resistance. With some exceptions, such as polyurethanes, most plastics are very stable and are not readily degraded when they enter the ground as waste, taking decades to biodegrade and therefore are major pollutants of terrestrial and marine ecosystems. During the last thirty years, extensive research has been conducted to develop biodegradable plastics as more environmentally benign alternatives to traditional plastic polymers. Polyvinyl alcohol (PVA) is a water-soluble polymer which has recently attracted interest for the manufacture of biodegradable plastic materials. PVA is widely used as a paper coating, in adhesives and films, as a finishing agent in the textile industries and in forming oxygen impermeable films. Consequently, waste-water can contain a considerable amount of PVA and can contaminate the wider environment where the rate of biodegradation is slow. Despite its growing use, relatively little is known about its degradation and in particular the role of fungi in this process. In this study, a number of fungal strains capable of degrading PVA from uncontaminated soil from eight different sites were isolated by enrichment in mineral salts medium containing PVA as a sole carbon source and subsequently identified by sequencing the ITS and 5.8S rDNA region. The most frequently isolated fungal strains were identified as Galactomyces geotrichum, Trichosporon laibachii, Fimetariella rabenhorsti and Fusarium oxysporum. G. geotrichum was shown to grow and utilise PVA as the sole carbon source with a mean doubling time of ca. 6-7 h and was similar on PVA with molecular weight ranges of 13-23 KDa, 30-50 KDa and 85-124 KDa. When solid PVA films were buried in compost, Galactomyces geotrichum was also found to be the principal colonizing fungus at 25°C, whereas at 45°C and 55°C, the principle species recovered was the thermophile Talaromyces emersonii. ESEM revealed that the surface of the PVA films were heavily covered with fungal mycelia and DGGE analysis of the surface mycelium confirmed that the fungi recovered from the surface of the PVA film constituted the majority of the colonising fungi. When PVA was added to soil at 25°C, and in compost at 25°C and 45°C, terminal restriction fragment length polymorphism (T-RFLP) revealed that the fungal community rapidly changed over two weeks with the appearance of novel species, presumably due to selection for degraders, but returned to a population that was similar to the starting population within six weeks, indicating that PVA contamination causes a temporary shift in the fungal community.

Cellulose is the most abundant and renewable biopolymer on earth. Generally, it is well known for its structure properties or its natural and industrial derivatives due to its biodegradability or easily controlled transformation. By contrast, non-biodegradable cellulose is relatively unusual and not intensively investigated. The discovery of well preserved Miocene fossil woods during a recent excavation from the Entre-Sambre-et- Meuse (ESEM) karsts (southern Belgium) gives a rare opportunity to investigate resistant cellulose. The wood specimens were examined using physicochemical and biochemical techniques in order to correlate the exceptional preservation of these fossilized remains after 15 million years to the non-biodegradability of their lignocellulose content. Structural and chemical changes were assessed by comparing the structural features of the fossil samples with those of their modern counterpart, Metasequoia. Solid state 13C nuclear magnetic resonance (NMR) and microscopic analysis showed good preservation of the cellulose structure in the fossil wood from the ESEM peat deposit. Moreover, there also appears to be a complete loss of hemicelluloses in the fossil wood structure maybe due to their branched structure and their lower molecular weight. According to several authors, the peatification, the initial biochemical stage of coalification, is characterized by a complete loss of hemicelluloses and a significant reduction in cellulose, which is completely degraded after a short span of geological time. However, the cellulose content, similar to that found in the living species Metasequoia, suggests that the ESEM fossil wood is at a very early stage of peatification. Compared to the lignin in modern Metasequoia, the lignin structure has undergone slight chemical alteration such as demethoxylation, cleavage of some b-O-4¢ linkages and alkylation of the resulting catechol-like structures. Furthermore, since uncondensed structures were degraded in the fossil woods, the condensed structures already present in the original lignin, became more dominant. Despite the substantial cellulose fraction available in the fossil tissue, degradation tests showed that the fossil cellulose could not be degraded by cellulases and microorganisms usually involved in the biodegradation of organic matter. This lack of bioavailability could be due to the structural features of the fossil biopolymers (cellulose, extractives and lignin) or the burial environment. In our work, we have analyzed the influence of the cristallinity, the size of crystallites, the type of crystal lattice and the ratio of two allomorphs I a/I b as well as the morphology of the cellulose microfibrilles on the cellulose digestibility. These cellulose structural features seemed to have no effect on cellulose biodegradability in the Miocene woods samples. On the other hand, the wood extractives (tannins, resin acids, terpenes,) may contribute to some resistance properties of the fossil cellulose but their action is not sufficient to explain the inhibition of the cellulose degradation. On the basis of our observations, we suggest that the presence of a modified lignin structure greatly influences cellulose biodegradability. Indeed, by altering the lignin structure with various delignification pretreatments and studying the effect of the resulting changes on enzymes efficiency, we showed that the disintegration of the condensed structures, linked to the drastic reduction of the uncondensed structures, could have significant impact on the major improvement of the cellulose bioavailability. In conclusion, the nature and/ or the proportion of intermolecular substructures could be the key of the cellulose protection. La cellulose est le biopolymère le plus abondant et le plus renouvelable sur terre. Généralement, il est reconnu pour ses propriétés structurales ou ses dérivés naturels et industriels, eux-mêmes caractérisés par leur biodégradabilité ou leur mise en uvre facilement contrôlée. Par contraste, une cellulose non-biodégradable est relativement inhabituelle et peu étudiée. La découverte de bois fossiles du Miocène extrêmement bien préservés au cours dexploitations récentes de karsts de lEntre-Sambre-et-Meuse (ESEM, Belgique) offre une rare opportunité détudier une cellulose résistante. Les spécimens de bois ont été examinés à laide de techniques physicochimiques et biochimiques en vue de corréler la préservation exceptionnelle de ces macrofossiles après 15 millions dannées à la non-biodégradabilité de leur contenu cellulosique. Les modifications structurales et chimiques ont été attribuées en comparant les caractéristiques des échantillons fossiles avec ceux de leur équivalent moderne, le Metasequoia. Les analyses microscopiques et par résonance magnétique nucléaire C13 de létat solide ont révélé une préservation importante de la structure cellulosique dans les bois fossiles des dépôts argilo-tourbeux de lESEM. De plus, la concentration en cellulose est proche de celle présente dans lespèce moderne, Metasequoia. Par contre, une perte complète des structures hémicellulosiques est constatée. Celle-ci pourrait être attribuée à leurs structures branchées et leurs faibles poids moléculaires. Ces observations suggèrent que les bois fossiles de lESEM se situent à un stade très précoce de la turbification. En effet, selon différents auteurs, la turbification, le stade biochimique initial de la coalification, est caractérisée par une perte complète des hémicelluloses et une réduction significative de la cellulose, qui est complètement dégradée après un très court intervalle de temps géologique. Par comparaison avec la lignine de lespèce moderne, la structure ligneuse fossile a subi de légères altérations chimiques telles que des déméthoxylations, le clivage de certains liens intermoléculaires (b-O-4¢ en majorité) et lalkylation des structures catéchols résultantes. En outre, les structures condensées déjà présentes dans la lignine dorigine, sont devenues plus dominantes par rapport aux structures non-condensées qui ont subi de fortes dégradations dans les bois fossiles. Malgré la fraction cellulosique substantiellement accessible dans les tissus fossiles végétaux, les tests de dégradation ont montré que la cellulose fossile ne pouvait être que faiblement dégradée par des cellulases et les microorganismes impliqués habituellement dans les processus de biodégradation de la matière organique. Ce manque de réactivité pourrait être lié aux caractéristiques structurales des biopolymères fossiles (cellulose, composés extractibles et lignine) ou à la matrice denfouissement. Limplication de celle-ci dans le processus de préservation de la cellulose a été écarté car les conditions environnementales présentes actuellement dans les karsts auraient du favoriser la biodégradabilité de ces bois. Au cours de nos recherches, nous avons analysé linfluence de la cristallinité, la taille des cristallites, le type de réseau cristallin et le rapport entre les allomorphes I a/I b ainsi que la morphologie des microfibrilles de cellulose sur la digestibilité de la cellulose. Ces paramètres structuraux associés à la cellulose ne semblent pas avoir deffet sur la biodégradabilité de la cellulose dans les échantillons de bois du Miocène. Dautre part, les composés extractibles (tannins, acides résiniques, terpènes,) peuvent contribuer à la capacité de résistance à la décomposition de la cellulose fossile mais leur action nest pas suffisante pour expliquer linhibition importante de la dégradation de la cellulose. Sur base de nos observations, nous suggérons que la présence dune structure ligneuse modifiée influence fortement la biodégradabilité de la cellulose. En effet, en altérant la structure ligneuse avec différents prétraitements de délignification et en étudiant limpact des transformations structurales sur lefficacité de la dégradation enzymatique, nous avons montré que la désagrégation des structures condensées, liée à la diminution drastique des structures non-condensées, pourrait avoir un impact significatif sur lamélioration majeure de la bioaccessibilité du substrat cellulosique. En conclusion, la nature et/ou la proportion des sous-unités intermoléculaires présentes dans la structure ligneuse pourrait être la clé de la protection exercée par la lignine.

The aim of this work is study of utilization of PHA producing bacteria in bioremediation technologies. For this study were used bacteria Pseudomonas putida KT2440 and two isolates from soil contaminated by petroleum - Pseudomonas gessardii (D2) a Pseudomonas fulva (D3). The experimental part describes especially study of feather biodegradation using selected microbial strains. All the tested bacterial strains were capable of feather degradation and utilization as the sole carbon source. During biodegradation experiment, we monitored weight loss of feather, protease and keratinase activity, concentration of bacterial biomass and PHA content as well as pH. The highest biodegradation ability and keratinase activity was observed in Pseudomonas putida. None of tested bacteria accumulated detectable amount of PHA during growth on waste feather, nevertheless, bacterial biomass grown during feather degradation can be used as an inoculum for PHA production on waste frying oil and octanoic acid. Using this experimental setup, high PHA content (54% of cell dry weight) was achiaved in Pseudomonas putida. Another part of the thesis deals with biodegradation of petroleum oil. The highest capability of growth on this carbon source were determined in Pseudomonas fulva.

The continuous demand on new residential and economic areas of the modern society has to face up with problems posed by polluted sites related to former industrial activities, typically located in suburbs areas. These sites, known as brownfields, are often located nearby navigable rivers to facilitate transport operations of industrial manufacturing, which increase their potential environmental threat due to the possible migration of pollutants in groundwater to surface water bodies through groundwater discharge. In this context, the objective of this research, performed in the scope of the FP6-IP AquaTerra project, was to contribute to a better assessment of the risk of groundwater contaminant dispersion for a brownfield located next to the Meuse River (Belgium), in a context where strong groundwater - surface water interactions prevail. The brownfield of interest corresponds to the site of the former coke factory of Flémalle. Resulting from industrial activities, soil and groundwater located in the alluvial aquifer are heavily contaminated with various types of organic (BTEX, PAHs, mineral oils...) and inorganic (As, Zn, Cd...) pollutants. To do so, detailed characterisation campaign was performed, consisting of, on the one hand, classical field experiments such as pumping tests, injection tests and tracer experiments; on the other hand, advanced and original field experiments such as detailed monitoring of groundwater - surface water interaction and dynamics, and the development and application of an innovative tracer technique, the Finite Volume Point Dilution Method (FVPDM), used to quantify and monitor groundwater fluxes. Monitoring and field works data was subsequently used to develop and calibrate a groundwater flow model using the finite difference code MODFLOW, with an automatic parameter estimation approach based on an original combined regional scale (zonation) and local scale (pilot points) approach. A transport model was also developed using MT3DMS and calibrated using tracer experiments performed in the brownfield. This groundwater flow and transport model was used to better quantify the dynamics of groundwater - surface water interactions and to model various scenarios of contaminant dispersion through the aquifer - river system. For these scenarios, benzene was considered because it is one of the main pollutants encountered in the site, its large solubility and mobility in groundwater and its acute toxicity. These scenarios were established considering various groundwater flow conditions (steady state vs. transient) and various hydrodispersive processes possibly affecting the mobility of benzene in groundwater, namely advection, hydrodynamic dispersion, sorption - desorption and, as evidenced by the research results of the University of Neuchâtel (Switzerland), benzene degradation under sulphate reducing conditions. These simulations indicate that benzene attenuation is mainly controlled by ongoing benzene degradation processes, aquifer heterogeneity and river stage fluctuations. Based on this analysis, the risk of benzene dispersion is low, and monitored natural attenuation (MNA) is a valuable option with (1) monitoring benzene at control planes downstream from the sources; (2) further investigation on risk of sulphate depletion in the alluvial aquifer; and (3) further investigation on mobilisation/immobilisation of heavy metals related to dynamics of organic pollutant plumes.