Ce travail a porté sur l'étude de la structure et de la dynamique des bio(géo)macromolécules de la tourbière des Sagnes (Limousin, France) à l'aide de méthodes de dégradation chimiques et thermochimiques. La première partie a été consacrée à l'application de méthodes de dégradation chimiques pour apprécier l'impact d'une perturbation argileuse contemporaine sur la tourbière étudiée dans le cadre d'un partenariat entre l'université de Poitiers et AREVA. La tourbière présente un fonctionnement normal : activité microbienne et apport de matière organique en surface (acrotelme et mésotelme) et état du sédiment profond (catotelme) non perturbés. La seule influence notable du dépôt argileux, c'est l'augmentation apparente de l'activité de bactéries sulfato-réductrices sous sa zone de dépôt. Une seconde partie de développement analytique a tout d'abord porté sur l'évaluation de la capacité de la thermochimiolyse préparative en présence de TMAH d'analyser en un temps court et simultanément plusieurs familles de biogéomarqueurs. Pour cela, les résultats obtenus ont été comparés avec ceux obtenus par les méthodes chimiques habituellement utilisées. Compte-tenu des différents mécanismes impliqués, la thermochimiolyse apparait plus comme une méthode complémentaire. Enfin, une nouvelle méthode d'analyse des sucres (utilisation du bromure d'acyle) a été développée. Cette méthode a permis l'analyse sélective des sucres de la fraction lignocellulosique (principalement xylose et arabinose). / The purpose of this work was to study the structural composition, and consequently, dynamics of bio(geo)macromolecules of the Sagnes peatland (Limousin, France) by chemical and thermochemical degradation techniques. The first part constituted a framework between the university of Poitiers and AREVA and aimed to present a molecular inventory of a peat disturbed by a clay deposit. Results show that the clay deposit has no significant impact on the peatland evolution: microbial activity and production in the upper layer (acrotelm and mesotelm) and no apparent modification of the deepest organic sediment in the bottom layer (catotelm). Only phospholipid analysis showed an increase in sulfate-reducing bacteria under the clay deposit zone. The second part consisted of comparative studies between the most used chemical and thermochemical techniques for molecular characterization. Thermochemolysis should be viewed and used as a pertinent and a complementary method for the analysis of complex environmental systems. Finally, a novel technique using acetyl bromide allows the specific analysis of ligno-cellulosic carbohydrates (especially xylose and arabinose).

Rapid, portable detection of biological threat agents such as Bacillus anthracis endospores (“spores”) is extremely important given the real and perceived threats of bioterrorism. Gas chromatography-mass spectrometry (GC-MS) is an excellent general means for chemical detection, although special sample preparation and specialized equipment are required to employ GC-MS for detecting biological agents such as spores in the field. A GC sample introduction probe consisting of a helical wire that can be retracted inside a syringe needle, called a coiled wire filament (CWF), was employed as a simple, passively-heated means to introduce the mixture of spores plus reagents for thermochemolysis methylation (TCM) into the pre-heated GC inlet. There, reactions between spore biomarkers and the TCM reagent mixture occur between 250-290°C. At these conditions, monomethyl sulfate salt mixtures are convenient and efficient TCM reagents for the rapid conversion of a key unique spore biomarker, dipicolinic acid (DPA), to its dimethyl ester (Me2DPA). By this process, Me2DPA yields from spores were quantitatively assessed for different combinations of tetramethylammonium hydroxide (TMA+OH−), sodium hydroxide (Na+OH−), and hydrogen monomethyl sulfate (H+MeSO4−). The best reagent mixture was found by varying the combinations of the ions within the neutral or basic system containing TMA+, Na+, OH−, and MeSO4− according to a novel scheme for design of experiments termed ionic mixtures design of experiments (IMDOE). A combination of the above ions was found that is near-quantitative in its methylation of DPA to Me2DPA; this mixture contained a 1:3:1:3 mole ratio of TMA+:Na+:OH−:MeSO4−. This yield of Me2DPA was approximately a ten-fold increase over the best performance observed at the same conditions with tetramethylammonium hydroxide alone, the TCM reagent widely-used for GC. The reactions involving MeSO4− and TMA+ as methylating reagents, plus relevant hydrolysis and methylation reactions involving acid and base plus water and methanol, were investigated. An overall model is presented and mechanisms are proposed for reasons why basic mixtures of MeSO4− salts are more effective in methylating DPA compared to TMA+ salts at the conditions employed.

A simple method was developed for detection and differentiation of five Tier 1 bacterial agents, including Bacillus anthracis, Francisella tularensis, Yersinia pestis, Burkholderia pseudomallei and Burkholderia mallei as well as their closely related near neighbors by gas chromatography-mass spectrometry (GC-MS). Generally, different classes of compounds can be used as biomarkers for biowarfare agent detection, including nucleic acids (i.e., DNA or RNA), proteins (i.e., antibodies), carbohydrates (i.e., sugars), lipopolysaccharides, lipids (i.e., fatty acids) and small molecules. One-step thermochemolysis (TCM) was developed to provide GC-MS detectable biomarker signatures, including sugars, fatty acids and small molecules. Solid phase micro-extraction (SPME) was used for biomarker extraction, concentration and introduction into the GC-MS. Statistical algorithms were constructed using a combination of biomarkers for the five agents, which were robust against different growth conditions (medium and temperature). A general GC-MS temperature program was developed for all five Tier 1 bacteria. The total analysis time, including TCM, SPME extraction and GC-MS, is approximately 40 min. The total-ion chromatograms are very different for the five species. The final goal of this research was to develop an accurate, fast, simple, robust and automated method for field application. Therefore, an automated sample preparation system was designed, constructed and tested. The system automatically controls the movement of sample vials from one position to another, crimping of septum caps onto the vials, precise delivery of reagents and TCM reaction times and temperatures. The specific operations of introduction of sample vials, SPME sampling, injection into the GC-MS system and ejection of used vials from the system were performed manually in this study, although they can be integrated into the automated system. Manual SPME sampling is performed by following visual and audible signal prompts for inserting the fiber into and retracting it from the sampling port. A rotating carousel design allows for simultaneous sample collection, reaction, biomarker extraction and analysis of sequential samples. Bacillus species were used to test this autoreactor, and 96% of the samples were correctly identified using a statistical algorithm. This research applies not only to the rapid identification of Tier 1 agents after a biological attack, but should also benefit clinical diagnosis, which is essential to effective treatment.

Cette étude porte sur l’influence de différents traitements sur les caractéristiques et l’évolution de la matière organique de boues de stations d’épuration (Step). Les échantillons étudiés proviennent de 3 stations municipales de la Vienne. Le premier volet développé au cours de l’étude a pour but la mise au point d’une méthode d’analyse qualitative et quantitative des constituants des boues de Step. Les résultats obtenus démontrent le potentiel de la thermochimiolyse-GCMS à caractériser, sans extraction préalable, la matière organique d’un échantillon brut. Le deuxième volet de cette thèse s’est intéressé à l’influence de différents traitements (séchages, accélérateur d’électrons et méthanisation) sur l’évolution de la matière organique. Ainsi, le séchage thermique provoque une fragilisation de la matière organique, le séchage solaire une complexification et les lits plantés de roseaux n’ont pas d’influence significative à court terme. L’application du procédé d’oxydation avancé sur une boue flottée a provoqué une acidification et des changements structuraux de la matière organique. Ainsi une faible dose (1,25 kGy) a conduit à une complexification de la matière organique alors qu’une plus forte dose (50 kGy) semble la fragiliser. La modification de la matière organique à l’issue du procédé de méthanisation concerne uniquement la fraction lipidique et notamment des acides gras. / This study investigated the influence of different treatments on the characteristics and evolution of organic matter of municipal wastewater sludge. For this, three municipal wastewater treatment plants of Vienne department were sampled.The first phase developed during the study focused on the improvement of a method for qualitative and quantitative analysis of the sewage sludge’s constituents. The presented results demonstrate the strong potential of the thermochemolysis-GCMS to characterize, without extraction, the organic matter of a raw sample.The second part of this Ph.D has focused on the influence of various treatments (drying, electron beam and methanation) on the evolution of organic matter. Thus, the thermal drying causes weakening of the organic matter while the solar drying induces its complexification and reed beds induce no significant change. The application of an advanced oxidation process on a floated sludge caused acidification and structural changes of the organic matter. A low dose (1.25 kGy) led to more complex organic matter, while a higher dose (50 kGy) seems to weaken it. The changes in organic matter induced by anaerobic digestion mainly concerned the lipid fraction, in particular fatty acids.

This master's thesis deals with the analysis of organic substances, which are present in biochar. Biochar contains a number of organic compounds, that can be toxic and also carcinogenic to living organisms. In order for biochar to be used to improve soil quality or to be used for other applications, its safety must be declared by European or international certifications. Relevant European and international standards have been created for the determination of organic substances, that could be present in biochar. The theoretical part of the thesis was focused on selected organic substances, the process of pyrolysis and thermochemolysis, biochar characterisation and the used analytical methods. The experimental part of the thesis consisted of extraction with toluene and after that the determination of concentration of the 38 polycyclic aromatic hydrocarbons was performed. Qualitative analysis was performed by two-dimensional gas chromatography. Biochar samples were also subjected to thermochemolysis method and then they were analysed on GC-MS again. In both analytical methods, a large number of substances from groups such as PAHs, carboxylic acids, amines, substituted naphthalenes etc. was found, as well as smaller number of eg. polychlorinated biphenyls.

Methods for fast, simple detection of biomarkers to detect and differentiate closely related Bacillus endospores including Bacillus anthracis (BA), Bacillus thuringiensis (BT), Bacillus atrophaeus (BG), and Bacillus cereus (BC) using thermochemolysis and methylation (TCM), coiled wire filament (CWF), solid phase micro extraction (SPME) and gas chromatography-mass spectrometry (GC-MS) were developed. The main biomarkers detected and used for differentiation include dipicolinic acid methyl ester (DPAME), fatty acid methyl esters (FAMEs), 3-methyl-2-butenoic acid methyl ester (3M2BAME), 2-butenoic acid methyl ester (2BAME), and several methylated sugars. TCM of endospores was performed based on hydrolysis and methylation at elevated temperature after the endospores were mixed with sulfuric acid (H2SO4) with or without addition of tetramethylammonium hydroxide (TMAH) in methanol (MeOH). TCM products were then introduced into a heated GC injector port using a coiled wire filament (CWF) or solid phase microextraction (SPME) for detection and differentiation of the endospores by GC-MS.The CWF, which consisted of a tiny platinum helical wire coil attached to a retractable plunger that moved the coil in and out of a syringe needle housing, allowed for sampling to be accomplished by dipping the CWF in an endospore sample suspension, evaporating the suspension liquid, and then introducing the CWF into the injection port to enable on-line TCM. New SPME techniques, including half-half extraction, coated-needle extraction (CNE), and a new home-made polymer coated needle were used to speed up solid phase micro extraction of biomarkers produced from TCM. These simplified the detection of anthrose and other biomarkers. TCM with a CWF and TCM with SPME produced high intensity profiles of DPAME, FAMEs, 2BAME, 3M2BAME and methylated sugars. While the presence of DPAME can be used for the general detection of endospores (Bacillus and Clostridium) and the presence of 3M2BAME for the detection of BA, specific saturated and unsaturated C15, C16, and C17 fatty acid methyl esters and methylated sugars provide additional information for differentiating various Bacillus species grown at different temperatures and in different media. DPAME was detected in samples containing as few as 2,500 and 6,000 endospores using TCM-CWF with and without a concentration step, respectively. GC-MS peak area percent reproducibility for FAMEs using TCM and CWF varied from 3 to 13% (RSD). Better than 97% correct predictability of Bacillus species identity was obtained from a blind experiment consisting of 145 samples using DPAME and specific FAMEs. Conventional SPME and a modified form of "in-needle" extraction allow for detection of the biomarkers in less than 35 min. The detection limits with SPME sample introduction injection were approximately 5 x 103 endospores.Using these approaches, differentiation of Bacillus endospores and other biological agents grown under different conditions were based on the following characteristics: (1) presence of DPAME and specific FAMEs (iso or anteiso C15:0 and iso or anteiso C17:0) in Bacillus endospores, (2) unique presence of 3M2BAME (anthrose by-product) in BA, (3) absence of 2-butenoic acid methyl ester in BG, and (4) presence and absence of specific methylated monosaccharides in various Bacillus species. Clostridium endospores and non-sporulating bacteria, such as Yersinia pestis (YP) and Francisella tularensis (FT) could also be easily distinguished from Bacillus endospores based on the presence and absence of several specific sugar derivatives and fatty acid methyl esters (FAMEs), such as iso or anteiso C15:0 and iso or anteiso C17:0, and > C18 FAMEs which were simultaneously produced during TCM.

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