Co-cultures of selected fermentative bacteria have been shown to improve rates of substrate conversion and yields of some fermentation end-products. We have tested this hypothesis using a co-culture of the mesophilic, cellulolytic bacterium Clostridium termitidis CT1112 and the mesophilic, saccharophylic bacterium, Clostridium intestinale URNW. C. termitidis can utilize cellulose as a sole carbon source. It releases glycosyl hydrolases that hydrolyze cellulose, cellobiose and glucose. C. intestinale URNW, which was isolated as a contaminant from a cellobiose stock bottle of C. termitidis (Ramachandran et al., 2011), is not capable of hydrolyzing cellulose, but can utilize the cellobiose and glucose released by cellulose hydrolysis to grow in co-culture with C. termitidis. Based on the faster doubling-time of C. intestinale URNW on cellobiose, it was expected that the “soluble sugar free” environment will stimulate C. termitidis to hydrolyse cellulose at faster rate and which in-turn will result in increased substrate utilization and end-product synthesis compared to the monoculture of C. termitidis. The designed co-culture was characterized in depth with the use of microbial quantification studies (multiplex quantitative Real Time Polymerase Chain Reaction – qPCR) and ‘Omics techniques to understand the population dynamics and gene product expression in each species in the co-culture versus their respective monocultures at the molecular level. Inoculation of co-culture with diffferent initial ratios of the C. termitidis and C. intestinale resulted a fixed ratio of approximately 13:1 (C. termitidis : C. intestinale) at 168 hour post-inoculation (h pi). A statistical difference in substrate utilization and total cell mass production, but not end-product cocentrations, was observed at 168 h pi in cultures with an initial C. termitidis : C. intestinale ratio of 1:1 and 1:0.2). No statisical differences in substrate utilization, biomass accumulation, or end-product synthesis concentrations were observed for all other initial C. termitidis : C. intestinale ratios, or for co-cultures in where the C. termitidis : C. intestinale ratio was 1:25. Thus, the hypothesis that synergistic interactions between species in co-cultures can stimulate substrate consumption and end-product synthesis was supported under very limited conditions in this co-culutre system. Unlike other co-cultures reported in literature, the co-culture of C. termitidis and C. intestinale did not show large increases in substrate utilization or end-product concentrations synthesized when cultured on 2 g/L α-cellulose. This may be due to the slower cellulose degradation ability of C. termitidis in the co-culture, (compared to the cellulose degraders in other co-cultures such as C. thermocellum), which is the main contributor to the growth of C. intestinale in the co-culture and the competition for the same substrate (cellobiose) by both the species in the co-culture. / October 2015

Estrogens are a group of endocrine disrupting compounds capable of causing abnormalities in the reproductive systems of the wildlife. Wastewater is a major source of environmental estrogens, in part due to incomplete removal of estrogens in biological wastewater treatment processes. This dissertation investigated factors affecting estrogen biodegradation in bioreactors. Specifically, research efforts were placed on characterization of several bacterial estrogen degraders (model strains: Aminobacter strains KC6 and KC7, and a Sphingomonas strain KC8) and examination of the effects of operating parameters on estrogen removal and estrogen-degrading microbial community structure. Sphingomonas strain KC8 can use 17beta-estradiol as a sole carbon source, suggesting that estrogen degradation by KC8 is a growth-linked, metabolic reaction; however, estrogen degradation by strains KC6 and KC7 might be a non-growth linked, cometabolic reaction. One important finding was that strain KC8 can also degrade and further utilize testosterone as a growth substrate. Strain KC8 was characterized in terms of its utilization kinetics toward estrogens and testosterone with the results that showed relatively smaller kinetic parameters than the typical values for heterotrophs in activated sludge. Strain KC8 can also grow on other organic constituents (glucose, succinate, and acetate). Strain KC8 retained its ability to degrade both 17beta-estradiol and estrone (after 15 d of growth on a complex nutrient medium without 17beta-estradiol). Effective removals (>98.7 %) of 17beta-estradiol with no significant differences were observed in sequencing batch reactors (SBRs) under three solid retention times (SRTs of 5, 10, 20 d). The population ratios of known estrogen degraders (strains KC8 and ammonia-oxidizing bacteria (AOB)) and amoA gene (associated with ammonia oxidation) to total bacteria decreased as SRT increased in SBRs. These observations correspond to the decreasing percentages of 17 beta-estradiol biodegraded in SBR when SRT increased from 5 to 20 d, when the sorption of 17 beta-estradiol onto biomass was considered. Real-time terminal restriction fragment length polymorphism showed that more ribotypes were observed in SBR-20d than SBR-5d. The species evenness (E) in microbial community structures in SBRs was not affected by SRT. However, diversity indices (Shannon-Weaver diversity index (H) and the reciprocal of Simpson?s index (1/D)) suggest that longer SRTs might lead to a more diverse microbial community structure.

Les hydrocarbures aromatiques polycycliques (HAP) sont des polluants organiques, ubiquistes, potentiellement toxiques et cancérigènes. Dans les sols, la dégradation des HAP est principalement due à l'activité microbienne. Certaines études ont montré que la biodégradation des HAP pouvait être augmentée dans la rhizosphère des plantes où le nombre et l'activité microbienne sont stimulés, grâce aux exsudats racinaires. Cependant les bénéfices des plantes ne sont pas toujours observés, et les exsudats pourraient aussi modifier la biodisponibilité des HAP. Les objectifs de ce travail ont été de mieux comprendre ces interactions sol-plante-microorganismes qui conditionnent le devenir des HAP dans la rhizosphère en suivant notamment (i) les bactéries possédant les gènes codant une HAP-dioxygènase, (ii) les espèces bactériennes impliquées dans la dégradation du phénanthrène, et (iii) la disponibilité et la biodégradation des HAP dans des terres industrielles historiquement contaminées.Les expériences ont été conduites dans des dispositifs à compartiments, lesquels permettent une diffusion des exsudats racinaires dans le sol tout en retenant physiquement les racines, puis en microcosmes avec un ajout d'exsudats racinaires naturels produits à partir d'une culture hydroponique de ray-grass (Lolium perenne, L). Les expériences ont été réalisées dans un premier temps avec du sable en ajoutant du phénanthrène (PHE) et un inoculum bactérien issu d'un sol d'une ancienne cokerie puis directement avec des sols historiquement contaminés en HAP. Les nombres de copies de gènes codant pour l'ADNr 16S et pour des HAP-dioxygènases ont été quantifiés par PCR en temps réel pour estimer la proportion de bactéries dégradantes. Les structures des communautés ont été comparées par électrophorèses (TTGE). En plus de l'analyse des 16 HAP totaux, une extraction non exhaustive des HAP a été réalisée à la cyclodextrine pour en estimer la disponibilité. L'utilisation de la méthode SIP (stable isotope probing) avec du 13C-phénanthrène a permis d'identifier les bactéries directement impliqués sa dégradation dans un sol historiquement contaminé. Les expériences en dispositifs à compartiments ont confirmé que la dissipation du phénanthrène est plus importante lorsque la distance aux racines est plus faible, et montrent que le nombre de copies de gène 16S et de gène de HAP-dioxygénase varie avec l'âge des plantes et du temps de contact des compartiments latéraux avec le tapis racinaire. Mais elles montrent aussi que la dissipation du phénanthrène n'est pas plus importante dans les pots plantés, tandis que dans les expériences en microcosmes une inhibition de la dissipation du PHE a même été observée en présence d'exsudats. La présence d'exsudats racinaires a profondément modifié la structure des communautés dégradant les HAP, et l'expérience SIP a permis d'identifier les bactéries directement impliquées dans la dégradation du 13C-phénanthrène et de montrer qu'elles étaient différentes en présence ou non d'exsudats. En présence d'exsudats, la proportion des bactéries dégradantes dans la population totale est passée de 1 % dans la terre d'origine et dans les traitements sans exsudats à plus de 10 %. Même si les exsudats racinaires ralentissent la dissipation du phénanthrène, en fournissant une source de carbone plus facilement métabolisable, ils ont augmenté la quantité de HAP extractibles à la cyclodextrine dans deux des trois sols historiquement contaminés, suggérant un effet de ceux-ci sur la biodisponibilité des HAP / Polycyclic Aromatic Hydrocarbons (PAH) are organics pollutants, ubiquitous, toxics and potentially carcinogenic. In soil, PAH degradation is mainly attributed to microbial organism. Several studies have thus reported enhanced PAH degradation in soil in the presence of plants. Rhizospheric soil increase the number et the activity of microorganisms in soil by the release of roots exudates. However, bene?cial effects of plants in the remediation are not always observed and roots exudates could be limited PAH biodegradation. The object of this study was to investigate the fate of PAHs in rhizosphere, following (i) the PAH-dioxygenase genes DNA to quantify the PAH-degrading bacteria, (ii) species implicated in phenanthrene biodegradation, and (iii) PAH availability and biodegradation from industrial soils.Different experimental devices have been designed to study detailed processes in the rhizosphere. First is a compartments devices were a nylon mesh permits diffusion of plant soluble substances towards the adjacent root free compartment as a rhizosphere. Secondly microcosms were enriched with natural roots exudates from hydroponic culture of ray-grass (Lolium perenne L.). In first time, experiments were conducted using sand and bacterial inoculum from an industrially PAH-contaminated soil and then directly with a soil historically contaminated by PAH. The Real-Time PCR quantification of 16S rRNA gene copy and of functional PAH-RHD? genes permitted to assess the proportion of a degrading bacteria. Bacterial community structure was approached from Temporal Thermal Gradient gel Electrophoresis (TTGE) fingerprinting, and bands sequencing. Nonexhaustive cyclodextrin-based extraction technique provided a estimate of the ?labile? or available pool of PAH in soil. Use of stable isotope probing (SIP) technique with [13C]phenanthrene allowed a bacterial identification of directly implicated in industrial soil.The presence of exudates modified microbial community of PAH-degrading bacteria. SIP experiment showed that 13C-labelled PHE-degrading bacteria was different depending on the exudates input. Many species having to degrade phenanthrene were able to use exudates. Presence of root exudates increased the proportion of PAH-RHD? genes compared to the bulk soil at the beginning and in microcosms without exudates (respectively 10% and 1 %). However, phenanthene dissipation in sand or soil were weaker with root exudates and aged PAH concentrations has not shifted during incubation time. Nevertheless, the root exudates increased the PAH labile fraction extract with cyclodextrin solution into two in three soils historically contaminated