Recent evaluations have demonstrated the ability of the bacteria Rhodococcus rhodochrous DAP 96253 to inhibit the growth of molds associated with plant and animal diseases as well as post-harvest loss of fruits, vegetables and grains. Pre-pilot-scale fermentations (20-30L) of Rhodococcus rhodochrous DAP 96253 were employed as a research tool with the goal of producing a practical biological agent for field-scale application for the management of white-nose syndrome (WNS) in bats and post-harvest fungal losses in several fruit varieties. Several key parameters within the bioreactor were evaluated for the potential to increase production efficiency as well as activity of the biocatalyst. These parameters included elapsed fermentation time, dissolved Oxygen, and carbohydrate concentration of which increased carbohydrate concentration at the time of harvest was shown to have a negative impact on the catalyst activity. In addition, process improvements including utilization of a liquid inoculum, an autoinduction feed strategy, and increased glucose concentration in the feed medium increased fermentation yields to 100-150g/L, while the biocatalyst efficiency was increased from previous work. To increase production efficiency, a multi-bioreactor scheme was developed that used a seed bioreactor and subsequent production tank, which doubled run yields per production cycle. Amidase, cyanidase, urease, and alkene-monoxygenase activity were monitored throughout the study as potential indicators for the multi-faceted mechanism of fungal antagonism. Of these amidase, cyanidase, and urease were demonstrated to be more elevated in cells that showed antifungal activity than those that did not. This study represents the first example of a reproducible pre-pilot plant-scale biomanufacturing process for a contact-independent biological control agent for established and emerging fungal pathogens of plants and animals, and facilitates large-scale production for broad application.

Rhodococcus equi (R. equi) is one of the most serious causes of pneumonia in young foals. The clinical disease is of great concern to breeding farms worldwide due to the impact of mortality on economic losses. While adult horses are resistant to R. equi, foals exhibit a distinct age-associated susceptibility. The mechanism underlying this susceptibility in foals is not well understood. Interferon-gamma (IFNg) plays an important role in the clearance of R. equi, but its expression is impaired in neonatal foals. Moreover, the regulation of this age-related IFNg expression in foals remains unknown. In humans, IFNg expression has been shown to be regulated by DNA methylation, lymphoproliferation, and influenced by environmental exposure. Therefore, we hypothesized that environmental exposure promotes IFNg expression through regulation of DNA methylation and lymphoproliferation. The objectives were: (1) to estimate the relevance of IFN-g production and R. equi infection in foals; (2) investigate the role of lymphoproliferation and DNA methylation in the regulation of IFN-g expression in foals; (3) to evaluate the effect of environmental exposure on IFN-g expression by housing foals in a barn environment verses pasture.; (4) to investigate the effect of environment exposure on antigen-presenting cells (APC), which sensor the environmental antigens and modulate IFN-g production by T cells. The results demonstrated that the IFN-g expression was inversely correlated with the age-related susceptibility to R. equi infection. lymphoproliferation promoted IFN-g expression in foals, whereas, DNA methylation repressed IFN-g expression. The IFN-g expression was augmented in foals exposed to the barn air which contained higher numbers of aerosol miroorganisms. DNA on the IFN-g promoter was demethylated and the lymphoproliferative activity was elevated in foals with barn-air exposure. The barn-air exposure also promoted the maturation and activation of APC to prime IFN-g expression by T cells in foals. Overall, this body of work demenstrated a relationship between IFN-g expression and R. equi infection, provided novel information on mechanisms that regulate IFN-g expression, and identified the effect of environment on mechanisms responsible for IFN-g expression.

La biodégradation du 2-aminobenzothiazole (ABT) a été comparée entre des bactéries en suspension dans l'eau et des bactéries immobilisées sur un support d'alginate. Trois processus de dégradation de l'ABT ont été étudiés : la photodégradation sous lumière solaire en présence du complexe Fe(III)-acide nitrilotriacétique (FeNTA), la biodégradation par la bactérie aérobie stricte Rhodococcus rhodochrous et la combinaison de ces deux processus. Le métabolisme de R. rhodochrous a été étudié par RMN in vivo du 31P et du 13C : des informations importantes sur le métabolisme phosphoré et carboné ont été obtenues. La réponse de la bactérie face à divers stress a été évaluée et a montré sa capacité d'adaptation aux variations environnementales. La spéciation du fer pour son rôle important dans l'activation de la biodégradation d'ABT a été etudiée : complexes organiques, oxydes et oxy(hydr)oxydes de fer ont été testés pour connaître les formes biodisponibles pour R. rhodochrous

Orientador: Marcio Garcia Ribeiro / Banca: José Paes de Almeida Nogueira Pinto / Banca: Rogerio Giuffrida / Resumo: A rodococose suína compreende doença infecciosa caracterizada por linfadenites piogranulomatosas. Diferentes fatores de virulência são reconhecidos na patogenicidade de Rhodococcus equi. A estrutura da parede celular bacteriana, a viabilidade no interior de fagócitos e na ausência de ferro, a produção de citotoxinas, a resistência aos antimicrobianos convencionais e, recentemente, a presença de proteínas associadas à virulência (Vap) reguladas por plasmídios, são considerados os principais mecanismos de virulência do microrganismo. Diferentes fatores de virulência foram avaliados em 23 (6,1%) linhagens de R. equi isoladas de 378 linfonodos submandibulares e mesentéricos de suínos e javalis (Sus scrofa). Foram realizados exames microbiológicos em 129 linfonodos apresentando lesões (linfadenite) e 129 sem lesões (controle) de suínos, e 60 linfonodos com lesões e 60 sem lesões de javalis. Dentre as 23 linhagens de R. equi, 19 (7,4%) foram isoladas de suínos, das quais 17 obtidas de linfonodos com lesões e duas sem lesões. Das 19 linhagens de suínos, 18 (94,7%) foram obtidos de linfonodos submandibulares e um (5,3%) de mesentérico. As quatro (3.3%) linhagens de R. equi isoladas de javalis foram obtidas exclusivamente de linfonodos com lesões. Destes, três foram obtidos de linfonodos submandibulares e um de mesentérico. Dentre nove antimicrobianos testados, azitromicina (100,0%), gentamicina (100,0%), levofloxacina (100,0%), vancomicina (100,0%), amoxicillina/ácido clavulânico (94,7%), eritromicina (94,7%) e rifampicina (94,7%) foram os fármacos mais efetivos. Baixa ocorrência de resistência aos antimicrobianos nos isolados de suínos foi observada contra os fármacos testados. A concentração inibitória mínima (MIC90) da azitromicina, eritromicina e rifampicina foi observada, respectivamente, em ≤2 µg/mL, ≤0,5 µg/mL and ≤1 µg/mL... (resumo completo, clicar acesso eletrônico abaixo) / Abstract: The rhodococcosis in swine comprise an infectious disease characterized by pyogranulomatous lymphadenitis. Different virulence factors are recognized in pathogenicity of the Rhodococcus equi. The structure of bacterial cell wall, the viability inside of phagocytes and in absence of iron, the production of cytotoxins, the resistance to conventional antimicrobials and recently, the presence of proteins associated to virulence (Vap) regulated by plasmids, are considered the most important virulence mechanisms of microorganism. Different virulence factors were evaluated in 23 (6.1%) R. equi strains isolated from 378 submandibular and mesenteric lymph nodes of swine and wild boars (Sus scrofa). Microbiological exams were performed in 129 lymph nodes presenting lesions (lymphadenitis) and 129 without lesions (controls) from swine, and 60 lymph nodes with lesions and 60 without lesions from wild boars. Among 23 R. equi strains, 19 (7.4%) were isolated from swine and, from these, 17 were obtained from lymph nodes with lesions and two without lesions. From 19 strains isolated from swine, 18 (94.7%) were obtained of submandibular lymph nodes and one (5.3%) from mesenteric. The four (3.3%) R. equi strains isolated from wild boars were obtained exclusively of lymph nodes presenting lesions. From these, three were obtained from submandibular lymph nodes and one of mesenteric. Among nine antimicrobials tested, azithromycin (100.0%), gentamicin (100.0%), levofloxacin (100.0%), vancomycin (100.0%), amoxicillin/clavulanic acid (94.7%), erythromycin (94.7%) and rifampin (94.7%) were the most-effective drugs. Low rates of resistance to antimicrobials in swine isolates were observed against drugs tested. The inhibitory minimal concentration of 90% of isolates (MIC90) with use of azithromycin, erythromycin and rifampin were observed respectively in 2 μg/mL, 0.5 μg/mL and 1 μg /mL... (Complete abstract click electronic access below) / Mestre

Rhodococci are notable for their ability to degrade a variety of natural and xenobiotic compounds. Recently, interest in Rhodococcus has increased due to the discovery of a large number of genes for secondary metabolism. Only a few secondary metabolites have been characterized from the rhodococci (including 3 recently described antibiotics). Twenty-four new Rhodococcus strains were isolated from soils in East Tennessee using acetonitrile enrichment culturing and identified using 16S rRNA analysis. Forty-seven Rhodococcus strains were screened for antibiotic production using a growth inhibition assay. One strain, MTM3W5.2, had 90% similarity to the Rhodococcus opacus 16S rRNA gene sequence and produced a large zone of inhibition against R. erythropolis and a large number of closely related species. The antimicrobial compound produced by MTM3W5.2 had a large MW of 911.5452 Da and acts much like a bacteriocin but no amino acids were detected in this molecule based on TLC analysis.

The soil bacterium Rhodococcus is a member of the phylum Actinobacteria and is related to Streptomyces, which is known for its production of many secondary metabolites. Recent genomic investigation of Rhodococcus has uncovered many silent gene clusters that appear to code for nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKS) of unknown function. Previous work, showed that Rhodococcus species strain KCHXC3 produces an inhibitory compound in agar culture extracts that displays prominent activity against several Gram positive and Gram negative species including the pathogens Rhodococcus equi, Shigella dysenteriae and Pseudomonas aeruginosa. Using the engineered Rhodococcus transposon vector, pTNR, the goal of this investigation is to screen random mutants of KCHXC3 for strains that no longer produce the inhibitory molecule. A library of 1825 random insertion mutants was produced via electroporation then screened for production of the inhibitory molecule by a disk diffusion assay against Shigella dysenteriae. From this screening, 7 mutants which no longer produce the compound of interest were identified.

The bacterium Rhodococcus is a potential source for novel antimicrobial metabolites. Recently, the Rhodococcus strain MTM3W5.2 was isolated from a soil sample collected from Morristown, East Tennessee and was found to produce an inhibitor molecule that is active against similar Rhodococcus species. The aim of this research is to extract, purify, and characterize the active compound. The compound was obtained from both agar and broth cultures of strain MTM3W5.2 and purified by primary fractionation of crude extract on a Sephadex LH-20 column, followed by semi-preparative reversed phase column chromatography. Final purification was achieved using multiple rounds of an analytical C18 HPLC column. Based on the results obtained from UV-Vis, FT-IR, and HR-MS, the molecule is a polyketide with a molecular formula of C52H78O13 and an exact mass of 911.5490 amu. The partial structure of this compound has been determined using 1D and 2D NMR spectroscopy.

Due to increases in antibiotic resistance stemming from the overuse of commercial antibiotics, the need to discover novel antibacterial compounds is becoming more urgent. A species of Rhodococcus, MTM3W5.2, has been discovered and was found to produce a metabolite with inhibitory activity against closely related species. The aim of this study is to elucidate the structure of the inhibitory metabolite by isolating and purifying it; then characterizing it using spectroscopic techniques. The compound was isolated from MTM3W5.2 RM broth cultures using n-butanol extraction, which yielded an active crude extract. The crude extract was then subjected to fractionation using a Sephedex LH-20 column with a 100% methanol solvent. The inhibitory activity of the fractions was tested through disk diffusion assay using Rhodococcus erythropolis as an indicator. Further preparation was completed using preparative reverse-phase high-performance liquid chromatography. Advanced purification was conducted by multiple rounds of analytical reverse-phase HPLC. Throughout the study the HPLC fractions were characterized and stability was monitored using UV-Visible spectroscopy. Two pure samples at 58.xx and 72.xx minutes from HPLC collections were selected for further structural identification and are currently being studied using spectroscopic techniques, most notably 2D NMR

Récemment, de nombreuses recherches ont été dévolues à la mise au point de réacteurs biphasiques, perçus comme une nouvelle technologie pour le traitement des polluants organiques dans les effluents gazeux. Ces réacteurs impliquent lutilisation dune seconde phase non aqueuse pour améliorer la solubilité et le transfert de masse des composés hydrophobes. Dans ce travail, nous avons développé un réacteur biphasique agité utilisant lhuile de silicone comme seconde phase. Initialement, Rhodococcus erythropolis T 902.1 a été sélectionné sur base de sa capacité à dégrader lisopropylbenzène (IPB), un composé choisi comme modèle représentatif de la famille du benzène. Deuxièmement, le transfert de masse de loxygène et de lIPB a été étudié en relation avec les conditions hydrodynamiques du réacteur et le type dhuile de silicone. Lutilisation dune proportion de 10 % dhuile de faible viscosité (10cSt) naffecte pas significativement le transfert de masse de loxygène. Cependant la grande solubilité de lIPB dans lhuile de silicone conduit à une forte augmentation du potentiel de transfert, spécialement pour les proportions en huile les plus élevées. Néanmoins, il ne semble pas utile de dépasser une proportion de 10 % car le KLaIPB et le KLaO2 diminuent drastiquement pour des proportions supérieures. Lexistence dune concentration optimale en élément biotique apparaît également. En effet, les concentrations optimales en biomasse (B) et extrait surfactant (ES) peuvent être évaluées à, respectivement 0,5 g/L et 0,7 g/L, elles assurent une valeur maximale du coefficient global de transfert de masse de loxygène (KLaO2). Plus spécifiquement, lES augmente laire interfaciale « a » en diminuant le diamètre des bulles tandis que la biomasse la diminue dès quune concentration de 1 g/L est atteinte. Au contraire, lES agit négativement sur le KL tandis que la biomasse laméliore globalement. En terme de performance, il est clairement montré que la taux de biodégradation de lIPB est davantage corrélé au débit gazeux de leffluent quà la concentration en polluant. Le réacteur biphasique a été suivi sur une période de 38 jours afin de caractériser son comportement à moyen terme pour différentes conditions opératoires. Lors dune phase dalimentation transitoire (10h/j), la capacité moyenne délimination est denviron 240 g/m3 pour une charge massique de 390 g/m3. Finalement, une approche originale a été développée en utilisant un bioréacteur de type scale-down pour reproduire les conditions hydrodynamiques rencontrées dans les réacteurs industriels. Il est clairement démontré que le polluant (IPB) affecte négativement lextrapolation en augmentant la vitesse de séparation de phase. Cependant cet impact négatif est largement compensé par la présence déléments biotiques qui stabilise fortement le système biphasique, rendant totalement envisageable lextrapolation à grande échelle. En conclusion, lutilisation dun réacteur biphasique eau-huile de silicone pour lélimination de concentrations élevées (~ 6g/m3) en polluants hydrophobes est adéquate. Le réacteur proposé présente de réelles opportunités pour le traitement biologique deffluents pollués par des composés hydrophobes. Son utilisation pourrait être envisagée lorsque loxydation thermique savère trop onéreuse ou lorsque les biofiltres classiques atteignent leurs limites ( >1 g/Nm3 et une charge volumique de 90m3/m3.h.)./Recently, a lot of research has been devoted to the study of two-phase partitioning bioreactors (TPPB) as new technology for xenobiotic degradation in gaseous effluents. These reactors involve the use of a second non-aqueous phase to improve the solubility and transfer of hydrophobic compounds. In this work, we have developed a stirred two-phase partitioning bioreactor using silicone oil as second phase. Initially, Rhodococcus erythropolis T 902.1 was selected on the basis of its capacity to metabolize isopropyl-benzene (IPB), used as representative of the benzene-containing compounds. Secondly, the mass transfer of both IPB and oxygen has been considered with relation to their influence on the hydrodynamics of the reactor and the type of silicone oil used. The addition of 10% low viscosity silicone oil (10 cSt) in the reactor does not significantly affect the oxygen transfer rate. The very high solubility of IPB in the silicone oil leads to an enhancement of the driving force term, especially when high proportion of silicone oil are used. However, it is not necessary to use a volume fraction higher than 10% since KLaIPB and KLaO2 decrease sharply at above such proportion. In addition, an optimal concentration appeared to exist for both biotic components, respectively 0,5 g/L and 0,7 g/L for biomass (B) and surfactant extract (SE) when the global mass transfer coefficient (KLa) of oxygen was measured in the TPPB. More specifically, SE improved the interfacial area a by decreasing the bubble diameter, while B reduced it at concentrations up to 1 g/L. In contrast, the SE concentration acted negatively on KL, while it was favoured by the B concentration. In term of performances, it was clearly shown that the biodegradation rate is more directly related to the inlet flow of IPB than to the concentration of IPB in the inlet gas. The TPPB was monitored for 38 days to characterise its behaviour under several operational conditions. During an intermittent loading phase (10 h/day), the average elimination capacity remained above 240 g/m3.h for an average IPB inlet load of 390 g/m3. h. Finaly, an original approach was developed using a scale-down bioreactor allowing to reproduce the hydrodynamics encountered under full scale TPPB. It was clearly shown that the IPB affects negatively the scaling-up of the process by increasing the speed of phase partitioning. However, this negative impact was strongly compensated by the presence of biotic compounds stabilizing the two phase system and rendering the scaling-up process feasible. In conclusion, the use of a water-silicone oil TPPB to remove a high inlet load of IPB was successful. The proposed reactor retains a high potential for the biological treatment of gas effluents polluted by hydrophobic aromatic compounds. The suggested process might be applied in the range of concentration and flow where thermal oxidation is too expensive (between 1 and 7 g/Nm3) or when the biofilters are usually limited, i.e. to treat a polluted effluent concentrated with > 1 g/Nm3 at a flow of 90m3/m3.h.

Lignin is the second most abundant biopolymer on Earth, right after cellulose, with a highly complex chemical structure that hinders its possible utilizations. Applications that utilize lignin in different manners are of great interest, due to its inexpensive nature. Present work is based on the notion of converting lignin into different biofuels that have only a few, however important, advantages over lignin as a direct energy source. The first part of current work (pyrolysis) details the analysis of lignin from a relatively new lignin isolation process called LignoBoost. It is obtained from the pulp and paper industry via CO₂ precipitation of lignin from black liquor (BL). This method is environment friendly, results lignin with minimal oxidation, eliminates the main bottleneck of the Kraft cycle (recovery boiler capacity), and yet leaves enough lignin in the process stream to recover pulping chemicals and generate energy for the pulp mill. Pyrolysis had converted this lignin into bio-oil with high aliphatic content and low oxidation level, all advantageous for application as liquid fuel. The second part of this dissertation proved the theory that lignin degradation and lipid accumulation metabolic pathways can be interconnected. Gram-positive Rhodococcus opacus species, DSM 1069 and PD630 were used to evaluate lignin to lipid bioconversion, starting with ethanol organosolv and Kraft lignin. This conversion is a first step in a multistep process towards biodiesel production, which includes transesterification, after lipids are extracted from the cells. Results clearly indicated that the lignin to lipid bioconversion pathway is viable, by cells gaining up to 4 % of their weight in lipids, while growing solely on lignin as a carbon and energy source.