Au cours des dernières décennies, la fermentation IBE (isopropanol, butanol and éthanol) a connu un regain d'intérêt en vue de la production de carburants ou de composés chimiques à partir de matériaux renouvelables. Dans cette étude, nous avons étudié la production d'IBE avec le producteur naturel Clostridium beijerinckii NRRL B593 et avec des souches modifiées de Clostridium. acetobutylicum ATCC 824. En culture discontinue, la souche C. beijerinckii NRRL B593 excrétait 13.2 g/L d'IBE (dont 4,5 g/L d'isopropanol). Afin d'améliorer la production d'IBE, le gène codant pour l'alcool déshydrogénase secondaire (s-Adh) de NRRL B593, ainsi que différentes combinaisons des gènes des enzymes actives de la conversion de l'acétoacétyl-CoA en acétone, c.-à-d. l'acétoacétyl-CoA acétate/butyrate transférase (ctfA et ctfB) et l'acétoacétate décarboxylase (adc), ont été exprimées dans la souche productrice d'ABE (acétone, butanol éthanol), C. acetobutylicum ATCC 824. Les résultats montrent que la sur-expression des gènes ctfA et ctfB augmentait significativement la productivité et les concentrations finales en IBE tandis que la surexpression du gène adc n'avait qu'un effet limité. Cultivée en discontinu, la meilleure souche, exprimant les gènes adh, ctfA, ctfB et adc a produit 24.4 g/L d'IBE dont 8.8 g/L d'isopropanol avec une productivité de 0.7 g/L h. Cultivée en mode continu à un taux de dilution de 0.1 h-1, la productivité en IBE a été portée à 1.7 g/L h. Puisque le mélange IBE est considéré comme un additif carburant de qualité, les transformants obtenus constituent une avancée réelle vers le développement d'un procédé IBE industriel de production de biocarburants. / Over the past decades, the IBE fermentation (isopropanol, butanol and ethanol) has received a renew interest for the production of fuels or biochemicals from renewable materials. In the present study, we have investigated the IBE fermentation using the natural producer C. beijerinckii NRRL B593 and genetically-modified strains of Clostridium acetobutylicum ATCC 824. In batch culture, C. beijerinckii NRRL B593 was found to excrete 13.2 g/L IBE of which 4.5 g/L was isopropanol. To increase IBE production, the gene coding the secondary alcohol dehydrogenase (s-Adh) of C beijerinckii NRRL B593 and different combinations of genes coding for enzymes active in acetoacetyl-CoA to acetone conversion i.e. acetoacetate decarboxylase (adc) and acetoacetyl-CoA: acetate/butyrate: CoA transferase subunits A and B (ctfA and ctfB) were expressed in the ABE (acetone, butanol ethanol) producer C. acetobutylicum ATCC 824. Results showed that the overexpression of the ctfA and ctfB genes significantly increased both speed and extent of the IBE production while the overexpression of the adc gene had only a little effect. In batch culture, the best mutant (expressing adh, ctfA, ctfB and adc) produced 24.4 g/L IBE (of which 8.8 g/L was isopropanol) and displayed an IBE productivity of 0.7 g/L h. Cultivated in continuous mode at the dilution rate of 0.1 h-1, IBE productivity was increased to 1.7 g/L h IBE. As the IBE mix has been considered as a valuable fuel additive, the transformants obtained are a real step forward towards the development of an industrial IBE process for biofuel production.

Per varie ragioni, tra cui le loro specifiche condizioni di crescita, la diagnosi di infezione e di contaminazione alimentare da clostridi presenta ancora numerose difficoltà sia a livello clinico-batteriologico che a livello molecolare. In questo lavoro di tesi si è cercato di ampliare lo spettro di conoscenze riguardo i clostridi e la loro diffusione; durante il primo anno di ricerca è stato studiato, applicando nuove tecniche di microscopia, il processo di germinazione di Clostridium tyrobutyricum, uno dei batteri maggiormente responsabili del gonfiore tardivo dei formaggi a pasta dura; l’applicazione di tecniche di Real-Time PCR ha nel contempo reso possibile una determinazione quantitativa dello stesso in latte. Successivamente, è stata condotta un’analisi di tipizzazione molecolare di clostridi nell’ambito di una filiera agro-zoo-casearia finalizzata alle matrici di processo al fine di individuare le possibili vie di diffusione dei microrganismi. La parte finale del lavoro è stata dedicata allo studio di espressione genica di un altro Clostridium responsabile di gonfiore ma scelto perché geneticamente indistinguibile da Clostridium botulinum, ovvero il Clostridium sporogenes; l’analisi trascrizionale dei suoi geni durante le fasi vegetativa, di sporulazione, germinazione ed esocrescita ha permesso di assegnare diverse funzioni a geni singoli o a gruppi di geni allo scopo di utilizzare queste informazioni per formulare ipotesi future anche su altre specie di clostridi patogeni. / For several reasons, including their specific growth requirements, the diagnosis of infections and food contamination caused by clostridia still presents much difficulty at the clinical, bacteriological and molecular levels. The main purpose of this work is to learn more about clostridia and their interactions with environment. First, new microscopy techniques have been used to study the germination process in Clostridium tyrobutyricum, an anaerobic bacterium responsible for late blowing defects during cheese ripening; meanwhile, the application of real-time PCR methods have been employed to enumerate C. tyrobutyricum cells and spores in milk. Then, a molecular genotyping has been set in order to identify the most common clostridia in a agro-dairy production aimed to detect the possible ways of diffusion of these microbial species. The last part concerns the study of expression patterns of Clostridium sporogenes, an apathogenic gram positive clostridium usually involved in food damage and frequently isolated from late bowled cheese; Clostridium sporogenes is genetically indistinguishable from Clostridium botulinum and is often used as a model for the toxic subtypes. The objective of this study is to use an array-based large-scale transcriptional analysis in order to study gene expression in four different steps of Clostridium sporogenes life cycle: vegetative cells, sporulating cells, dormant spores and germinating ones. Our aims includes being able to relate gene-expression patterns to specific phenotypes and to discover gene expression divergences between the different phases of living, germination and outgrowth of spore-forming bacteria. An important aim is to assign functions to groups of or individual C. sporogenes genes and use this information to formulate specific hypotheses for further testing also on pathogenic clostridia types.

Antibiotics are important to equine medicine, but can cause detrimental side-effects including reduced feed intake, allergic reactions, and diarrhea. Antibiotic-associated diarrhea (AAD) is attributed to disruption of the hindgut microflora, permitting proliferation of pathogenic microbes. The objectives were to evaluate the effects of antibiotics on beneficial fecal bacteria, AAD-associated pathogens, microbial species richness and fermentation. Horses were assigned to treatment groups: control (no antibiotics, n=6), trimethoprim-sulfadiazine (oral, n=6), or sodium ceftiofur (IM, n=6). Fecal samples were taken during adaptation (3 wk), antibiotic challenge (1 wk), and withdrawal (1 wk). Fecal cellulolytics decreased by >99% during challenge and did not recover during withdrawal (P < 0.0001). Lactobacilli decreased by >60% during challenge (P = 0.0453). Salmonella spp. increased 94% with trimethoprim-sulfadiazine challenge (P = 0.0115). There was no detectable Clostridium difficile during adaptation or in any control horse. C. difficile increased (P < 0.0001) when horses were challenged, and remained elevated 7 d after withdrawal. There was no effect of challenge on in vitro digestibility or microbial species richness as evaluated by denaturing gradient gel electrophoresis (P > 0.05). These results indicate that antibiotics can disrupt the normal flora and allow proliferation of pathogens, even without affecting digestibility and causing AAD.

O objetivo desse trabalho foi descobrir melhores estratégias de controle de perdas em silagens de cana-de-açúcar, utilizando combinações de aditivos químicos e microbianos ou estratégias de vedações. No primeiro experimento o intuito foi estudar estratégias de controle de clostrídios em silagens de cana-de-açúcar aditivadas com cal virgem. Os tratamentos foram: 1) controle; 2) 1,5% cal virgem (Cal); 3) 1,5% cal virgem + 5×105 ufc/g Lactobacillus plantarum Ma 18/5U (Cal+LP); 4) 1.5% cal virgem + 5×105 ufc/g Lactobacillus buchneri 40788 (Cal+LB); 5) 1,5% cal virgem + 0,07% nitrito de sódio (Cal+Nitrito); 6) 1,5% cal virgem + 0,15% benzoato de sódio (Cal+Benzoato). Como silos experimentais foram utilizados baldes plásticos com capacidade de 20 L. A cal não foi eficiente em diminuir perdas em relação ao controle (20,6% e 19,1%, respectivamente) já as combinações cal+Benzoato, cal+Nitrito, cal+LB e cal+LP, diminuíram perdas, apresentando os valores de 11,2%, 14,1%, 14,1% e 13,9%, respectivamente. A estabilidade aeróbia das silagens tratadas com cal (172 h), cal+Benzoato (155 h) e cal+Nitrito (223 h), foram maiores em comparação às tratadas com cal+LB (49 h), cal+LP (48,4 h) e controle (51,9 h). A silagem controle apresentou menores contagens de clostrídios (3,26 log ufc/g) e os tratamentos Cal e Cal+LB apresentaram as contagens mais altas (6,74 log ufc/g e 5,96 log ufc/g, respectivamente), enquanto Cal+nitrito (5,24 log ufc/g), Cal+LP (4,63 log ufc/g) e Cal+benzoato (4,41 log ufc/g) apresentaram contagens intermediárias. Nenhum tratamento imposto foi capaz de controlar o crescimento de clostrídios estimulado pela cal virgem. No segundo ensaio o objetivo foi comparar filmes plásticos na vedação de silagens de cana-de-açúcar. Os tratamentos impostos foram: 1) filme de poliamida 45?m, recoberta com manta de polietileno para proteção contra raios ultravioleta e danos físicos (PA45+M); 2) filme de polietileno coextrusado com poliamida, dupla face, 125?m (PA125); 3) filme de polietileno, dupla face, 200?m (DF). Três silos tipo trincheira foram preenchidos simultaneamente e durante o abastecimento a cana-de-açúcar picada foi tratada com benzoato de sódio na dose 0,15% da matéria natural. Decorridos 90 dias de armazenamento, os silos foram abertos e as silagens utilizadas como ingredientes para alimentação de vacas em lactação. Quinze vacas Holandesas (615 kg PV) foram alocadas aleatoriamente em três Quadrados Latinos 3x3, com períodos de 18 dias. O consumo de matéria seca (CMS). Amostras de leite foram coletadas para análises de proteína, gordura, lactose, caseína, ácidos graxos livres, sólidos totais, Nuréico e contagem de células somáticas. O óxido de cromo foi utilizado como marcador externo para estimar a produção fecal. As diferentes estratégias de vedação não influenciaram a qualidade das silagens e, consequentemente, não afetaram o CMS 19,9 kg/dia, a produção 25,1 kg/dia e a composição do leite (gordura 3,5%, proteína 3,3%, lactose 4,4%), das vacas. As diferentes estratégias de vedação utilizadas não influenciam a qualidade das silagens de cana-de-açúcar e o desempenho de vacas leiteiras e se mostraram efetivas para vedação de silos horizontais. No presente estudo, tanto a utilização de aditivos como a adoção de estratégias de vedação não foram efetivas em controlar perdas em silagens de cana-de-açúcar. / The aim of this study was to discover the best strategies to control losses in sugarcane silages, using combinations of chemical and microbial additives or covering strategies. In the first trial the aim was to compare strategies to prevent clostridium grow in sugarcane silages added with lime. Treatments were Control: no additives; L: 1.5% lime; L + LP: 1.5% lime + Lactobacillus plantarum Ma 18/5U (5×105 cfu/g fresh forage); L + LB: 1.5% lime + Lactobacillus buchneri 40788 (5×105 cfu/g fresh forage); L + N: 1.5% lime + 0.07% sodium nitrite; L + B: 1.5% lime + 0.15% sodium benzoate. As experimental silos were used plastic buckets with a capacity of 20 L. Mini bags were prepared with the following treatments 1) control, 2) 1.5 % of lime and 3) 1.5 % of lime + 5 × 105 cfu/g of L. plantarum, in order to analyze the pH drop at the onset of fermentation. The lime was not effective in decreasing losses compared to control silages (20.6% and 19.1%, respectively) however the treatments lime+Benzoate, lime+nitrite, lime+LP and lime+LB, decreased losses, showing lower values: 11.2%, 14.1%, 14.1% and 13.9%, respectively. The aerobic stability of silages treated with lime (172 h), lime+Benzoate (155 h) and lime+Nitrite (223 h) were higher compared to those treated with lime+LB (49 h), lime+LP (48.4 h) and control (51.9 h). The control treatment showed the lowest counts of clostridia (3.26 log cfu/g) and treatment with lime and lime+LB had the highest counts (6.74 log cfu/g and 5.96 log cfu/g, respectively), while lime+nitrite (5.24 log cfu/g), lime+LP (4.63 log cfu/g) and lime+benzoate (4.41 log cfu/g) showed intermediate counts of clostridia. None of additives combined with lime were able to provide butyric acid free silages. In the second trial the aim was to compare plastic films for sealing sugarcane silages. Treatments were: 1) polyamide film 45?m plus a protection against physical damage and ultraviolet light (PA45 + P), 2) polyethylene film coextruded with polyamide, black-on--white, 125?m (PA125), 3) polyethylene film, blackon- white 200?m (BW). Three trench silos were filled out simultaneously with chopped sugarcane treated with 0.15% of sodium benzoate (as fed basis). After 90 days of storage, the silos were opened and silages fed to lactating cows as total mixed rations. . Fifteen Holstein cows (615 kg BW) were randomly allocated into 3x3 Latin square with periods of 18 days. Dry matter intake, diet apparent digestibility, milk yield and milk composition were evaluated on days 14 to 18 in each period. Chromium oxide was used as external marker to estimate fecal excretion. Sealing strategies had no influence on silage quality and cows performance. Dry matter intake (19.9 kg /day), milk yield (25.1 kg/day), and milk composition (fat 3.5%, 3.3% protein, and 4.4% lactose) were quite similar across treatments. The different sealing strategies do not affect sugarcane silage quality and dairy cows performance and proven effective for sealing horizontal silos. In the present study, neither additives nor covering strategies were effective in reducing losses in sugarcane silages.

Ziel der Arbeit war es, die Besonderheiten des Gärungsverlaufes bei der Silierung von nitratarmem Grünfutter aufzuklären. Dazu wurden sechs Silierversuche zur Untersuchung des Gärungsverlaufes mit unterschiedlichem Clostridiensporenbesatz durchgeführt. Dabei wurde auch die Wirkung eines Zusatzes von 0,05 bzw. 0,1 % N / TS als Nitrat und Nitrit sowie Zusätze von Inoculantien und Ameisensäure geprüft. Weiterhin wurde untersucht, ob die unter Laborbedingungen gefundenen Auswirkungen des Fehlens von Nitrat ebenso bei Grünfutter, das unter praxisnahen Bedingungen geerntet wurde, auftreten. Zur Fragestellung, welche Siliermittel bei nitratarmem Grünfutter eingesetzt werden können, wurde auch hier der Zusatz von zwei MSB-Präparaten und Ameisensäure, sowie eines nitrithaltigen Siliermittels bei geringem und erhöhtem Clostridiensporengehalt des Siliergutes geprüft. Als Ergebnis dieser mehrjährigen, umfangreichen Untersuchungen mit verschiedenen Futterpflanzen und unterschiedlicher Clostridiensporenbelastung hatte sich gezeigt, daß der Verlauf der Stoffumsetzungen und das Gärproduktmuster am Ende der Gärung in Abhängigkeit vom Nitratgehalt wesentlich unterschiedlich ist. In Silagen aus nitratarmem Grünfutter trat Buttersäure bereits von Gärbeginn und parallel zur Milchsäuregärung auf. Die Essigsäuregehalte waren stets sehr niedrig. Anaerob stabile und instabile Silagen aus nitratarmem Grünfutter weisen in allen Stadien des Gärungsprozesses ein völlig anderes Verhältnis zwischen Buttersäure und den übrigen Merkmalen des unerwünschten Stoffabbaus - Essigsäure, Ammoniak, pH - auf als Silagen aus nitrathaltigem Grünfutter. Für die Einschätzung der Vergärbarkeit sind außer TS und Z/PK auch der Nitratgehalt sowie epiphytischer Keimbesatz und Clostridiensporengehalt des AM zu berücksichtigen. Bei Fehlen von Nitrat besteht, unabhängig von der nach TS und Z/PK vorhergesagten Vergärbarkeit, ein besonderes Risiko für das Auftreten von Buttersäure. In Abhängigkeit vom Aufwuchs war die Einschätzung der Vergärbarkeit des Grünfutters verschieden und es trat eine unterschiedliche Gärqualität der Silagen auf. Bei nitratarmem Grünfutter ist der strategische Einsatz von MSB-Präparaten zu empfehlen. Das nitrithaltige Siliermittel hatte sich insbesondere bei erhöhter Clostridiensporenbelastung und/oder niedrigem TS-Gehalt des Grünfutters als sehr wirksam erwiesen. Bei Anwendung des derzeit gültigen, für Silagen aus nitratreichem Grünfutter entwickelten DLG-Beurteilungsschlüssels auf Silagen aus nitratarmem Ausgangsmaterial ist mit einer Fehlbewertung zu rechnen. / The object of this work is to explain the distinctive feature of ensilage of green forage low in nitrate. The fermentation process of ensiling green forage low in nitrate was proved in 6 experiments with different content of spores of clostridia. In all experiments the green forage was ensiled with following treatments: without additives (control), with 0,05 and 0,1% N / DM as nitrate or nitrite, with lactic acid bacteria and formic acid. Furthermore the effects of absence of nitrate, proved under laboratory conditions, has been investigated in experiments with green forage produced under practical conditions. The treatments were the same as mentional above. As a result of this several years and extensive investigations with different green fodder and different content of spores of clostridia it was shown that metabolism during fermentation process and pattern of fermentation products in the end of fermentation are significant different depending on content of nitrate in green forage. In ensiling material low in nitrate butyric acid was formed already at the beginning of the fermentation process, parallel to the lactic acid fermentation. The content of acetic acid was always extremely low. In comparison with silages from green forage high in nitrate anaerobe stable or unstable silages show a different ratio between butyric acid and other characteristics of undesirable decomposition during fermentation - acetic acid, ammonia, pH - in equal fermentation stages. In summary, one can say that epiphytic lactic acid bacteria, content of clostridia spores and nitrate of herbage take into account to judge the fermentability more than previous, together with DM and WSC/BC. Furthermore the judgement of herbage fermentability and the fermentation quality of silages were different depending on number of growth. In silages low in nitrate is an especially risk for occurrence of butyric acid, independing on judgement of fermentability on the basis of DM and ratio of watersoluble carbohydrates to buffering capacity. It`s advisable to use lactic acid bacteria additives (inoculants) always for ensiling green forage low in nitrate. The additive with nitrite has proved as most effectively especially for green forage high in clostridia spores and/ or low content of dry matter of green forage. It is very probably, that the use of current DLG- evaluation system to estimate the fermentation quality, developed for silages from green forage high in nitrate, is not correct and leads to error of judgement of silages from green forage low in nitrate.

O objetivo desse trabalho foi descobrir melhores estratégias de controle de perdas em silagens de cana-de-açúcar, utilizando combinações de aditivos químicos e microbianos ou estratégias de vedações. No primeiro experimento o intuito foi estudar estratégias de controle de clostrídios em silagens de cana-de-açúcar aditivadas com cal virgem. Os tratamentos foram: 1) controle; 2) 1,5% cal virgem (Cal); 3) 1,5% cal virgem + 5×105 ufc/g Lactobacillus plantarum Ma 18/5U (Cal+LP); 4) 1.5% cal virgem + 5×105 ufc/g Lactobacillus buchneri 40788 (Cal+LB); 5) 1,5% cal virgem + 0,07% nitrito de sódio (Cal+Nitrito); 6) 1,5% cal virgem + 0,15% benzoato de sódio (Cal+Benzoato). Como silos experimentais foram utilizados baldes plásticos com capacidade de 20 L. A cal não foi eficiente em diminuir perdas em relação ao controle (20,6% e 19,1%, respectivamente) já as combinações cal+Benzoato, cal+Nitrito, cal+LB e cal+LP, diminuíram perdas, apresentando os valores de 11,2%, 14,1%, 14,1% e 13,9%, respectivamente. A estabilidade aeróbia das silagens tratadas com cal (172 h), cal+Benzoato (155 h) e cal+Nitrito (223 h), foram maiores em comparação às tratadas com cal+LB (49 h), cal+LP (48,4 h) e controle (51,9 h). A silagem controle apresentou menores contagens de clostrídios (3,26 log ufc/g) e os tratamentos Cal e Cal+LB apresentaram as contagens mais altas (6,74 log ufc/g e 5,96 log ufc/g, respectivamente), enquanto Cal+nitrito (5,24 log ufc/g), Cal+LP (4,63 log ufc/g) e Cal+benzoato (4,41 log ufc/g) apresentaram contagens intermediárias. Nenhum tratamento imposto foi capaz de controlar o crescimento de clostrídios estimulado pela cal virgem. No segundo ensaio o objetivo foi comparar filmes plásticos na vedação de silagens de cana-de-açúcar. Os tratamentos impostos foram: 1) filme de poliamida 45?m, recoberta com manta de polietileno para proteção contra raios ultravioleta e danos físicos (PA45+M); 2) filme de polietileno coextrusado com poliamida, dupla face, 125?m (PA125); 3) filme de polietileno, dupla face, 200?m (DF). Três silos tipo trincheira foram preenchidos simultaneamente e durante o abastecimento a cana-de-açúcar picada foi tratada com benzoato de sódio na dose 0,15% da matéria natural. Decorridos 90 dias de armazenamento, os silos foram abertos e as silagens utilizadas como ingredientes para alimentação de vacas em lactação. Quinze vacas Holandesas (615 kg PV) foram alocadas aleatoriamente em três Quadrados Latinos 3x3, com períodos de 18 dias. O consumo de matéria seca (CMS). Amostras de leite foram coletadas para análises de proteína, gordura, lactose, caseína, ácidos graxos livres, sólidos totais, Nuréico e contagem de células somáticas. O óxido de cromo foi utilizado como marcador externo para estimar a produção fecal. As diferentes estratégias de vedação não influenciaram a qualidade das silagens e, consequentemente, não afetaram o CMS 19,9 kg/dia, a produção 25,1 kg/dia e a composição do leite (gordura 3,5%, proteína 3,3%, lactose 4,4%), das vacas. As diferentes estratégias de vedação utilizadas não influenciam a qualidade das silagens de cana-de-açúcar e o desempenho de vacas leiteiras e se mostraram efetivas para vedação de silos horizontais. No presente estudo, tanto a utilização de aditivos como a adoção de estratégias de vedação não foram efetivas em controlar perdas em silagens de cana-de-açúcar. / The aim of this study was to discover the best strategies to control losses in sugarcane silages, using combinations of chemical and microbial additives or covering strategies. In the first trial the aim was to compare strategies to prevent clostridium grow in sugarcane silages added with lime. Treatments were Control: no additives; L: 1.5% lime; L + LP: 1.5% lime + Lactobacillus plantarum Ma 18/5U (5×105 cfu/g fresh forage); L + LB: 1.5% lime + Lactobacillus buchneri 40788 (5×105 cfu/g fresh forage); L + N: 1.5% lime + 0.07% sodium nitrite; L + B: 1.5% lime + 0.15% sodium benzoate. As experimental silos were used plastic buckets with a capacity of 20 L. Mini bags were prepared with the following treatments 1) control, 2) 1.5 % of lime and 3) 1.5 % of lime + 5 × 105 cfu/g of L. plantarum, in order to analyze the pH drop at the onset of fermentation. The lime was not effective in decreasing losses compared to control silages (20.6% and 19.1%, respectively) however the treatments lime+Benzoate, lime+nitrite, lime+LP and lime+LB, decreased losses, showing lower values: 11.2%, 14.1%, 14.1% and 13.9%, respectively. The aerobic stability of silages treated with lime (172 h), lime+Benzoate (155 h) and lime+Nitrite (223 h) were higher compared to those treated with lime+LB (49 h), lime+LP (48.4 h) and control (51.9 h). The control treatment showed the lowest counts of clostridia (3.26 log cfu/g) and treatment with lime and lime+LB had the highest counts (6.74 log cfu/g and 5.96 log cfu/g, respectively), while lime+nitrite (5.24 log cfu/g), lime+LP (4.63 log cfu/g) and lime+benzoate (4.41 log cfu/g) showed intermediate counts of clostridia. None of additives combined with lime were able to provide butyric acid free silages. In the second trial the aim was to compare plastic films for sealing sugarcane silages. Treatments were: 1) polyamide film 45?m plus a protection against physical damage and ultraviolet light (PA45 + P), 2) polyethylene film coextruded with polyamide, black-on--white, 125?m (PA125), 3) polyethylene film, blackon- white 200?m (BW). Three trench silos were filled out simultaneously with chopped sugarcane treated with 0.15% of sodium benzoate (as fed basis). After 90 days of storage, the silos were opened and silages fed to lactating cows as total mixed rations. . Fifteen Holstein cows (615 kg BW) were randomly allocated into 3x3 Latin square with periods of 18 days. Dry matter intake, diet apparent digestibility, milk yield and milk composition were evaluated on days 14 to 18 in each period. Chromium oxide was used as external marker to estimate fecal excretion. Sealing strategies had no influence on silage quality and cows performance. Dry matter intake (19.9 kg /day), milk yield (25.1 kg/day), and milk composition (fat 3.5%, 3.3% protein, and 4.4% lactose) were quite similar across treatments. The different sealing strategies do not affect sugarcane silage quality and dairy cows performance and proven effective for sealing horizontal silos. In the present study, neither additives nor covering strategies were effective in reducing losses in sugarcane silages.

Ziel der vorliegenden Arbeit war zum einen die Ermittlung des im Grünfutter notwen-digen Mindest-Nitratgehaltes zur Erzielung buttersäurefreier Silagen, wobei TS-Gehalt und Z/PK-Quotient des Ausgangsmaterials sowie dessen Belastungsgrad mit Clostridiensporen berücksichtigt wurden. Dazu wurden zwei mehrfaktorielle Laborsi-lierversuche durchgeführt, bei denen eine Variationsreihe des Nitratgehaltes (0,01...0,3 % NO3-N in TS) mit Variationsreihen des TS-Gehaltes (ca. 14...40 %) und des Z/PK-Quotienten (1,5...3,1) systematisch kombiniert wurden. Jede Wertekombi-nation wurde sowohl mit sauber geerntetem Grünfutter als auch mit Clostridienspo-ren kontaminiertem Grünfutter geprüft. Die Silagen wurden nach 180 Tagen Lage-rungsdauer untersucht. Zum anderen wurde die Dynamik der Clostridienentwicklung im Gärungsverlauf in Abhängigkeit von TS-Gehalt, Säuerungsintensität und Nitratge-halt geprüft. Jede Stufe des TS-Gehaltes (20, 30, 40 und 50 %) wurde mit Zusätzen von Nitrat, Milchsäurebakterien (MSB) bzw. MSB + Glucose und MSB + Nitrat ange-setzt. Das Ausgangsmaterial (Dac. glomerata, nitratfrei) war durchgehend mit Clostridiensporen kontaminiert. Die Untersuchung der Silagen erfolgte 3, 7, 14, 28, 56 und 180 Tage nach dem Ansatz. Der Konservierungserfolg bei der Silierung hängt nicht nur vom TS-Gehalt und Z/PK-Quotienten sondern auch vom Nitratgehalt und Clostridiensporenbesatz des Aus-gangsmaterials ab. Bei sehr niedrigem Nitratgehalt des Grünfutters liegt ein erhöhtes Risiko für die Entstehung von Buttersäure in der Silage vor, auch bei dem als leicht vergärbar geltendem Grünfutter (VK > 45) und auch dann, wenn es im sauber geern-tetem Zustand einsiliert worden ist. Bei weiter erhöhten VK-Werten, > 45 (durch Er-höhung des TS-Gehaltes und/oder Z/PK-Quotienten), wird die Höhe der Buttersäure-gehalte zwar eingeschränkt. Zur sicheren Ausschaltung von Buttersäuregärung ist jedoch auch hier ein gewisser Nitratgehalt notwendig. Bei der Silierung nitratfrei-en/nitratarmen Grünfutters nimmt das Fehlgärungsrisiko mit dem Belastungsgrad an Clostridiensporen zu. Der notwendige Mindest-Nitratgehalt (MNG) hängt sowohl vom VK-Wert als auch vom Kontaminationsgrad des Grünfutters mit Clostridiensporen ab. Er ist um so niedriger, je höher der VK-Wert und geringer der Kontaminationsgrad ist, und umgekehrt. (MNG (% NO3-N in TS) für sporenarmes Grünfutter = 0,24 - 0,0035 . VK MNG (% NO3-N in TS) für sporenreiches Grünfutter = 0,20 - 0,0021 . VK) Hohe Clostridiensporengehalte lagen vor allem in buttersäurehaltigen Silagen vor und insbesondere dann, wenn das Grünfutter sehr geringe Nitratgehalte aufwies. Zwischen der Höhe der Buttersäuregehalte und dem Clostridiensporengehalt besteht jedoch kein direkter Zusammenhang. Erhöhung des TS-Gehaltes bewirkt eine Ein-schränkung der Clostridienentwicklung. Ein Rückgang des Sporengehaltes im Ver-gleich zum Ausgangsmaterial (nitratfrei) lag aber erst bei einem TS-Gehalt von etwa 50 % vor. Durch Zusätze von MSB sowie MSB + Glucose konnte die Milchsäuregä-rung deutlich intensiviert werden. Ein sehr geringer pH-Wert war schon am 3. oder 7. Lagerungstag erreicht. Buttersäuregärung war aber erst bei TS-Gehalten > 40 % ausgeschaltet. Eine Einschränkung der Sporenbildung lag ebenfalls erst bei TS-Gehalten über 40 % vor. Bei Nitratzusatz blieben die Silagen aller TS-Stufen bis zur Auslagerung buttersäurefrei. Die Sporengehalte gingen in allen TS-Stufen während des Gärungsverlaufes kontinuierlich zurück. Bei steigenden TS-Gehalten war der Rückgang der Sporengehalte verlangsamt. Durch die Kombination von MSB und Nit-rat konnte nicht nur ein sicherer Erfolg bei der Unterbindung von Buttersäurebildung und Laktatabbau sondern auch eine starke Verminderung der Sporengehalte erreicht werden. / The first aim of this work was to determine the minimum content of nitrate (MCN) which is required to get silage free of butyric acid. For it, two multi-factorial experi-ments with orchardgrass were carried out under laboratory condition. In this experi-ments, nitrate content (0.01 ... 0.3 % N in DM) was systematically combined with staggered levels of the dry matter (DM) content (14 ... 40 %) and of the ratio of water-soluble carbohydrate to buffering capacity (WSC/BC: 1.5 ... 3.1). All variants were tested with forage without or with addition of clostridial spores. The silages were ana-lysed after 180 days of incubation. The second aim of this study was to explain the dynamic of clostridial development during ensilage, depending on DM content, intensity of lactic acid formation, and ni-trate content. orchardgrass (free of nitrate) with 4 levels of DM (20, 30, 40, and 50 %) was firstly contaminated with clostridial spores about 104 / g FM. Then it was ensi-laged with following treatments: without additives, with inoculation of lactic acid bac-teria (LAB) alone or in combination with 2 % Glucose in FM (LAB+G), with nitrate addition (0.1 and 0.15 % N in DM), and with LAB plus nitrate 0.1 % N in DM. The si-lages were analysed 3, 7, 14, 28, 56, and 180 days after ensiling. Results showed that silage quality not only depends on DM content and ratio of WSC to BC, but also depends on nitrate content and extent of clostridial spores in forage. With an extremely low content of nitrate a high risk of butyric acid formation is given in silages, even if the ensiling material had a high ensilability (FC ³ 45) and a very low content of clostridial spores. The butyric acid concentration decreased with in-creasing DM content from 14 to 40 % or with increasing WSC/BC-ratio. But to get the silages free of butyric acid, a certain amount of nitrate was required. By adding clos-tridial spores in fodder the risk of butyric acid formation was increased, especially in case of material lacking in nitrate. The value of MCN depends on ensilability of the forage, as measured by DM-content and WSC/BC-ratio or by fermentability coefficient (FC) = DM+8WCS/BC, as well as depends on content of clostridial spores in the material used. The higher the FC-value and the lower the spores content is, the less nitrate is required to get silage free of butyric acid. MNC (% N in DM) for very low contaminated forage = 0.24 - 0.0035.FC MNC (% N in DM) for high contaminated forage = 0.20 - 0.021.FC High content of clostridial spores was especially found in silages containing butyric acid, which were made from forage with very low nitrate content (£ 0.02 % N in DM). But a strong relationship was not found between butyric acid and spores content. By increasing DM content the development of clostridia during ensiling was limited. A continuous decrease of spores content, in comparison with the forage before ensiled, was observed at first by increasing DM content to 50 %. By inoculation with LAB or LAB+G the lactic acid formation was strongly stimulated. A very low pH was reached 3 or 7 days after ensiling. But the butyric acid formation could be firstly prevented by increasing DM content to over 40 %. For all levels of DM, by nitrate addition the silages remained no butyric acid during the whole period of incubation. The concentration of clostridial spores decreased continuously during ensilage. This decrease was slower with increasing DM content . By combination of LAB with nitrate a reliable prevention of butyric acid formation and a fast decrease in spores concentration were reached.

Reducing the production cost of industrial biofuels will greatly facilitate their proliferation and co-integration with fossil fuels. The cost of feedstock is the largest cost in most fermentation bioprocesses and therefore represents an important target for cost reduction. Meanwhile, the biorefinery concept advocates revenue growth through complete utilization of by-products generated during biofuel production. Taken together, the production of biofuels from low-cost crude glycerol, available in oversupply as a by-product of bioethanol production, in the form of thin stillage, and biodiesel production, embodies a remarkable opportunity to advance affordable biofuel development. However, few bacterial species possess the natural capacity to convert glycerol as a sole source of carbon and energy into value-added bioproducts. Of particular interest is the anaerobe Clostridium pasteurianum, the only microorganism known to convert glycerol alone directly into butanol, which currently holds immense promise as a high-energy biofuel and bulk chemical. Unfortunately, genetic and metabolic engineering of C. pasteurianum has been fundamentally impeded due to a complete lack of genetic tools and techniques available for the manipulation of this promising bacterium. This thesis encompasses the development of fundamental genetic tools and techniques that will permit extensive genetic and metabolic engineering of C. pasteurianum. We initiated our genetic work with the development of an electrotransformation protocol permitting high-level DNA transfer to C. pasteurianum together with accompanying selection markers and vector components. The CpaAI restriction-modification system was found to be a major barrier to DNA delivery into C. pasteurianum which we overcame by in vivo methylation of the recognition site (5’-CGCG-3’) using the M.FnuDII methyltransferase. Systematic investigation of various parameters involved in the cell growth, washing and pulse delivery, and outgrowth phases of the electrotransformation procedure significantly elevated the electrotransformation efficiency up to 7.5 × 104 transformants µg-1 DNA, an increase of approximately three orders of magnitude. Key factors affecting the electrotransformation efficiency include cell-wall-weakening using glycine, ethanol-mediated membrane solubilization, field strength of the electric pulse, and sucrose osmoprotection. Following development of a gene transfer methodology, we next aimed to sequence the entire genome of C. pasteurianum. Using a hybrid approach involving 454 pyrosequencing, Illumina dye sequencing, and single molecule real-time sequencing platforms, we obtained a near-complete genome sequence comprised of 12 contigs, 4,420,100 bp, and 4,056 candidate protein-coding genes with a GC content of 30.0%. No extrachromosomal elements were detected. We provide an overview of the genes and pathways involved in the organism’s central fermentative metabolism. We used our developed electrotransformation procedure to investigate the use of established clostridial group II intron biology for constructing chromosomal gene knockout mutants of C. pasteurianum. Through methylome analysis of C. pasteurianum genome sequencing data and transformation assays of various vector deletion constructs, we identified a new Type I restriction-modification system that inhibits transfer of vectors harboring group II intron gene knockout machinery. We designated the new restriction system CpaAII and proposed a recognition sequence of 5’-AAGNNNNNCTCC-3’. Overcoming restriction by CpaAII, in addition to low intron retrohoming efficiency, allowed the isolation of a gene knockout mutant of C. pasteurianum with a disrupted CpaAI Type II restriction system. The resulting mutant strain should be efficienty transformed with plasmid DNA lacking M.FnuDII methylation. Lastly, we investigated the use of plasmid-based gene overexpression and chromosomal gene downregulation to alter gene expression in C. pasteurianum. Using a β-galactosidase reporter gene, we characterized promoters corresponding to the ferredoxin and thiolase genes of C. pasteurianum and show that both promoters permitted high-level, constitutive gene expression. The thiolase promoter was then utilized to drive transcription of an antisense RNA molecule possessing complementarity to mRNA of our β-galactosidase reporter gene. Our antisense RNA system demonstrated 52-58% downregulation of plasmid encoded β-galactosidase activity throughout the duration of growth. In an attempt to perturb the central fermentative metabolism of C. pasteurianum and enhance butanol titers, we prepared several antisense RNA constructs for downregulation of 1,3-propanediol, butyrate, and hydrogen production pathways. The resulting downregulation strains are expected to exhibit drastically altered central fermentative metabolism and product distribution. Taken together, we have demonstrated that C. pasteurianum is amendable to genetic manipulation through the development of methods for plasmid DNA transfer and gene overexpression, knockdown, and knockout. Further, our genome sequence should provide valuable nucleotide sequence information for the application of our genetic tools. Thus, the genome sequence, electrotransformation method, and associated genetic tools and techniques reported here should promote extensive genetic manipulation and metabolic engineering of this biotechnologically important bacterium.

Soil microbial communities are affected extensively by addition of amendments to their environment. Of particular concern is the addition of poultry litter, which contains a substantial C, energy, and nutrient supply, but also antibiotic resistance genes (ARG), antimicrobials, and a multitude of microbial species. This project seeks to primarily assess if there is a change in bacterial community structure in response to poultry litter amendments to pasture land across geographically independent land across northern Georgia. It may be that changes in the relative abundance of bacterial communities also result in alteration in ARGs, and the community resistance to antibiotics (“resistome”) which in turn increases the potential threat of antibiotic resistance genes. While another part of this study will determine changes in integrons and specific ARGs, this project will focus on changes in bacterial communities and the potential functional changes in the community, which in turn have consequences for ARG levels and its horizontal transfer to various members of the soil community. Addition of waste from livestock is a historical method for increasing nutrients needed in the soil for the cultivation of crops, and in turn causes pronounced shifts in soil microbial communities due to the addition of large amounts of carbon, nutrients, foreign microbes, and other material. This study is unique because it utilizes a novel and relatively large landscape-scale to determine if there are discernable and repeatable patterns of bacterial community structure change in response to amendment regardless of exact soil type or source of chicken litter amendment. In the future, these data can also provide insight into the changes in the relative abundance antibiotic related genes associated with community change. / M.S. / Soil is complicated, both in terms of its physical makeup and the organisms that live inside of it. Predicting changes in soil based on the addition of foreign material such as chemicals or biological waste is not an easy process, and whether or not it is even possible to reliably predict those changes is a matter of some dispute. This study is designed to illustrate that such changes can in fact be reliably and consistently predicted even with regard to the addition of complicated materials to the soil. In this study, specifically, the material in question is chicken litter. A mix of the bedding and waste produced by chickens, litter is commonly handled by composting and is added to soil in farms as a fertilizer rich in organic matter. It is possible to point at specific elements of the soil such as the chemistry and bacteria and see how it is changed with the addition of chicken litter, which allows us to determine the nature and extent of the change that chicken litter has on soil. This study is conducted on a larger scale than similar experiments conducted in the past, making it apparent that these relationships exist on a repeated basis. It is the object of this study to pave the way and make it easier for scientists in the future to determine these relationships in other unique contexts.

Dans le cadre des législations européennes relatives au traitement des déchets et aux énergies renouvelables, la méthanisation apparaît comme une alternative prometteuse pour la stabilisation et la valorisation des Ordures Ménagères Résiduelles (OMR). D'un point de vue opérationnel l'hétérogénéité et les difficultés de mise en mouvement d'une matrice aussi complexe que les OMR sont à l'origine de pertes de rendement voire de l'arrêt d'installations de méthanisation. Les performances de méthanisation sont en particulier limitées par l'étape d'hydrolyse des fractions lignocellulosiques qui représentent la majorité du potentiel méthanogène des OMR. Dans ce contexte, l'objectif principal du travail de thèse, était l'étude d'un procédé de percolation dans lequel le déchet n'est pas mis en mouvement. Au travers de ce travail nous avions également pour ambition de produire des connaissances à caractère plus générique sur l'hydrolyse afin d'en améliorer les performances. Des expériences préliminaires ont d'abord permis la définition d'un système expérimental adéquat pour l'étude à l'échelle laboratoire de l'hydrolyse des OMR. La représentativité d'un déchet reconstitué, reproductible et d'utilisation aisée, a notamment été vérifiée en termes de potentiel méthanogène, de profil hydrolytique et de flore microbienne. Suite à la définition de ce système expérimental, son comportement hydrolytique a été comparé à celui d'un test de lixiviation de référence (NF EN 12457-4) afin de valider l'intérêt opérationnel de la percolation pour l'hydrolyse des OMR. De façon inattendue, l'extraction de 38,90% de la matière carbonée initiale du déchet a ainsi été mise en évidence lors de l'hydrolyse par percolation contre 17,84% lors de l'hydrolyse par lixiviation, renforçant l'intérêt suscité par la percolation pour l'hydrolyse des OMR. L'optimisation des performances d'hydrolyse par percolation a ensuite été réalisée par le criblage de huit paramètres opérationnels afin de déterminer leur influence sur les performances d'hydrolyse des OMR, au travers de deux plans d'expérience. L'ajout d'alcalinité (12 gHCO3-.L-1) et la recirculation du percolat pendant 6 h par jour ont ainsi permis d'augmenter significativement les performances d'hydrolyse, passant de 17 à 43% d'extraction de la matière organique (DCO) initiale du déchet (autrement dit de 26 à 69% de la matière biodégradable initiale). L'étude des communautés microbiennes et de leur activité a également été réalisée. Le séquençage des pyrotags d'ADNr 16S a ainsi permis de mettre en évidence le caractère dominant des Classes Clostridia et Bacteroidia au sein des communautés hydrolytiques. Le couplage de cette démarche qualitative à une approche quantitative par qPCR sur une série de biomarqueurs taxonomiques et fonctionnels a permis de montrer qu'il existe une corrélation positive entre l'ajout de carbonates, la neutralisation du pH, la quantité de matière hydrolysée à 14 jours et soit l'abondance de la Classe Bacteroidia soit celle des gènes de la famille hydA, impliqués dans la fermentation. Finalement, l'analyse microbiologique a été approfondie au jour 4, c'est-à-dire durant la phase d'hydrolyse intense, grâce à une approche de métatranscriptomique. L'analyse des transcrits fonctionnels indique que l'alcalinité influence l'activité des microorganismes de la Classe Clostridia dès le jour 4 des essais d'hydrolyse. Plus spécifiquement, l'ajout de carbonates semble corrélé à une modification du métabolisme des sucres chez des microorganismes non cultivables apparentés à Clostridium cellulolyticum et à l'augmentation de l'expression de l'opéron nif, impliqué dans la fixation de l'azote, chez différents groupes de microorganismes. / In the framework of the European green policy, anaerobic digestion appears as a promising technology for stabilization and valorization of Municipal Solid Waste (MSW). In practice, mechanical mixing of a complex and heterogeneous matrix such as MSW induces major operational constraints. Anaerobic digestion performances are especially limited by hydrolysis of lignocellulosic fractions which represent the main part of MSW methanogenic potential. In this context, this PhD project was aiming to characterize and optimize of a percolation process in which MSW stands still. Preliminary experiments were conducted in order to define an experimental system suitable for lab-scale study of MSW hydrolysis. Therefore, the representativeness of an easy-to-use and reproducible reconstituted waste was verified in terms of methanogenic potential, hydrolytic profiles and associated microbial communities. Following system definition, hydrolysis behavior by percolation was compared to a reference lixiviation test (NF EN 12457-4). Surprisingly, hydrolysis by percolation permitted the extraction of 39% of carbonated matter initially contained in waste whereas 18% were extracted during hydrolysis by lixiviation, thus validating operational benefit of percolation for MSW hydrolysis. Optimization of hydrolysis performance was then conducted through the screening of eight operational parameters for their influence on MSW hydrolysis performances thanks to two Designs Of Experiment (DOE). Cumulative effect of alkalinity addition (12 gHCO3-.L-1) and percolate recirculation (6 hour.day-1) significantly improved hydrolysis yield, from 17 to 43% of extracted organic matter compared to the initial content of waste (corresponding to an extraction of 26 and 69% of biodegradable matter). Structure and activity of hydrolytic microbial communities were also studied. 16S rDNA-pyrotags sequencing brought out the dominance of classes Clostridia and Bacteroidia. Additionally, a quantitative approach led by qPCR revealed a correlation between carbonates addition, pH neutralization, amounts of hydrolyzed matter at day 14 and either class Bacteroidia or genes from hydA family, involved in fermentation. Finally, metatranscriptomic approach was conducted at day 4 in order to further study microbial activity during the intense hydrolysis phase. According to functional analysis, alkalinity seems have positive influence on class Clostridia activity. More specifically, carbonates addition seems correlated to a modification of carbohydrates metabolism of organisms affiliated to Clostridium cellulolyticum and to transcriptional up-regulation of nif operon, involved in nitrogen fixation, among various types of microorganisms.