Techn. Universiẗat, Diss., 2002--Berlin.

To study the regulation of gene expression, a good reporter system is very useful. The lack of a good reporter system for the solvent-producing clostridia hindered the progress of research in this area. The objective of this study was to develop a reporter system to facilitate the elucidation of the control mechanism for the expression of solvent-producing genes. A potential reporter gene was found in Clostridium beijerinckii NRRL B593, which contains an adh gene encoding a primary-secondary alcohol dehydrogenase and this adh gene is not present in Clostridium acetobutylicum ATCC 824 and Clostridium beijerinckii NRRL B592. The adh gene was cloned into the E. coli -Clostridium shuttle vectors to generate plasmids. An electro-transformation procedure was developed for C. beijerinckii NRRL B592. Shuttle plasmids were transformed into C. beijerinckii NRRL B592 or C. acetobutylicum ATCC 824. The copy number of the plasmids in C. beijerinckii was 4. Isopropanol production suggested that the adh gene was expressed in transformants of C. acetobutylicum ATCC 824 and C. beijerinckii NRRL B592. Northern analysis indicated that the expression of the adh gene was regulated at the transcriptional level in the transformants of C. beijerinckii. The transcriptional start site for the adh gene was identified by the primer extension method. A promoter-probing vector was constructed and tested with the promoter from the ferredoxin(fer) gene. The expression of the adh gene under the control of the fer promoter was at a low and similar level during acidogenesis and solventogenesis. The expression pattern of the adh gene under the control of the promoter of the adh gene differed from that under the control of the promoter of the fer gene. / Ph. D.

Clostridium beijerinckii is a spore-forming, obligate anaerobe that is capable of producing butanol, acetone and isopropanol. These industrial chemicals are traditionally known as solvents. The regulation of solventogenic fermentation is linked to the onset of sporulation, so that by the time the organism begins to produce solvents, it is also entering into spore formation and metabolic slowdown. The goal of this research project was to study the effect of placing the solvent-production genes from C. beijerinckii under the control of constitutive promoters from other genes, in an attempt to allow an earlier start of butanol production during the growth phase than is the case with the wild-type cells. The aldehyde dehydrogenase from C. beijerinckii NRRL B593 (ald) and alcohol dehydrogenase from C. beijerinckii NRRL B592 (adhA) were placed under the control of the promoter from the acid-producing operon (the BCS operon) in one vector, and under the control of the promoter from the ferredoxin gene in another. In both cases, aldehyde dehydrogenase activity was produced earlier in the growth phase in transformed cells, but alcohol dehydrogenase activity was not. The adhA gene from C. beijerinckii NRRL B592 was paired with the adhB gene from the same organism in a third vector, both under the control of the promoter from the BCS operon. In cells transformed with this vector, alcohol dehydrogenase activity was observed earlier in the growth phase than it was in wild-type NRRL B592 cells. / Ph. D.

Hohenheim, Univ., Diss., 2006.

University, Diss., 2003--Konstanz.

abstract: Reductive dechlorination by members of the bacterial genus Dehalococcoides is a common and cost-effective avenue for in situ bioremediation of sites contaminated with the chlorinated solvents, trichloroethene (TCE) and perchloroethene (PCE). The overarching goal of my research was to address some of the challenges associated with bioremediation timeframes by improving the rates of reductive dechlorination and the growth of Dehalococcoides in mixed communities. Biostimulation of contaminated sites or microcosms with electron donor fails to consistently promote dechlorination of PCE/TCE beyond cis-dichloroethene (cis-DCE), even when the presence of Dehalococcoides is confirmed. Supported by data from microcosm experiments, I showed that the stalling at cis-DCE is due a H2 competition in which components of the soil or sediment serve as electron acceptors for competing microorganisms. However, once competition was minimized by providing selective enrichment techniques, I illustrated how to obtain both fast rates and high-density Dehalococcoides using three distinct enrichment cultures. Having achieved a heightened awareness of the fierce competition for electron donor, I then identified bicarbonate (HCO3-) as a potential H2 sink for reductive dechlorination. HCO3- is the natural buffer in groundwater but also the electron acceptor for hydrogenotrophic methanogens and homoacetogens, two microbial groups commonly encountered with Dehalococcoides. By testing a range of concentrations in batch experiments, I showed that methanogens are favored at low HCO3 and homoacetogens at high HCO3-. The high HCO3- concentrations increased the H2 demand which negatively affected the rates and extent of dechlorination. By applying the gained knowledge on microbial community management, I ran the first successful continuous stirred-tank reactor (CSTR) at a 3-d hydraulic retention time for cultivation of dechlorinating cultures. I demonstrated that using carefully selected conditions in a CSTR, cultivation of Dehalococcoides at short retention times is feasible, resulting in robust cultures capable of fast dechlorination. Lastly, I provide a systematic insight into the effect of high ammonia on communities involved in dechlorination of chloroethenes. This work documents the potential use of landfill leachate as a substrate for dechlorination and an increased tolerance of Dehalococcoides to high ammonia concentrations (2 g L-1 NH4+-N) without loss of the ability to dechlorinate TCE to ethene. / Dissertation/Thesis / Ph.D. Microbiology 2013

Clostridium difficile (CD) is the leading cause of bacterial hospital-associated infection in North America. How CD colonizes the human host, including its response to the innate immune system and other stresses, is poorly understood. This work considers CD's defenses against two stresses found in the host - the antimicrobial peptide LL-37 and reactive oxygen species (ROS). LL-37 had bactericidal activity against CD. CD strains varied in their sensitivity to the peptide, and epidemic-associated strains were more resistant to LL-37 than others. CD became more resistant to LL-37 following exposure to sub-lethal concentrations of the peptide, suggesting the presence of inducible resistance mechanisms. A quantitative proteomics analysis revealed definite alterations in CD protein expression caused by LL-37. Specific changes included increased expression of DltB, a protein previously reported to confer resistance against other antimicrobial peptides. Notably, disruption of individual LL-37-induced genes did not sensitize CD to the peptide. This suggests functional redundancy, and that LL-37 may cause global changes in protein expression, not limited to antimicrobial peptide resistance determinants. One of the proteins most strongly induced by LL-37 was a predicted superoxide reductase (SOR). As CD is considered a strict anaerobe, expression of a predicted antioxidant protein was an interesting finding. Heterologous expression of CD SOR in a superoxide dismutase-deficient E. coli strain confirmed its action as a superoxide scavenger. Insertional inactivation of SOR rendered CD more sensitive to oxygen and ROS-generating compounds, indicating that SOR contributes to antioxidant defense in CD. SOR mutants were impaired in their ability to cause disease in hamsters, indicating a role for this protein in infection.

Vid tillverkning av massa och papper förorenas årligen 505 miljoner kubikmeter vatten som måste renas innan det släpps tillbaka till omgivningen. Vid reningen avskiljs först stora partiklar som sedan avvattnas och förbränns. Vattnet som blir kvar genomgår ytterligare en rening, varvid det bildas bioslam. Bioslammet innehåller mycket intracellulärt vatten, vilket gör det kostsamt och energikrävande att avvattna. Det är även sedan 2005 förbjudet att dumpa organiskt material, varför en mer ekonomiskt attraktiv behandling av slammet är anaerob nedbrytning. I denna nedbrytning omvandlas det organiska materialet till metan och koldioxid där metanet är den eftertraktade gasen. Bioslammet innehåller dock partiklar såsom träfiberrester och mikroorganismer med komplex struktur och är näringsfattigt. Någon form av sönderdelande förbehandling underlättar därför rötningsprocessen. I detta projekt undersöktes termisk förbehandling i kombination med kemisk förbehandling på bioslam från Stora Enso Skoghalls bruk på Hammarö. Själva rötningen skedde i två omgångar varav den första omgången med termisk förbehandling vid 70C och den andra vid 140C. Den kemiska förbehandlingen skedde med tillsats av lut (natriumhydroxid), kalk (kalciumhydroxid) och syra (fosforsyra) vid pH 9 och 11 för baserna och pH 2 och 4 för syran. Även neutrala prov (endast värmebehandling) och ett blankprov (ingen förbehandling) gjordes. Bioslammet ympades med kommunalt slam från Fiskartorpets reningsverk i Kristinehamn som har en mesofil bakteriekultur. Rötningen varade i 19 dagar per omgång i en temperatur på 35C och skedde satsvis i E-kolvar försedda med påsar för gasuppsamling. Totalt rötades 42 prov per omgång som utgjordes av sju mätpunkter á sex replikat för goda statistiska underlag. Resultaten gav en indikation för högst metanproduktion för proven behandlade med kalk vid 140C och för provet utan kemisk förbehandling vid 140C. Lägst produktion hade det kalkbehandlade provet vid pH 9 och 70C följt av blankprovet. Lutproven gav lägre metanproduktion vid 140C än vid 70C och fosforsyran hade så gott som oförändrad produktion mellan temperaturerna. Gemensamt för alla prover som behandlats vid 70C var att de fick en högre procentandel metan då de behandlats vid 140C. De resultat som erhållits är dock osäkra då det i vissa fall var stor spridning mellan provens biogasproduktion inom de enskilda förbehandlingsområdena. / In the pulp and paper process 505 million tons of water are polluted annually, which has to be purified before it is returned to the surrounding lakes. When the water is treated bigger particles are first separated to form sludge, then dewatered and finally incinerated. The excess water is further treated were a type of sludge  bio sludge  is formed. The bio sludge contains high concentration of intracellular water, why it is expensive and energy demanding to dewater. It is also forbidden to dump organic waste since 2005, why a more economically attractive treatment of the water is anaerobic digestion. In the digestion organic compounds is converted into methane and carbon dioxide where the methane is the desired gas. The bio sludge also contains fiber residues and microorganisms with complex structure and is nutrient-poor, which makes it hard to digest. Some kind of disintegrating pretreatment is needed and co-digestion with a more nutrient-rich sludge to facilitate the digestion process. In this project thermal pretreatment in combination with chemical pretreatment was examined on bio sludge from Stora Enso Skoghalls bruk at Hammarö. The anaerobe digestion was done by two rounds whereof the first round thermal pretreated at 70C and the second at 140C. The chemical pretreatment was done by additive of sodium hydroxide, calcium hydroxide and phosphoric acid at pH 9 and 11 for the bases and pH 2 and 4 for the acid. Also neutral samples (no chemical pretreatment) and a reference sample (no pretreatment) were done. The bio sludge were co-digested with municipal sludge from Fiskartorpets reningsverk in Kristinehamn which has a mesophilic bacterial culture. The anaerobic digestion lasted for 19 days per round at a temperature of 35C and were done batch wise in E-flasks provided with a small bag for gas collection. Totally 42 samples were made per round which consisted of seven measurement points and six replicates each for a good statistical basis. The results gave an indication of the highest methane production for the samples treated with calcium hydroxide at 140C and the neutral sample treated at 140C. The sample treated with calcium hydroxide at pH 9 and 70C gave the lowest production of methane followed by the reference sample. The samples treated with sodium hydroxide gave a lower methane production at 140C than at 70C while the acid treated samples had almost the same production at the two different temperatures. All the samples had in common a higher proportion of methane in the biogas when treated at 140C than at 70C. The results should be taken with caution since the distribution amongst the samples within the same pretreatment method sometimes is very high.

3-Methyl indole (3-MI) is a malodorant in food and animal waste and Clostridium scatologenes ATCC 25775 is the model organism for the study of 3-MI production. 3-MI is an anaerobic degradation product of L-tryptophan and can cause pulmonary disorders and death in cattle and goats. To elucidate the 3-MI biosynthesis pathway and the underlying genes, it is necessary to develop a system to allow genetic modification in Clostridium scatologenes ATCC 25775. Bacteriophages and transposons are useful tools to achieve this goal. Isolation of Clostridium scatologenes ATCC 25775 bacteriophage was attempted by prophage induction and enrichments using environmental sources. To induce prophages, cultures of Clostridium scatologenes ATCC 25775 were exposed to an effective concentration of mitomycin C at 2μg/ml and 5μg/ml. Induction with temperature was performed at 42ºC and 55ºC. Bacteriophage liberation, determined by a decrease in optical density was not observed in response to mitomycin C or by different growth temperatures. Nineteen environmental samples were tested for the presence of a bacteriophage that could infect Clostridium scatologenes ATCC 25775. The first cycle of enrichments suggested a decrease in cell density, consistent with the presence of a bacteriophage but this was not observed in further iterations. Plaque assays were performed to confirm the presence of phage, but no plaques were observed. Although, different experimental conditions were tested, a transducing bacteriophage capable of infecting Clostridium scatologenes ATCC 25775 was not isolated. Transposons have been successfully used to generate mutants in Clostridium difficle. Therefore, we attempted to introduce transposons Tn5 and Tn916 into Clostridium scatologenes ATCC 25775 using electroporation. Transposon mutagenesis using Tn916 did not yield antibiotic resistant colonies. In contrast, commercially available transposon Tn5 gave antibiotic resistant colonies. However, further screening of the colonies using transposon specific primers in PCR reactions, did not yield any PCR product. We were unsuccessful in developing a genetic modification system in Clostridium scatologenes ATCC 25775 using bacteriophage or transposons.

Die vorliegende Arbeit befaßt sich mit der anaeroben Entfärbung und aeroben Reinigung von Textilabwasser durch biologische Verfahren. Dazu wurde über mehrere Jahre eine Versuchskläranlage im Pilotmaßstab betrieben und die dabei gewonnenen Erkenntnisse in einer Betriebskläranlage umgesetzt. Die Pilotanlage bestand aus einer anaeroben Vorlage und einem Reaktor, der nach dem SBR-Verfahren (sequentielles biologisches Reinigungsverfahren) betrieben wurde. Das Reaktorvolumen beider Behälter betrug 1 m³. Mit dieser Anlage wurden in der Anaerobstufe farbige Abwässer und gezielt verschiedene Farbmittel entfärbt und in der anschließenden aeroben Belebung die organischen Belastungen und andere Verschmutzungen bis auf die vom Gesetzgeber geforderten Grenzwerte reduziert. Die Versuche ergaben, daß es unter anaeroben Bedingungen möglich ist, auch aerob nicht eliminierbare Färbemittel abzubauen und damit das Gesamtabwasser zu entfärben. Für die Versuche kamen nur „reale“ Abwässer zum Einsatz. Durch die umfangreichen Untersuchungen und Anpassungen an der Versuchskläranlage konnte das Verfahren für die Textilveredlung Erzgebirge technisch so weiterentwickelt werden, daß solide Bemessungsgrundlagen für die großtechnische Realisierung in Form einer Betriebskläranlage vorlagen. Das Reaktorvolumen der beiden parallel geschalteten SBR-Behälter beträgt jeweils 1200 m³. Neben der umfassenden Darstellung und Auswertung der Forschungsergebnisse und Praxiserfahrungen an der Pilotanlage in zahlreichen Abbildungen und Tabellen ist auch die Dokumentation des mehrjährigen Betriebs der Großanlage Gegenstand dieser Arbeit. Am Gesamtabwasserstrom eines Textilveredlungsbetriebes konnte die Eignung einer Kombination aus anaerober biologischer Entfärbung und aerober biologischer Reinigung nachgewiesen werden.