Experiments were conducted to examine the cause of lag-phase growth during phenol degradation by mixed microbial cultures that had been acclimated to one of four substrates. Four aerated Imhoff cones were inoculated with wastewater sludge and fed one of four substrates: acetate, egg albumin, vegetable oil, or phenol. Inocula from these cones were injected into batch reactors containing phenol. Time-dependent growth was measured by two methods: most probable number (MPN) and epifluorescence microscopy (EM). The MPN technique was used to distinguish two cell concentrations: total cells and a phenol-degrading community within the total; EM was also used to count total cells. The results indicated that a lag in phenol utilization for all cultures, except the phenol-acclimated cultures, was a result of growth of a phenol-degrading subpopulation, and not due to enzyme induction of the existing population. Similar experiments were conducted using 2,4-dichlorophenol (2,4-DCP), which resulted in no growth and no degradation of 2,4-DCP.

Copies of author's previously published articles inserted. Bibliography: leaves 175-194. Eikenella corrodens, a Gram-negative rod, is a normal inhabitant of the human oral cavity. It is one of the most commonly detected cultivable bacteria from sub- and supra-gingival plaque and is often isolated in elevated proportions from sites exhibiting destructive periodontal disease compared with healthy sites. The primary aim of this study was to investigate the metabolism of E. corrodons, with particular reference to energy generation, and to determine the effect of physical environmental parameters on its growth.

Graduation date: 2006

The 1,1-dichloroethene (1,1-DCE) cometabolic transformation abilities of indigenous and bioaugmented microorganisms were compared in microcosms constructed with groundwater and aquifer solids from the Moffett Field site, CA. Microbial community structure in the microcosms and possible community shifts due to 1,1-DCE transformation stress was evaluated by terminal restriction fragment length polymorphism method (T-RFLP). An existing biotransformation model was used to simulate the experimental data using parameter values determined by Kim et al. (2002) and Rungkamol (2001) with small adjustments to the parameter values. The laboratory microcosm studies showed that both indigenous and bioaugmented butane utilizers were capable of transforming 1,1-DCE when fed butane as a primary substrate. A butane-grown enriched culture was bioaugmented into the microcosms and exposed to several repeated additions of butane and/or 1,1-DCE, ranging from 7.1 to 76 ��mol and from 0.17 to 1.99 ��mol, respectively. The bioaugmented butane-utilizers showed a reduced lag period compared to the indigenous butane-utilizers. The greatest ability to transform 1,1-DCE was observed in bioaugmented microcosms, simultaneously exposed to butane and 1,1-DCE. Very little 1,1-DCE was transformed in the bioaugmented microcosms that were not fed butane, presumably due to lack of reductant supply and/or product toxicity of 1,1-DCE transformation. Microbial community analyses revealed similar results for replicate microcosms and differences in the community structure in microcosms subjected to different patterns of substrate addition and 1,1-DCE cometabolism. 1,1-DCE transformation resulted in temporal fluctuations in specific bacterial groups in the bioaugmented microcosms. It could be inferred that microorganisms, correlated with the T-RFL of 183 base pair (bp) were generally predominant in butane-fed bioaugmented microcosms simultaneously exposed to 1,1-DCE. Bioaugmented microcosms that were pre-exposed to 1,1-DCE for 29 days in the absence of growth substrate, followed by the addition of butane showed a significantly different microbial community from bioaugmented microcosms fed butane and 1,1-DCE simultaneously. Microorganisms with T-RFL of 179 or 277.8 bp dominated in these microcosms. These differences were possibly the result of extensive 1,1-DCE transformation product toxicity during the pre-exposure phase of the tests. A model developed by Kim et al. (2002) was used to mathematically describe the rate and extent of butane utilization and the cometabolic transformation of 1,1-DCE in the microcosm tests. Using the kinetic parameter values previously determined by Kim et al. (2002) and Rungkamol (2001), heuristic fits were obtained between the experimental data and model simulations. The model successfully predicted the trend of the butane utilization and 1,1-DCE transformation. The model outputs were statistically quantified for their fit to the experimental data by estimating Standard Error of Estimate (SEE). A reasonable fit between model predictions and experimental observations was achieved. A significant contribution of this study was developing the laboratory methods to evaluate the microbial abilities to cometabolize 1,1-DCE and determining the communities of microorganisms correlated with those biotransformation activities. Furthermore, the model comparison to experimental data indicated that there was a potential in using the existing model to predict and improve bioremediation strategies. The results showed the successful bioaugmentation of a butane-utilizing culture to improve transformation performance. / Graduation date: 2003

Natural nitro compounds with diverse structures and biological functions are produced by bacteria, fungi, plants and animals. Little is known about the behavior of such compounds in natural ecosystems. The lack of accumulation in the biosphere implies that they are biodegraded. Microbial strategies for biodegradation of synthetic nitro compounds are well established; however only two pathways are known for degradation of natural nitro compounds. The research described here examines the genes that encode the key enzymes required for biodegradation of 3-nitrotyrosine (3NTyr) and 1-nitro-2-phenylethane (NPE). 3NTyr is a biological marker for disease and inflammation in plants and animals. A 3NTyr degrading microbe, Variovorax sp. JS669 was isolated from soil. We identified the JS669 denA, which encodes an enzyme that catalyzes denitration of 4-hydroxy-3-nitro-phenylacetate, the key step in metabolism of 3NTyr. The isolation of 3NTyr degraders and development of molecular probes specific to denA revealed that 3NTyr degradation is a widespread phenomena in natural habitats and the compound is metabolized by phylogenetically diverse bacteria. Phylogenetic analysis of the 4-hydroxy-3-nitro-phenylacetate denitrase from JS669 revealed it to be the first functionally annotated protein in a clade of unidentified Class A flavin monooxygenases. NPE has been identified from several plants, yet the biodegradation of the compound remained a mystery. Here we report the degradation of NPE and its analog 2-nitropropylbenzene. Discovery of the metabolic pathway revealed a novel microbial strategy to use a meta-ring fission degradation pathway to cleave an undesirable side chain from an aromatic compound and use the remainder of the compound as a carbon and energy source. Two genes that encode enzymes in the biodegradation pathway were identified and both are deeply branched within their respective phylogenetic trees, indicating that both represent highly specialized microbial enzymes. Furthermore, microbial degradation of NPE resulted in the production of 3-nitropropionic acid, a natural toxin that inhibits succinate dehydrogenase and is responsible for livestock illness and death. This is the first report of bacterial production of 3-nitropropionic acid, and might represent a significant source of 3-nitropropionic acid in natural habitats. The findings from these studies contribute to the overall understanding of microbial metabolism. Specifically, this research reveals genes that encode novel enzymes and strategies for the biodegradation of two natural nitro compounds. Furthermore, discovery of mechanisms for the biodegradation of such compounds reveals novel microbial metabolic diversity and provides insight into the evolution of degradation pathways for synthetic compounds.

Thesis (doctoral)--Helsinki University of Technology, 2009. / Includes bibliographical references. Also available on the World Wide Web.

Zymomonas mobilis is a non-pathogenic, facultatively-anaerobic Gram-negative bacterium, which has historically been used for the fermentation of alcoholic beverages in many tropical/sub-tropical countries. Due to its excellent ethanol-producing capabilities, significant effort has been undertaken over recent years to utilize it for industrial ‘bioethanol’ production. Its physiological and metabolic properties indicate that it may also be an excellent organism for the bio-production of many different types of organic molecules. Consequently, the aim of my thesis was to develop new molecular methodologies that would enable Z. mobilis to be ‘engineered’ for use in future ‘bioproduction’ endeavours. In the first part of my study, I analyzed the native (cryptic) plasmids present within a variety of Z. mobilis strains, including two poorly-studied Z. mobilis strains: NCIMB 11163 and NCIMB 8227. Several plasmid libraries containing restriction-digested fragments of Z. mobilis cryptic plasmid DNA were prepared, and their inserts were sequenced. This enabled the complete DNA sequences of three small (non-integrating, double-stranded DNA) cryptic plasmids to be determined: pZMO1A and pZMO7 from NCIMB 11163, and pZMO1B from NCIMB 8227. Their DNA sequences were analyzed using bioinformatic approaches, to identify open reading frames, and regions of DNA that were putatively involved in transcription or DNA replication. In the second part of this thesis, the minimally-replicating region from plasmid pZMO7 was used to construct a series of Escherichia coli-Z. mobilis shuttle vectors. These vectors were found to be stable within several Z. mobilis strains for over 60 generations without antibiotic selective pressure. A reliable and reproducible method based on quantitative real time PCR (Q-RT-PCR) was developed to accurately determine the copy number of cryptic plasmids and shuttle-vectors present in Z. mobilis cultures. The pZMO7-based shuttle vectors exhibited good compatibility with cryptic plasmids as well as the widely-used pZM2-based shuttle vectors. Genes encoding glutathione S-transferase (GST) as well as green and red fluorescent protein (GFP and RFP) reporters were cloned into various shuttle vector constructs; placing them under the control of endogenous (Ppdc) or exogenous (Plac and Ptac) promoters. Promoter strength was evaluated by quantifying the reporter gene expression. The plasmid-based expression of GFP and RFP was visualized within planktonic and biofilm cultures using confocal laser scanning microscopy (CLSM). Shuttle vector-based GST pull-down experiments were used to study intracellular protein-protein binding interactions. In the third part of my thesis, I explored the potential use of Z. mobilis for the bioproduction of isoprenoid (terpenoid) compounds. Five predicted sesquiterpene synthases (terpene cyclases) of unknown function from the dimorphic fungus Penicillium marneffei, and several terpene cyclases from several other bacteria, fungi and plants were initially functionally-analyzed in E. coli. Several cyclase genes were cloned into E. coli-Z. mobilis shuttle vectors for expression trials within Z. mobilis cells. In summary, this thesis describes the development of a variety of novel methodologies and genetic ‘tools’ that may be used to express heterologous genes within Z. mobilis cells. These will be invaluable for future studies concerned with exploring the biology and industrial applications of this ‘microbial cell factory’. / published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

No description available.

Five strains (9L, 642A, 714, 5688T and 3384Y) of Histophilus ovis were investigated with respect to iron acquisition. All strains used ovine, bovine and goat, but not porcine or human, transferrins (Tfs) as iron sources for growth. In solid phase binding assays, total membranes from only two (9L and 642A) of the five strains, grown under iron-restricted conditions, were able to bind Tfs (ovine, bovine and goat, but not porcine or human). However, when the organisms were grown under iron-restricted conditions in the presence of bovine Tf, total membranes from all strains exhibited Tf binding (as above); competition experiments demonstrated that all three Tfs (ovine, bovine and goat) were bound by the same receptor(s). An affinity isolation procedure allowed the isolation of two putative Tf-binding polypeptides (78 and 66 kDa) from total membranes of strains 9L and 642A grown under iron-restricted conditions, and from membranes of all strains if the growth medium also contained Tf. A gene encoding a Pasteurella multocida TbpA homologue was shown to be present in each of two representative strains (9L and 3384Y); these genes were sequenced and determined to be the structural genes encoding the 78-kDa Tf-binding polypeptides. The identification of a fur homologue and a Fur box within the promoter region of tbpA in both strains indicated that Fur (and iron) is responsible for the iron-repressible nature of Tf-binding activity. Although tbpA transcripts were detected by reverse transcription (RT)-PCR with RNA isolated from strains 9L and 3384Y grown under iron-restricted conditions, with strain 3384Y, and depending on the primer pair, tbpA transcripts were detected by RT-PCR predominantly when the RNA was isolated from cells grown under conditions of iron-restriction in the presence of Tf. The presence of an additional G in the tbpA gene of strain 3384Y grown under iron-replete conditions, compared to organisms grown under iron-restricted conditions plus bovine Tf, is

No description available.