Culture independent studies have revealed a greater diversity of Archaea than the two kingdoms initially defined by Carl Woese. Culturing species from the newly discovered archaeal lineages, as with the majority of microbes, has been difficult. To overcome the culturing dilemma, metagenomics is being used to reconstruct environmental genomes. Two metagenomic studies are presented in this thesis, with the aim of recovering newly proposed archaeal genomes from the Nanoarchaeota and Korarchaeota. In the first study, a sediment sample, NZ13, was collected from a terrestrial New Zealand hot spring. Along with the sediment sample, two complex enrichments were sequenced on an Illumina MiSeq platform. Assembly and differential binning recovered two nearly complete genomes of a nanoarchaeote and a korarchaeote. The NZ13 nanoarchaeote is similar to other terrestrial nanoarchaeotes, which lack an ATP synthase and encode genes for glycolysis/gluconeogenesis and archaella. One notable difference is the NZ13 nanoarchaeote contains CRISPR genes, which are absent in other terrestrial nanoarchaeotes, although present in a marine nanoarchaeote, Nanoarchaeum equitans. The NZ13 korarchaeote mirrors Candidatus Korarchaeum cryptofilum, lacking genes for de novo synthesis of purines and several cofactors, while containing an abundance of peptide transporters and amino acid fermentation pathways. The second study focused on sulfide samples collected from deep-sea hydrothermal vent fields in southwestern Pacific Ocean along the Eastern Lau Spreading Center. Ten sulfide samples were sequenced on an Illumina HiSeq platform. Small subunit ribosomal RNA genes were extracted from the metagenome reads and aligned against the SILVA Ref NR 99 123 database. The preliminary results identified which samples could be prioritized for genome reconstruction of uncultured bacterial and archaeal lineages. Three uncultured bacteria, candidate division SR1, Gracilibacteria (GN02), and Parcubacteria (OD1) were identified in several samples. Many uncultured deep-sea hydrothermal archaeal lineages were identified in all samples. In particular, korarchaeotal sequences were in high relative archaeal abundances in two samples, ABE 1 and Vai Lili-2, while few nanoarchaeotal reads were classified.

Thesis (Ph.D.)--Montana State University--Bozeman, 2006. / Typescript. Chairperson, Graduate Committee: C. Martin Lawrence. Includes bibliographical references (leaves 146-159).

DNA is the central storage molecule for genetic information in the cell. Therefore, the DNA must be protected from damage that will otherwise be passed on to future generations as deleterious mutations. Although many different pathways have evolved for repairing different classes of damage there are certain features that are common to all repair pathways. Generically, for DNA damage to be repaired it must first be recognized, then excised and replaced with undamaged DNA. DNA damage recognition is highly varied since specific interactions are required between the protein and the damaged DNA. DNA damage repair, paradoxically, requires the action of highly processive nucleases. The nucleases may digest hundreds if not thousands of nucleotides, sometimes for the repair of a single mutant nucleotide. We have chosen to focus on Exonuclease VII (ExoVII), one of the processive nucleases that have been implicated in the process of Mismatch Repair (MMR). ExoVII is a hetero-pentameric enzyme composed of one large subunit (XseA) and four small subunits (XseB). It has been previously characterized as a processive, single-strand specific nuclease able to digest DNA in either the 5'->3' or 3'->5' direction by a metalindependent mechanism. Early studies have shown that although ExoVII is a hydrolytic nuclease it was completely active in the presence of large amounts of EDTA and was strongly stimulated by phosphate. This feature is unusual because hydrolytic DNA nucleases typically function by a mechanism that requires coordination of a divalent cation. To further our understanding of the mechanism ExoVII we have identified and characterized the ExoVII homolog from Thermotoga maritima (T. maritima, Tm), a hyperthermophilic bacterium. The genes responsible for Tm ExoVII (TM1768 and TM1769) were cloned into an overexpression construct and the resulting proteins were overexpressed, co-purified and characterized. Consistent with previous studies, we found that Tm ExoVII is a processive, single-strand specific nuclease. Surprisingly, unlike Ec ExoVII, the T. maritima homolog was found to have an absolute requirement for the divalent cation magnesium and was strongly inhibited by the presence of either phosphate or sulfate in the reaction buffer. Using multiple sequence alignments of the large subunit we have identified a conserved core present within the C-terminal ExoVII_Large domain. This conserved core, RGGGx27GHx2Dx4Dx9P, although unique among nucleases, is reminiscent of a metal-coordinating hydrolytic active site. We have tested this putative active site using site-directed mutagenesis to create the TmD235A/TmD240A double mutant. This mutant protein was purified and the resulting protein was found to be inactive. We propose that this conserved core represents the metal-coordinating active site of all ExoVII homologs and that the group of E. coli-like homologs are unique in their EDTA resistance and anion (phosphate and sulfate) stimulation. Since ExoVII is a bi-directional nuclease (both 5'->3' and 3'->5' activity), and MMR is a bi-directional process, our model was that ExoVII was the primary nuclease associated with MMR. To test this model and determine if, in fact, a minimal conserved MMR pathway can be defined, we performed an analysis of the genomic occurrence profiles for the genes involved in MMR. To do this we have developed a bioinformatic application, Magma, which assists in the creation of a searchable relational database. Using Magma we have found that MutH, the enzyme responsible for generating a nick that directs MMR to excise the newly synthesized DNA strand including a DNA mispair, is only present in E. coli and a subset of gamma-proteobacteria, suggesting that MutH is not a core component of MMR. Instead, most organisms employ a nicking activity found in the MutL subunit. We also show that, although four nucleases have been implicated as having "redundant" roles in bacterial mismatch repair, RecJ is the primary nuclease responsible for degrading the mutated DNA strand and that 5'->3' single-strand exonuclease activity is a core MMR component. From this analysis, it appears that prokaryotic mismatch repair is more similar to eukaryotic mismatch repair than was previously thought, from the genetic and biochemical work done in E. coli. We offer a model for a universal minimal MMR system.

El consum energètic representa un 30 % dels costos d'operació en sistemes intensius de tractament d'aigües residuals urbanes. En depuradores convencionals que utilitzin un sistema de fangs activats, entorn al 15-20 % de l'energia és consumida en la línia dels fangs, que inclou el bombeig, l'espessiment, l'estabilització i la deshidratació. Per tant, la optimització de la gestió dels fangs pot contribuir substancialment en la reducció dels costos de tractament d'aigües residuals. La digestió anaeròbia termofílica és més eficient que la mesofílica i pscicrofílica, en termes de producció de biogàs i metà, eliminació de sòlids volàtils (SV) i destrucció de patògens. El procés es pot accelerar mitjançant el pre¬tractament dels fangs, afavorint la seva solubilització i hidròlisi. L'objecte d'aquesta Tesi Doctoral fou estudiar l'impacte dels paràmetres del procés en la digestió anaeròbia termofílica dels fangs de depuradora urbana, avaluar l'efecte del pre-tractament tèrmic dels fangs a baixa temperatura, i valorar processos alternatius des del punt de vista energètic. Els resultats experimentals presentats s'obtingueren mitjançant l'operació de dos reactors de laboratori durant prop de dos anys. En aquest període es va estudiar l'efecte de la temperatura del procés, del temps de retenció dels fangs (TRF), de la velocitat de càrrega orgànica (VCO) i del pre-tractament a 70 ºC en la digestió anaeròbia dels fangs de depuradora. El procés fou avaluat en termes de la producció d'energia (biogàs i metà) i de la qualitat del fang digerit (contingut de SV i d'àcids grassos volàtils (AGV), facilitat de deshidratació i higienització). S'analitzà l'estabilitat del procés a mesura que es reduïa el TRF i s'incrementava la VCO, i es comparà l'eficiència en períodes d'estabilitat corresponents a les diferents condicions operacionals. Finalment, s'avaluaren els resultats des del punt de vista energètic, mitjançant el càlcul de balanços i ratis energètics teòrics, que es compararen amb els resultats obtinguts a partir de dades experimentals d'altres estudis. També s'utilitzà un model cinètic de primer ordre. Les conclusions que es desprenen d'aquest treball es resumeixen a continuació: Durant la digestió anaeròbia dels fangs, la transició d'un reactor mesophilic (43 ºC) a termofílic (50 ºC) es podria dur a terme sense alterar el procés, treballant a TRF elevats (≥ 30 dies) i VCO baixes (≤ 0.5 kg SV m-3reactor d-1). En aquestes condicions, les principals diferències entre reactors termofílics (50-55 ºC) i mesofílics (38-43 ºC) fan referència a una certa acumulació d'AGV (0.5-2.5 g L-1) i millora de la destrucció de patògens (E. coli ≤ 102 UFC mL-1). La digestió termofílica a 50 ºC i 55 ºC dóna lloc a resultats similars pel que fa a la producció de biogàs, estabilització, higienització i facilitat de deshidratació de l'efluent, si no varien els altres paràmetres operacionals. La producció de metà tendeix a incrementar proporcionalment a la VCO, és a dir al TRF i el contingut de SV als fangs alimentats. Així mateix, la qualitat de l'efluent (contingut de SV i AGV, facilitat de deshidratació dels fangs) també depèn de la VCO. D'acord amb els resultats obtinguts a 55 ºC, la producció de metà s'incrementà 2-3 vegades (de 0.2 a 0.4-0.6 m3CH4 m3reactor d-1) en disminuir el TRF de 30 a 15-10 dies, incrementant la VCO de 0.5 a 2.5-3.5 kg SV m3reactor d-1. En canvi, el procés es desestabilitzà amb la reducció del TRF a 6 dies i VCO per sobre de 5 kg SV m3reactor d-1. Les següents concentracions poden ser útils per detectar i prevenir la desestabilització d'un digestor termofílic de fangs: AGV totals (2.5 g L-1), acetat (0.5 g L-1), rati acetat/propionat (0.5), alcalinitat intermèdia (1.8 g CaCO3 L-1), rati alcalinitat intermèdia/alcalinitat parcial (0.9), rati alcalinitat intermèdia/alcalinitat total (0.5), contingut de metà al biogàs (55 %). El pre-tractament a 70 ºC afavoreix la solubilització dels fangs, incrementant la proporció de matèria orgànica soluble respecte la matèria orgànica total del 5 % al 50 % en 9-24 h; seguit d'una progressiva generació d'AGV després de 24h. Durant la subseqüent digestió anaeròbia de fangs pre¬tractats (9-48 h), s'incremetà la producció de biogàs en un 30-40 %, treballant a 55 ºC i 10 dies de TRF. El rendiment de producció de biogàs fou un 30 % superior amb fangs pre-tractats (0.28-0.30 vs. 0.22 L·gVS¬1) i el contingut de metà al biogàs també fou superior (69 % vs. 64 %). La digestió anaeròbia termofílica de fangs pot donar lloc a una producció neta d'energia, durant estacions fredes i càlides, si s'utilitzen reactors amb aïllament tèrmic de les parets i amb recuperació energètica a partir del biogàs i dels fangs digerits. En aquest cas, l'eficiència energètica de reactors termofílics treballant a la meitat de TRF (10-15 dies) que reactors mesofílics (20-30 dies) seria similar, per la qual cosa el cabal diari podria ser doblat, o el volum del reactor reduït, amb el conseqüent estalvi en el cost de tractament dels fangs. A més, un sistema en dues etapes (70/55 ºC) produiria més energia neta que un sistema en una sola etapa (55 ºC) amb un TRF de 10 dies. De totes maneres, la quantitat d'energia neta generada augmenta amb el volum del digestor donat que, malgrat la disminució en la producció de metà a TRF creixents, la producció d'energia segueix essent superior al consum, i per tant com més quantitat de fangs hi hagi al digestor, més energia es produirà. / Energy consumption accounts for some 30 % of the total operating costs of intensive sewage treatment systems. In conventional wastewater treatment plants employing an activated sludge process, around 15-20 % of this energy is used in the sludge treatment line, including sludge pumping, thickening, stabilisation and dewatering. Therefore, optimisation of sludge management can substantially contribute in the reduction of wastewater treatment costs. Thermophilic anaerobic digestion is more efficient than mesophilic anaerobic digestion, in terms of biogas production, volatile solids (VS) removal and pathogens destruction. The process might be further accelerated by sludge pre-treatment, promoting sludge solubilization and hydrolysis. The aim of this PhD Thesis was to study the impact of process parameters on the thermophilic anaerobic digestion of sewage sludge, to evaluate the effect of implementing a low temperature pre¬treatment step, and to assess alternative processes from an energy perspective. The experimental results presented were obtained by operating two lab-scale reactors for almost two years. During this period, the effect of process temperature, sludge retention time (SRT), organic loading rate (OLR) and 70 ºC sludge pre-treatment on the anaerobic digestion of sewage sludge was studied. The process was evaluated in terms of energy production (i.e. biogas and methane production) and the quality of the effluent sludge (i.e. VS and volatile fatty acids (VFA) content, sludge dewaterability and hygienisation). Focus was put on the stability of the process at decreasing SRT and increasing OLR. Process efficiency during stable performance under each operating condition assayed was compared. Finally, the results were assessed from an energy perspective, by means of theoretical energy balances and ratios; and compared to the results obtained with experimental data from other studies. A first order kinetic model was also used. The conclusions drawn from the different issues dealt in this work are summarised as follows: During anaerobic sludge digestion, the transition from a mesophilic (43 ºC) to a thermophilic operation (50 ºC) may be carried out without disturbing the process, by operating the reactors at high SRT ( ≥ 30 days) and low OLR (≤ 0.5 kg VS m-3reactor d-1). Under such conditions, some VFA accumulation (0.5-2.5 g L-1) and enhanced pathogen destruction (residual E. coli ≤ 102 CFU mL-1) would be the main differences of thermophilic (50-55 ºC) compared to mesophilic (38-43 ºC) reactors. Thermophilic sludge digestion at 50 ºC and 55 ºC should be similar in terms of biogas production and effluent stabilisation, hygienisation and dewaterability; provided that other process parameters are the same. Methane production rate tends to increase proportionally to the OLR, thus to the SRT and VS concentration in the feed sludge. Similarly, the quality of the effluent sludge (VS content, VFA content and sludge dewaterability) is also affected by the OLR. According to the results obtained at 55 ºC, methane production rate increased by 2-3 times (from 0.2 to 0.4-0.6 m3CH4 m3reactor d-1) by decreasing the SRT from 30 to 15-10 days; increasing the OLR from 0.5 to 2.5-3.5 kg VS m3reactor d-1. However, process unbalance resulted from SRT reduction to 6 days, with OLR above 5 kg VS m3reactor d-1. The following concentrations might be useful to detect and prevent digester failure during thermophilic sludge digestion: total VFA (2.5 g L-1), acetate (0.5 g L-1), acetate/propionate ratio (0.5), intermediate alkalinity (1.8 g CaCO3 L-1), intermediate alkalinity/partial alkalinity ratio (0.9), intermediate alkalinity/total alkalinity ratio (0.5), methane content in biogas (55 %). The 70 ºC sludge pre-treatment may initially promote sludge solubilization, increasing the concentration of soluble to total organic matter from 5 to 50 % within 9-24 h; which is followed by a progressive VFA generation after 24 h. Subsequent anaerobic digestion of pre-treated sludge samples (9¬48 h) could increase biogas production by 30-40 % working at 55 ºC with a SRT of 10 days. Biogas yield is some 30 % higher with pre-treated sludge (0.28-0.30 vs. 0.22 L·gVSfed-1) and methane content in biogas is also higher with pre-treated sludge (69 vs. 64 %). Thermophilic anaerobic sludge digestion would result in net energy production, during cold and warm seasons, provided that digesters with wall insulation and with energy recovery from both the biogas produced and the effluent sludge are used. In this case, the energetic efficiency would be similar for thermophilic digesters working at half the SRT (10-15 days) of mesophilic digesters (20-30 days), meaning that the sludge daily flow rate could be doubled, or the reactor volume reduced, with subsequent savings in terms of sludge treatment costs. Furthermore, two-stage systems (70/55 ºC) may result in higher net energy production compared to single-stage systems (55 ºC) at 10 days SRT. However, the amount of surplus energy generated increases with digester volume. In spite of the decrease in methane production rate at increasing SRT, energy production is still higher than energy consumption, and therefore the bigger the amount of sludge in the digester, the higher the energy production.

This study explored the use of a biosecure, static composting structure to inactivate MAP. In Experiment #1, it was concluded that composting is unlikely to achieve temperatures necessary to inactivate MAP associated with cattle mortalities and that M. smegamatis is an unlikely surrogate for MAP. This study also used the same system to explore changes in the microbial community in mortality compost after exposure to thermophilic temperatures. As high-throughput sequencing technologies advance, it is possible to characterize microbial communities in environments with a high degree of resolution. In Experiment #2, as members of Clostridia were present at temperatures > 55°C, it appears that anaerobic conditions existed within regions of the compost. Extreme temperatures and non-homogeneous high moisture conditions resulted in spatial distribution of temperature in a biosecure, static composting system, which failed to meet conditions necessary for complete composting and pathogen reduction.

This study explored the use of a biosecure, static composting structure to inactivate MAP. In Experiment #1, it was concluded that composting is unlikely to achieve temperatures necessary to inactivate MAP associated with cattle mortalities and that M. smegamatis is an unlikely surrogate for MAP. This study also used the same system to explore changes in the microbial community in mortality compost after exposure to thermophilic temperatures. As high-throughput sequencing technologies advance, it is possible to characterize microbial communities in environments with a high degree of resolution. In Experiment #2, as members of Clostridia were present at temperatures > 55°C, it appears that anaerobic conditions existed within regions of the compost. Extreme temperatures and non-homogeneous high moisture conditions resulted in spatial distribution of temperature in a biosecure, static composting system, which failed to meet conditions necessary for complete composting and pathogen reduction.

Thesis (M.S.)--University of Nevada, Reno, 2006. / "December, 2006." Includes bibliographical references (leaves 39-44). Online version available on the World Wide Web. Library also has microfilm. Ann Arbor, Mich. : ProQuest Information and Learning Company, [2006]. 1 microfilm reel ; 35 mm.

Thesis (Ph.D. Biological Sciences)--University of Waikato, 2007. / Title from PDF cover (viewed March 6, 2008) Includes bibliographical references (p. 111-134)

Thesis (Ph. D.)--Michigan State University. Program in Cell and Molecular Biology , 2006. / Title from PDF t.p. (viewed on June 19, 2009) Includes bibliographical references. Also issued in print.

Thesis (M.S.)--University of North Carolina Wilmington, 2008. / Appendixes (p. 93-117) Title from PDF title page (viewed September 22, 2008) Includes bibliographical references (p. 90-92)