Dissertations / Theses on the topic 'Thermophilic'

Molecular systematic methods were applied in a series of studies designed to resolve the taxonomic relationships of thermophilic actinomycetes known to be difficult to classify using standard taxonomic procedures. The test strains included representatives of clusters defined in an extensiven umerical phenetic survey of thermophilic streptomycetesa nd twelve marker strains. The resultant genotypic data together with the results of corresponding phenotypic studies were used to highlight novel taxa and to improve the circumscription of validly described species. The most comprehensive study was undertaken to clarify relationships within and between representative alkalitolerant, thermophilic and neutrophilic, thermophilic streptomycetes isolated from soil and appropriate marker strains. The resultant data, notably those from DNA: DNA relatedness studies, supported the taxonomic integrity of the validly described species Streptomyces thermodiastaticus, Streptomyces thermoviolaceus and Streptomyces thermovulgaris. However, the genotypic and phenotypic data clearly show that Streptomyces thermonitrificans Desai and Dhala 1967 and Streptomyces thermovulgaris (Henssen 1957) Goodfellow et al. 1987 represent a single species. On the basis of the priority, Streptomyces thermonitrificans is a later subjective synonym of Streptomyces thermovulgaris. Similarly, eight out of eleven representative alkalitolerant, thermophilic isolates and three out of sixteen representative neutrophilic, thermophilic isolates had a combination of properties consistent with their classification as Streptomyces thermovulgaris. One of the remaining alkalitolerant, thermophilic isolate, Streptomyces strain TA56, merited species status. The name Streptomyces thermoalcalitolerans sp. nov. is proposed for this strain. A neutrophilic, thermophilic isolate, Streptomyces strain NAR85, was identified as Streptomyces thermodiastaticus. Four other neutrophilic thermophilic isolates assigned to a numerical phenetic cluster and a thermophilic isolates from poultry faeces were also considered to warrant species status; the names Streptomyces eurythermophilus sp. nov. and Streptomyces thermocoprophilus sp. nov. are proposed to accommodate these strains. It was also concluded that additional comparative taxonomic studies are required to clarify the relationships between additional thermophilic streptomycete strains included in the present investigation. A corresponding polyphasic approach was used to clarify the taxonomy of six thermophilic isolates provisionally assigned to either the genera Amycolatopsis or Excellospora. Two of the isolates, strain NT202 and NT303, had properties consistent with their classification in the genus Amycolatopsis. However, the genotypic and phenotypic data also showed that these strains formed a new centre of taxonomic variation for which the name Amycolatopsis eurythermus sp. nov. is proposed. Similarly, the four remaining strains formed two new centre of taxonomic variation within the genus Excellospora. It is proposed that isolates TA113 and TA114 be designated Excellospora alcalithermophilus sp. nov. Similarly, the name Excellospora thermoalcalitolerans sp. nov. is proposed for strains TA86 and TA111. An emended description is also given for the genus Excellospora.

La température est un paramètre crucial dans le fonctionnement du monde vivant, notamment de la machinerie moléculaire (les protéines) dont la stabilité et l’activité en dépendent sensiblement. Celles-ci sont souvent considérées comme étant équivalentes : si une protéine fonctionne, c’est qu’elle est stable, et vice-versa. Cependant, les protéines des organismes thermophiles, qui prolifèrent dans de températures élevées, sont stables à température ambiante, mais y présentent une faible activité. Cette dernière est optimale à la température de croissance de l’organisme hôte. Lorsqu’on parle de stabilité et d’activité protéique, la rigidité mécanique est souvent utilisée comme paramètre pertinent, offrant une explication simple et attractive à la fois pour la stabilité thermodynamique à haute température et au manque d’activité à des températures plus modérés. La réalité s’avère souvent plus complexe, et les mécanismes moléculaires reliant rigidité/flexibilité avec la stabilité et l’activité sont encore mal compris. Dans ce travail, nous abordons le problème au travers de trois systèmes. Nous avons examiné l’activation thermique des modes fonctionnels du domaine G de la protéine EF ainsi que les homologues mésophiles et thermophiles de la déshydrogénase Lactate/Malate. Par ailleurs, nous avons mis en évidence l’existence d’un paramètre unique (la moyenne des fluctuations atomiques) permettant d’expliquer la dynamique de la protéine lysozyme près de son point de fusion, et ce quelle que soit la nature de l’environnement autour de la protéine (qui décale le point de fusion). Nos conclusions se basent principalement sur une approche in silico où la dynamique moléculaire et des techniques d’échantillonnage améliorées sont utilisées et sont complémentées par des expériences de diffraction de neutrons
Temperature is one of the major factors governing life as demonstrated by the fine tuning of stability and activity of the molecular machinery, proteins in particular. The structural stability and activity of proteins have been often presented as equivalent. However, the thermophilic proteins are stable at ambient condition, but lack activity, the latter recovered only when the temperature increases to match that of the optimal growth condition for the hosting organism. In discussing the protein stability and activity, mechanical rigidity is often used as a relevant parameter, offering a simple and appealing explanation of both the extreme thermodynamic stability and the lack of activity at low temperature. The reality, however, illustrates the complexity of the rigidity/flexibility trade off in ensuring stability and activity through intricate thermodynamic and molecular mechanisms. Here we investigate the problem by studying three study cases. These are used to relate the thermal effects on mechanical properties and the stability and activity of the proteins. For instance, we have probed the thermal activation of functional modes in EF G-domain and Lactate/Malate dehydrogenase mesophilic and thermophilic homologues and verified a “universal” scaling of atomistic fluctuation of the Lysozyme approaching the melting in different environmental conditions. Our conclusions largely rest on an in silico approach, where molecular dynamics and enhanced sampling techniques are utilized, and are often complemented with neutron scattering experiments

La fermentation sombre et les piles à combustible microbiennes (MFC) sont deux technologies émergentes respectivement pour la conversion biologique de l'énergie chimique des composés organiques en hydrogène (H2) et en électricité. En raison des avantages cinétiques et thermodynamiques, la température élevée peut être la clé pour augmenter à la fois la production d'H2 de fermentation sombre et la production d'électricité dans les MFC. Par conséquent, cette thèse se concentre sur la manière dont la température influence la production biologique de H2 et d'électricité à partir d'eaux usées contenant du carbone organique. Deux inocula traités thermiquement (à boues activées fraîches et digérées) ont été comparés pour la production de H2 à partir de xylose à 37, 55 et 70 °C. A la fois à 37 et 55 °C, on obtient un meilleur rendement en H2 par les boues activées fraîches comparé aux boues digérées tandis qu'un très faible rendement en H2 est obtenu par les deux inocula à 70 °C. Ensuite, quatre prétraitements d'inoculum différents (chocs acides, alcalins, thermiques et de congélation) ont été évalués pour créer une efficace communauté productrice de H2 mésophile (37 °C) ou thermophile (55 °C). Les chocs acides et alcalins ont sélectionné des micro-organismes producteurs de H2, appartenant aux Clostridiaceae, au détriment des bactéries produisant du lactate, ce qui a donné respectivement le rendement en H2 le plus élevé à 37 et 55 °C. Bien que le choc thermique ait abouti à un faible rendement en H2 dans un seul lot, il a été montré que la production de H2 par les boues activées fraîches traitées thermiquement augmentait dans l'expérience avec quatre cycles consécutifs. Des boues activées fraîches et traitées thermiquement ont été sélectionnées comme inoculum pour la production continue de H2 à partir d'une eau usée synthétique contenant du xylose dans un réacteur à lit fluidisé (FBR) mésophile (37 °C) et thermophile (55-70 °C, augmenté par étapes). Un rendement en H2 plus élevé a été obtenu dans le FBR thermophile que dans le FBR mésophile. En outre, la production de H2 à 70 °C, qui a échoué dans l'étude précédente, a été couronnée de succès dans le FBR, avec un rendement stable de 1.2 mol H2 mol-1 xylose. La température de fonctionnement de 70 °C s'est également révélée optimale pour la production de H2 à partir d'eaux usées thermomécaniques (TMP) dans un incubateur à gradient de température, car la culture en batch à 70 ° C. Une approche de l'ARN a été utilisée pour étudier la structure et le rôle des communautés microbiennes attachées à l'anode, attachées à la membrane et planctoniques dans un MFC mésophile (37 °C) et thermophile (55 °C) alimenté au xylose. Une communauté anodine dominée par Geobacteraceae a soutenu la production d'électricité à 37 °C, alors que l'établissement de micro-organismes méthanogènes et H2 oxydants a entraîné une faible production d'électricité à 55 °C. Cependant, le développement d'une communauté exoélectrogène thermophile peut être favorisé en appliquant une stratégie de démarrage qui comprend l'imposition d'un potentiel négatif à l'anode et l'inhibition chimique des méthanogènes. Une communauté exoélectrogénique mésophile a également été montré pour produire de l'électricité à partir d'eaux usées de TMP dans un MFC à flux ascendant exploité à 37 °C. En conclusion, une production de H2 plus élevé et plus stable peut être obtenu dans une fermentation sombre thermophile plutôt que mésophile. La fermentation sombre à 70 °C est particulièrement appropriée pour le traitement des eaux usées de TMP car elle est libérée à haute température (50-80 °C) et pourrait être traitée sur site. Les eaux usées de TMP peuvent également être utilisées comme substrat pour la production d'électricité dans les MFC mésophiles. La production d'électricité dans les MFC thermophiles est faisable, mais l'enrichissement des micro-organismes exoélectrogènes thermophiles peut nécessiter une longue période de démarrage
Dark fermentation and microbial fuel cells (MFCs) are two emerging technologies for biological conversion of the chemical energy of organic compounds into hydrogen (H2) and electricity, respectively. Due to kinetic and thermodynamic advantages, high temperature can be the key for increasing both dark fermentative H2 production and electricity production in MFCs. Therefore, this thesis focuses on delineating how temperature influences biological production of H2 and electricity from organic carbon-containing wastewaters. Two heat-treated inocula (fresh and digested activated sludge) were compared, for H2 production from xylose at 37, 55 and 70 °C. At both 37 and 55 °C, a higher H2 yield was achieved by the fresh than digested activated sludge, whereas a very low H2 yield was obtained by both inocula at 70 °C. Then, four different inoculum pretreatments (acidic, alkaline, heat and freezing shocks) were evaluated for creating an efficient mesophilic (37 °C) or thermophilic (55 °C) H2 producing community. Acidic and alkaline shocks selected known H2 producing microorganisms belonging to Clostridiaceae at the expenses of lactate producing bacteria, resulting in the highest H2 yield at 37 and 55 °C, respectively. Although a heat shock resulted in a low H2 yield in a single batch, H2 production by the heat-treated fresh activated sludge was shown to increase in the experiment with four consecutive batch cycles.Heat-treated fresh activated sludge was selected as inoculum for continuous H2 production from a xylose-containing synthetic wastewater in a mesophilic (37 °C) and a thermophilic (55-70 °C, increased stepwise) fluidized bed reactor (FBR). A higher H2 yield was obtained in the thermophilic than in the mesophilic FBR. Furthermore, H2 production at 70 °C, which failed in the earlier batch study, was successful in the FBR, with a stable yield of 1.2 mol H2 mol-1 xyloseadded. Operation temperature of 70 °C was also found optimal for H2 production from thermomechanical pulping (TMP) wastewater in a temperature gradient incubator assay.A RNA approach was used to study the structure and role of the anode-attached, membrane-attached and planktonic microbial communities in a mesophilic (37 °C) and a thermophilic (55 °C) two-chamber, xylose-fed MFC. An anode attached community dominated by Geobacteraceae sustained electricity production at 37 °C, whereas the establishment of methanogenic and H2 oxidizing microorganisms resulted in a low electricity production at 55 °C. However, the development of a thermophilic exoelectrogenic community can be promoted by applying a start-up strategy which includes imposing a negative potential to the anode and chemical inhibition of methanogens. A mesophilic exoelectrogenic community was also shown to produce electricity from TMP wastewater in an upflow MFC operated at 37 °C. In conclusion, a higher and more stable H2 yield can be achieved in thermophilic rather than mesophilic dark fermentation. Dark fermentation at 70 °C is particularly suitable for treatment of TMP wastewater as it is released at high temperature (50-80 °C) and could be treated on site. TMP wastewater can be also used as substrate for electricity production in mesophilic MFCs. Electricity production in thermophilic MFCs is feasible, but enrichment of thermophilic exoelectrogenic microorganisms may require a long start-up period

A reverse transcription reaction allows the production of complementary DNA (cDNA) using an RNA template and relies on polymerases displaying reverse transcriptase (RT) activity. This process, with major applications in both research and in medical diagnostics, is often limited by the nature of the RTs available. RNA secondary structure can prove problematic where mesophilic retroviral RTs are used while the alternative approach, using thermophilic DNA polymerases with RT activity, often results in error-prone cDNA production.
This project recognised the need to study other possible sources of thermophilic RTs and outlines the study of four previously uncharacterised Group II Intronencoded proteins (IEP), with RT domains, from thermophilic bacteria. While cloning of the IEP genes and their expression on a small scale proved successful, difficulties were encountered when attempting purification. Despite a lack of overall purity, samples containing IEPs from Thermosinus carboxydivorans and Petrotoga mobilis were shown to have RT activity but characterisation of these IEPs was not carried out. However, an IEP from Bacillus caldovelox proved to be an excellent candidate for characterisation as successful purification was achieved. Enzyme engineering was also performed, fusing a Sac7d domain onto the C-terminus of this protein. These enzymes were shown to have optimum RT activity at 54ºC with activity still being displayed at 76ºC. Other studies on these enzymes showed that, unlike the retroviral RTs, the IEPs displayed no DNA-dependent DNA polymerase activity. The Sac7d fusion protein was also studied in terms of possible enhancements to the RT activity of an IEP. However, preliminary studies showed that, although this domain did not prove to be detrimental to the enzyme, it had little effect on improving the processivity of the RTs.
Although this class of RT looks promising in terms of use as an alternative thermophilic RT, the IEPs studied in this report did incur major limitations during cDNA synthesis, which included lower than expected optimum reaction temperatures, very low fidelity and an inability to synthesise cDNA using complex RNA templates.

There is a requirement in the European Union to divert organic wastes from landfill because of the risk of methane emissions. One alternative is anaerobic digestion of organic wastes, such as food waste, to stabilise them whilst at the same time recovering the energy from them. One problem with this approach is that the high nitrogen content of food waste may lead to ammonia inhibition. A solution to this has been found for mesophilic digestion but had not been attempted in thermophilic digestion where ammonia toxicity is known to be more acute. The work was carried out in laboratory-scale semi-continuous digesters operated over long time periods to provide maximum opportunity for acclimatisation, and in duplicate to give an indication of reproducibility. A series of experimental runs were undertaken at thermophilic temperatures to assess the influence of trace element (TE) addition on the digestion process. These were carried out at organic loading rates (OLR) of 2, 3 and 4 g volatile solids (VS) l-1 day-1 against unsupplemented controls at OLR 2 g VS l-1 day-1. Although TE addition could offset the accumulation of VFA which occurred in response to an increasing concentration of total ammonical nitrogen (TAN), it could not prevent this. The high alkalinity resulting from ammonia, however, allowed the digesters to continue to produce methane until VFA had accumulated to high concentrations before eventual failure due to a rapid drop in pH. To determine the threshold inhibitory ammonia concentration in thermophilic digestion, one pair of digesters was run on synthetic low nitrogen food waste (low-N food waste) at an OLR 2 g VS l-1 day-1 and compared to a control pair running on domestic food waste at the same loading. All four digesters received TE supplementation. The digesters fed with low-N food waste showed consistently stable performance with pH ~7.4, IA/PA ratio ~0.4-0.5, SMP 0.39 l CH4 g-1 VS, 52-55% CH4, total VFA <500 mg l-1 and 88% VS destruction whereas the controls showed signs of failure after 112 days and irreversible VFA accumulation at a TAN concentration >3.5 g N l-1. One of the low N digesters was supplemented with urea slowly and one by a shock dose: both showed signs of VFA accumulation at TAN >2.5 gl-1 and, again, an irreversible trend in propionic acid build-up when TAN >4 g N l-1. Long term operation showed meta-stable conditions when the digesters were operated at TAN between 2.5 - 3.5g l-1 with oscillations in VFA (especially propionic acid) concentration. Mesophilic digestion at 37oC with TE addition showed very stable performance with pH ~8, IA/PA ≤ 0.3, SMP ~0.48 l CH4 g-1 VS, 55-60% CH4, total VFA < 300 mg l-1 and VS destruction ~75-78% with a final total ammonia nitrogen (TAN) concentration of 4.5 g N l-1. As the temperature in digesters was raised from 35 to 43 oC in 1oC steps a change in performance was noted when the temperature reached 40 oC. Above this temperature VFA concentrations rose above 4000 mg l-1 and biogas and methane production fluctuated. It is probable that the higher temperature increased the concentration of free ammonia nitrogen (FAN) to ~800 g N l-1 at the measured TAN concentration ~5.5-6.0 g N l-1 and this was sufficient be inhibitory even with TE dosing. Fluorescent in situ hybridisation (FISH) was used to identify the methanogenic populations in some of the trials over selected time periods. This showed changes in population structure both in relation to temperature (mesophilic or thermophilic) and also in response to increasing concentrations of TAN. At high TAN concentrations Methanomicrobiales was the dominant methanogenic group and under mesophilic conditions this proved to be extremely ammonia tolerant. A 14C radio-labelling assay confirmed the dominant pathway to methane formation was by the hydrogenotrophic route which reflected the known metabolic pathway of this methanogen. It was concluded that thermophilic digestion of source segregated domestic food waste would lead to instability and failure of the process unless measures were introduced to reduce the TAN concentration to < 3.5 g N l-1, and preferably to < 2.5 g N l-1. Keywords: Anaerobic digestion, food waste, biogas, VFA accumulation, ammonia inhibition, Fluorescent in situ Hybridisation (FISH).

In this thesis, the anaerobic co-digestion of thickened waste activated sludge (TWAS) and, fat, oil and grease (FOG) was investigated as a method for TWAS:FOG treatment, stabilization, reduction and conversion to bio-methane gas as a valuable source of renewable energy. In the first phase, thermophilic and hyper-thermophilic anaerobic co-digestion of TWAS and FOG were investigated and compared. 20 – 80%FOG (based on total volatile solids) were tested using two sets of biochemical methane potential assays (BMP). Hyper-thermophilic co-digestion of TWAS with up to 60%FOG was shown to significantly increase the methane production and VS reduction as compared to the thermophilic co-digestion of the same TWAS:FOG mixture and as compared to the control (TWAS thermophilic mono-digestion). Both linear and non-linear regression models were used to represent the co-digestion results. In the second phase, the feasibility of the thermophilic and hyper-thermophilic co-digestion of TWAS and FOG were more investigated using lab scale semi-continuous reactors. The dual stage hyper-thermophilic reactor was introduced for the first time in this work for co-digesting TWAS and FOG. The dual stage co-digestion reactor was shown to significantly outperform the single-stage thermophilic mono-digestion reactor (the control) and the single-stage thermophilic co-digestion reactor at all three hydraulic retention times (HRTs) considered in the study namely, 15, 12 and 9 days. The dual-stage hyper-thermophilic co-digester digested up to 70%FOG at 15 days HRT without any stressing signs and produced a methane yield that was 148.2% higher compared to the control methane yield at the same HRT. It also produced a class A effluent at all three tested HRTs and positive net energy for 15 and 12 days HRT. The effects of microwave (MW) pretreatment, and combined alkaline-MW pretreatment on the co-digestion of TWAS:FOG mixtures with 20, 40 and 60% FOG were investigated in the third phase of this study. MW pretreatment at a high temperature of 175ᵒC was shown to be the most effective MW pretreatment option in solubilizing TWAS:FOG mixtures and in boosting the methane yield. It resulted in maximum solubilization for the 20%FOG samples and maximum methane yield for samples with 60%FOG. The combined alkaline-MW (NaOH-MW) pretreatment at a pH 10 showed to be an ineffective option for TWAS:FOG pretreatment before the anaerobic co-digestion process. In the fourth phase, the effects of the three selected pretreatments on the solubilization of TWAS and 20%FOG mixture on the molecular scale were investigated. The pretreatments used included: (i) MW pretreatment at 175ᵒC (since this was the best MW pretreatment condition according to the results of phase 3), (ii) hyper-thermophilic stage @ 70ᵒC and 2days HRT (effectively used in phases 1 and 2), and (iii) conventional heat at 70ᵒC. The analysis involved separation of the solubilized substrates after pretreatment using ultrafiltration (UF) at four different sizes (1, 10, 100 and 300 kDa). The results showed that each pretreatment method uniquely changed the particle size distribution. These changes showed to affect the biodegradability of substrates with different class size. Finally, two brief studies were performed using BMP tests to investigate the feasibility of FOG addition as a biogas booster in TWAS anaerobic digestion. First, the effect of FOG addition on TWAS and organic fraction of municipal solid waste (OFMSW) co-digestion was tested using hyper-thermophilic BMP tests. The addition of 30% FOG (based on total volatile solids) was shown very effective in improving the methane yield. The 30% FOG addition to TWAS:OFMSW mixture resulted in 59.9 and 84.4% higher methane yield compared to the methane yields of TWAS:OFMSW and TWAS samples, respectively. Second, the feasibility of using the soluble part of FOG (L-FOG) as a co-digestion substrate to increase the biogas production from the thermophilic digestion of TWAS was investigated. The results showed that co-digestion of TWAS and 20 to 80% (based on total VS) of L-FOG using a substrate to inoculum ratio (S/I) of 1 improved the biogas yield by 13.5 to 83.0%, respectively. No inhibition was reported at high L-FOG %.

Double-stranded DNA viruses including bacteriophage and herpesviruses package their genomes into empty capsids during viral replication, using an ATP-driven motor. The capsid expands to accommodate the full genome and maintain viable DNA densities. Structural and functional studies of the mechanism of DNA packaging and capsid expansion are required to understand the molecular basis of these processes, and how these steps are coordinated. To advance such research, this study focused on bacterial virus P23-45, which infects thermophilic bacterium Thermus thermophilus. It is shown that empty capsids can be packaged with DNA in vitro in the presence of the packaging ATPase. Both the unexpanded procapsid and expanded capsid were packaged when ATP was provided. Optimal packaging was observed at 50–65 ◦C, consistent with the system’s thermophilic origin. Expanded capsids were more active at packaging DNA than procapsids. Cryo-electron microscopy reconstructions determined at 3.7 Å and 4.4 Å resolution for the expanded capsid and procapsid respectively, revealed capsids had T=7 laevo quasi-symmetry. Capsids possessed a “supersized” capsid lattice spacing of ~17 nm, ~20 % larger than the 13–14 nm spacing observed in other bacteriophage and herpesviruses. Consequently, P23-45 capsids are larger and can accommodate twice the expected length of DNA for a capsid of this protein fold and symmetry. This is demonstrative of an additional evolutionary strategy available to viruses in modifying capsid size. The unique vertex of the icosahedral capsids containing the portal protein was characterised in both capsids, revealing a spacious interface between the 5-fold symmetrical vertex and the 12-fold portal protein. Parallel studies on the evolutionarily related encapsulin compartment from Myxococcus xanthus revealed a decameric oligomer of ferritin-like protein EncB is responsible for mineralisation of iron in the encapsulin core. These cores are likely similar in composition and structure to those of ferritin.

Thermal stability of theG37 tRNA methyltransferase proteins from Thermotoga maritima and Aquifex aeolicus have been compared using Differential Scanning Calorimetry. It was shown that the Thermotoga protein is remarkably stable and is denatured at temperatures in excess of 100 degrees Centigrade. The Aquifex aeolicus protein was less stable, denaturing broadly at temperatures between 55oC and 100oC. In contrast, the mesophilic E. coli protein was completely denatured at 55oC. Enzymatic activity of the proteins was measured at various temperatures. Both the Thermotoga and Aquifex enzymes are active at ambient temperatures, and display a significant decrease in activity when the temperature is raised above 50oC. This may relate to subtle changes in protein structure causing an effect on the tRNA based assay. Both enzymes contain inter subunit disulfide bonds which might contribute to thermal stability. Assays of the enzymes in the presence of high concentrations of Dithiothreitol (DTT) did not significantly reduce activity at higher temperatures, but did stimulate activity at lower temperatures. Site directed mutagenesis of non -conserved protein sequences within Thermotoga maritima were initiated in order to determine what structures might confer heat stability on the protein. Alanine mutagenesis of lysine residues 103,104 led to reduced catalytic activity, but did increased activity at higher temperatures. Aspartate is the most common residue at the relative position 166 in the variable loop of most TrmD genes. It has been shown that in E. coli this is essential for catalytic activity and possibly the residue which carries out N1 deprotonation on residue G37 in tRNA. In Thermotoga glutamate is present at this position. Alanine mutagenesis of this residue did not eliminate activity suggesting another nearby residue may function in this capacity in the Thermotoga TrmD protein.

The Nanoarchaeota were proposed as the fourth archaeal sub-division in 2002, and the only fully characterized nanoarchaeon was found to exist in a symbiotic association with the crenarchaeote, Ignicoccus sp. This nanoarchaeote, named Nanoarchaeum equitans could not be detected with &ldquo
universal&rdquo
archaeal 16S PCR primers and could only be amplified using specifically designed primers. In order to identify and access a wide diversity of archaeal phylotypes a new set of &ldquo
universal&rdquo
archaeal primers A571F (5&rsquo
-GCY TAA AGS RIC CGT AGC-3&rsquo
) and UA1204R (5&rsquo
-TTM GGG GCA TRC IKA CCT-3&rsquo
) was designed, that could amplify the 16S rRNA genes of all four archaeal sub-divisions. Using these primers community DNA was amplified from Chinese and New Zealand hydrothermal systems.

Alternative sources of fuel are required worldwide, and bio-ethanol is the leading candidate. Lignocellulosic biomass, a waste component of the agricultural industry, is a promising renewable source. Due to its complex structure it is highly recalcitrant, requiring the synergistic action of a battery of enzymes to achieve complete digestion. These enzymes include cellulases, hemicellulase and the accessory enzymes acetyl xylan esterase (AXE) and ferulic acid esterase (FAE). Thermpohilic Actinomyces isolates with the ability to hydrolyze xylan were screened for esterase activity. Two isolates (ORS10 and GSIV1), identified as Streptomyces spp, were positive for AXE activity. A cosmid library representative of isolate ORS10 was composed and screened for AXE activity using -naphthyl acetate as substrate. An 18 kb cosmid clone, 18D7, tested positive for AXE activity. Intracellular fractions extracted from ORS10 were precipitated with ammonium sulphate and partially purified 161-fold. Specific activity was measured after dialysis and ion-exchange chromatography. Overall yield of the partially purified enzyme was 34 %. Two protein bands of molecular masses 40 kDa and 60 kDa have been subjected to trypsin digestion and MALDI-TOF mass spectrometry analysis. The partially purified AXE displayed optimum activity at pH 9 and at 50°
C. AXE activity was stable for at least 1.5 hours between 30°
C and 40°
C, and for 24 hours between pH 6-9. The kM and Vmax values were 16.93 mg/ml and 1645 units/mg enzyme, respectively. The stability of the partially purified AXE at 30°
C-40°
C suggests potential for industrial applications that utilise mesophilic fermentations.

To ensure the sustainability of bioethanol production, major attention has been directed to develop feedstocks which provide an alternative to food-crop biomass. Lignocellulosic (LC) biomass, which is chiefly composed of industrial plant residues, is a carbon-rich reservoir that is presently attracting much attention. However LC material is highly recalcitrant to bioprocessing and requires a mixture of physical and enzymatic pretreatment in order to liberate fermentable sugars. Thermostable enzymes are extremely desirable for use in thermophilic fermentations due to their inherent stability. Hemicellulose, a core constituent of LC, requires a cascade of hemicellulases to stimulate the depolymerisation of its xylan backbone. &alpha
-L-arabinofuranosidase (AFase) increases the rate of lignocellulose biodegradation by cleaving arabinofuranosyl residues from xylan thereby increasing the accessibility of other hemicellulases. Twenty thermophilic Actinomycete isolates were screened for AFase activity using pnp-arabinofuranoside as the substrate. Three strains (ORS #1, NDS #4 and WBDS #9) displayed significant AFase activity and were identified as Streptomyces species with 16S rRNA gene sequence analysis. Genomic DNA was isolated from these strains and a cosmid library constructed in the shuttle vector pDF666. Subsequent functional and PCR-based screening revealed no positive clones.

Microbial biogeography explores the spatial and temporal distribution of microorganisms at multiple scales and is influenced by environmental selection and passive dispersal. Understanding the relative contribution of these factors can be challenging as their effects can be difficult to differentiate. Dormant thermophilic endospores in cold sediments offer a natural model for studies focusing on passive dispersal. Understanding distributions of these endospores is not confounded by the influence of environmental selection; rather their occurrence is due exclusively to passive transport. Sediment heating experiments were designed to investigate the dispersal histories of various thermophilic spore-forming Firmicutes in the River Tyne, a tidal estuary in North East England linking inland tributaries with the North Sea. Microcosm incubations at 50-80°C were monitored for sulfate reduction and enriched bacterial populations were characterised using denaturing gradient gel electrophoresis, functional gene clone libraries and high-throughput sequencing. The distribution of thermophilic endospores among different locations along the estuary was spatially variable, indicating that dispersal vectors originating in both warm terrestrial and marine habitats contribute to microbial diversity in estuarine and marine environments. In addition to their persistence in cold sediments, some endospores displayed a remarkable heat-resistance surviving multiple rounds of autoclaving. These extremely heat-resistant endospores are genetically similar to those detected in deep subsurface environments, including geothermal groundwater investigated from a nearby terrestrial borehole drilled to >1800 m depth with bottom temperatures in excess of 70°C. The ability of these endospores to survive extreme temperatures whilst in a dormant state may enable them to withstand adverse conditions for long periods of time and then germinate in response to changing surroundings. This was investigated further in the context of seawater injection during secondary oil recovery, where cold seawater is injected into hot oil reservoirs, resulting in a cooler reservoir temperature near the injection well bore. Microcosm experiments designed to simulate this showed that cooling triggered the germination of endospores of sulfate-reducing Desulfotomaculum leading to the onset of souring in this model system. The results presented here, indicate that bacterial endospores are transported between terrestrial and marine, surface and subsurface environments. Their survival and distribution therefore has ii relevance to understanding deep biosphere processes, and factors shaping microbial diversity in the marine environment.

The aim of this study was to investigate the iron- and sulphur-oxidizing activities of thermophilic bacteria with reference to the possible use of such bacteria in the extraction of metals from mineral sulphides. The initial characterization of a range of isolates was based on growth studies with iron and sulphur substrates and on the comparison of whole cell protein electrophoresis patterns. Three groups of bacteria were isolated and studied: moderately thermophilic iron- and mineral sulphide-oxidizing bacteria, moderately thermophilic sulphur oxidizers and extremely thermophilic Sulfolobus-like organisms. Both moderately and extremely thermophilic acidophiles were isolated from hot spring and coal pile samples. The moderately thermophilic iron-oxidizing bacteria and the extreme thermophiles which were examined were sub-divided into three and four sub-groups respectively. In a comparative study of continuous flow iron-oxidation reactors, moderate thermophiles did not produce higher rates of ferric iron production than the mesophile T. ferrooxidans but iron oxidation was less sensitive to inhibition by chloride in a vessel containing a thermophile than in a vessel operating with the mesophile. Iron oxidation during autotrophic growth of moderately thermophilic acidophiles and the rapid dissolution of mineral sulphides during the autotrophic growth of both the moderate and the extreme thermophiles were demonstated, thus considerably increasing the potential industrial significance of these bacteria. The yield of soluble copper from a chalcopyrite concentrate was shown to increase with temperature from relatively low yields with the mesophile T. ferrooxidans, through moderate yields with the moderately thermophilic bacteria to almost complete mineral solubilization with the newly isolated Sulfolobus strains.

The Old Yellow Enzyme (OYE) family of enzymes has been shown to reduce industrially important chemicals and has been used to study quantum tunnelling during enzymatic hydrogen transfer. Though extensively studied, only mesophilic homologues have been studied within the enzyme family. This thesis discusses the characterisation of Thermophilic Old Yellow Enzyme (TOYE), from Thermoanaerobacter pseudethanolicus, and provides the first published crystal structure of a thermophilic OYE-family member. In addition to increased thermostability compared to mesophilic homologues, thermophilic enzymes are important for use in industrial as often they are more stable towards organic solvents used in industry than their mesophilic homologues while catalysing the same reactions. This makes thermophiles and hyperthermophiles interesting targets for investigating the importance of enzyme dynamics during catalysis. They have also been used to study the linking of protein motion to quantum tunnelling during hydrogen transfer in other enzyme systems. In the work for this thesis, the basic characteristics of TOYE were examined. Thermal stability up to 70 °C was shown by CD and fluorescence studies and the preference towards reductive coenzyme was analysed by stopped-flow studies. Structural studies were conducted using X-ray crystallography, electron microscopy and sedimentation velocity studies. The crystal structure revealed a tetrameric enzyme with a relatively large active site. Evidence for higher oligomeric states was also obtained. The potential use of TOYE as a biocatalyst was explored by steady-state reaction, biotransformation and organic solvent resistance studies. The temperature dependences of kinetic isotope effects were used to examine the presence of tunnelling and importance active site geometry during catalysis and compared to previously described enzymes. These studies introduce a new and unique OYE-family member, allowing for more in-depth analysis of TOYE.

As part of a large study aimed at searching for patterns of diversity in the genus Persephonella along the north to south geochemical gradient of the ELSC, ten novel strains of Alphaproteobacteria were isolated unexpectedly. Using defined media under microaerophilic conditions to enrich for Persephonella from chimney samples collected at the seven vent fields on the ELSC and the dilution to extinction by serial dilution method to purify cultures, a total of ten strains belonging to the Alphaproteobacteria were isolated. Two of these isolates, designate MN-5 and TC-2 were chosen for further characterization and are proposed as two new species of a novel genus to be namedThermopetrobacter. Both strains are aerobic, capable of chemoautotrophic growth on hydrogen and grow best at 55°C, pH 6 and 3.0% NaCl. Strain MN-5 is capable of heterotrophic growth on pyruvate and malate and TC-2 is only able to grow heterotrophically with pyruvate. The GC content of MN-5 is 69.1 and TC-2 is 67 mol%. GenBank BLAST results from the 16S rRNA gene reveal the most closely related sequence to MN-5 is 90% similar and the most closely related sequence to strain TC-2 is 89% similar. Sampling at a shallow marine vent on the coast of Vulcano Island, Italy in 2007 led to the isolation of a novel species of Hydrogenothermus, a genus within the Hydrogenothermaceae family. This isolate, designated NV1, represents the secondHydrogenothermusisolated from a shallow marine vent. NV1 cells are rod-shaped, approximately 1.5μm long and 0.7μm wide, motile by means of a polar flagellum and grow singularly or in short chains. Cells grow chemoautotrophically using hydrogen or thiosulfate as electron donors and oxygen as the sole electron acceptor. Growth was observed between 45 and 75°C with an optimum of 65°C (doubling time 140 min), pH 4.0-6.5 and requires NaCl (0.5-6.0% w/v). The G+C content of total DNA is 32 mol%.

Dissertation (PhD)--University of Stellenbosch, 2003.
ENGLISH ABSTRACT: The moderately thermophilic (45 to 50DC), highly acidophilic (pH 1.5 to 2.5), chemolithoautotrophic Acidithiobaci/lus caldus strain "f' was isolated from a biooxidation process used to treat nickel ore concentrates. Trans-Alternating Field Electrophoresis (TAFE) analysis of total DNA from the At. caldus cells revealed two plasmids of approximately 14 and 45-kb. The 14-kb plasmid, designated pTC-F14, was cloned and shown by replacement of the cloning vector with a kanamycin resistance gene to be capable of autonomous replication in Escherichia coli. Autonomous replication was also demonstrated in Pseudomonas putida and Agrobacterium tumefaciens LBA 4404 which suggested that pTC-F14 was a broad host-range plasmid. Sequence analysis of the pTC-F14 replicon region revealed five open-reading frames, and a replicon organization like that of the broad host-range IncQ plasmids. Three of the open-reading frames encoded replication proteins with amino acid sequence identities similar to that of the IncQ-like plasmid pTF-FC2 (RepA, 81%; RepB, 78%; RepC, 74%). This high level of relatedness suggested that the two replicons had evolved from a common ancestor. Since closely related replicons are usually incompatible, the compatible replicons of pTC-F14 and pTFFC2 raised the question of how the replicons of the two sister plasmids had evolved such that they can now co-exist in the same host cell line. Further incompatibility testing with the IncQ-like plasmid pIEll08 and the IncQ prototype plasmid RSF10101R11621R300B determined that pTC-F14 was compatible with pIEI108, but incompatible with the IncQ prototype plasmid. It was found that the RepB and RepA replication proteins ofpTF-FC2 and pIEll08 were able to complement the pTC-FI4 orthologs only ifpTC-F14 RepC was present in trans. The RepC protein ofpTC-F14 was thus plasmid-template specific, while the RepA and RepB proteins were less plasmid-template specific. A five nucleotide possible iteron-discriminating region in the direct repeats of IncQ-like plasmid oriV regions has been identified (Tietze, E. (1998) Plasmid 39: 165-181). The iteron sequence ofpTC-F14 differs from pTF-FC2 and pIE 1108 by three nucleotides in this iteron-discriminating region. It was therefore proposed that co-evolution of the iterons and the RepC protein to a point where the RepC protein no longer recognizes the iteron sequence of a closely related sister plasmid is the mechanism by which replicons evolve to become compatible in the same host cell. The incompatibility determinant of the IncQ prototype plasmid RSFlOlOIR11621R300B was also sought, and subsequently localized to the region encoding the IncQ prototype plasmid's repAC genes. Interference with the initiation of pTC-F14 replication by the IncQ prototype plasmid was demonstrated by growth inhibition of a replication-deficient M13 bacteriophage into which oriVpTC-F14 had been cloned. Secondly, the IncQ prototype derivative pKE462 displaced a ColEloriVpTC- F14 construct in complementation assays, and a construct containing only the pTC-F14 repBAC genes similarly displaced the pKE462 plasmid. As the oriVRSFIOIO region was not incompatible with a pTC-F14 replicon, this suggested that it was not the oriV region which was expressing incompatibility, but the products of the IncQ prototype plasmid repAC genes. It is proposed that incompatibility between pTC-F14 and the IncQ prototype plasmid was the consequence of the repAC gene products binding to the iterons of the related rep licon, and that these products are unable to initiate replication. The compatible phenotypes expressed by members of the IncQ plasmid family indicates the inadequacy of using plasmid incompatibility as a classification system. Alignment of the amino acid sequences of the three replication protein orthologs clearly showed that the IncQ plasmid family was divided into two groups. To account for replication protein relatedness and the incompatibility phenotype expressed, it is now proposed that that members of the IncQ family be classified into subdivisions that reflect the different IncQ-like replicons identified in this study. Investigation of pTC-F14 replicon regulation identified a putative promoter sequence which is believed to regulate the initiation of a 5.l-5.7-kb polycistronic transcript that encodes all the replication proteins of the pTC-F14 replicon and the MobB and MobA proteins of the IncP-type mobilization module. The large polycistronic transcript appears to regulated by the RepB protein of the pTC-F14 replicon, and is not subject to cross-regulation by related IncQ plasmids. This suggested that the RepB primase function was not plasmid specific, but that its regulatory function was replicon specific. A second putative promoter sequence identified upstream of the pTC-F14 pasAB operon was, however, cross-regulated by the closely related pTF-FC2 plasmid. The pTC-F14 pas operon encodes two proteins with high amino acid sequence identity (PasA, 81 %; PasB, 72 %) to the plasmid addiction system ofpTF-FC2. This is the second time a plasmid addiction system of this type has been found on an IncQ-like plasmid.
AFRIKAANSE OPSOMMING: Die matig termofiliese (45 to 50°C), hoogs asidofiliese (pH 1.5 to 2.5), chemolitooutotrofiese Acidithiobaci/lus caldus ras "f' is geïsoleer vanaf 'n biooksiderende proses wat gebruik word om gekonsentreerde nikkel-erts te behandel. Trans- Afwisselende Veld Elektroforese (TAVE) analise van totale DNA vanaf die At. caldus selle, het twee plasmiede van ongeveer 14 en 45-kb. onthul. Die 14-kb plasmied, genaamd pTC-F14, is gekloneer en deur vervanging van die kloneringsvektor met 'n kanamisien weerstandsgeen is daar gewys dat hierdie plasmied in staat is tot outonome replikasie in Escherichia coli. Outonome replikasie is ook gedemonstreer in Pseudomonas putida en Agrobacterium tumefaciens LBA 4404 wat suggereer dat pTC-F14 'n wye gasheer-reeks plasmied is. Volgorde analise van die pTC-F14 replikon area het vyf oop leesrame onthul, en 'n replikon organisasie soortgelyk aan dié van die wye gasheer-reeks IncQ plasmiede. Drie van die oop leesrame kodeer vir replikasie proteïene met aminosuur volgordes ooreenstemmend met dié van die IncQ-tipe plasmied pTF-FC2 (RepA, 81%; RepB, 78%; RepC, 74%). Hierdie hoë vlak van verwantskap stel voor dat die twee replikons vanaf 'n gemeenskaplike voorouer ontwikkel het. Aangesien naby-verwante replikons gewoonlik onverenigbaar is, het die verenigbaarheid van die replikons van pTC-F14 en pTF-FC2 die vraag laat onstaan van hoe die replikons van twee susterplasmiede ontwikkel het, sodat hulle nou gelyktydig in dieselfde gasheer sellyn kan voortbestaan. Verdere onverenigbaarheid toetsing van die IncQ-tipe plasmied pIE1108 en die IncQ prototipe plasmied RSF10101R11621R300B, het bepaal dat pTCF14 verenigbaar is met pIE1108, maar onverenigbaar met die IncQ prototipe plasmied. Daar is gevind dat die RepB en RepA replikasie proteïene van pTF-FC2 en pIE1108 in staat was om die pTC-F14 ortoloë te komplementeer, slegs as pTC-F14 RepC in trans teenwoordig was. Die RepC proteïen van pTC-F14 is dus plasmiedtemplaat spesifiek, terwyl die RepA en RepB proteïene minder plasmied-templaat spesifiek is. 'n Moontlike iteron-onderskeidende vyf-nukleotied area in die direkte herhalings van die IncQ-tipe plasmied oril/ areas, is geïdentifiseer (Tietze, E. (1998) Plasmid 39: 165-181). Die iteron volgorde van pTC-F14 verskil van pTF-FC2 en pIEll08 met drie nukleotiedes in hierdie iteron-onderskeidende area. Om hierdie rede is daar voorgestel dat ko-evolusie van iterons en die RepC proteïen, tot by 'n punt waar die RepC proteïen nie meer die iteron volgorde van 'n naby-verwante susterplasmied herken nie, die meganisme is waardeur replikons ontwikkel om verenigbaar te word in dieselfde gasheersel. Die onverenigbaarheidsbepaler van die IneQ prototipe plasmied RSFIOIOIR11621R300B is ook ondersoek en gelokaliseer tot die area wat kodeer vir die IneQ prototipe plasmied se repAC gene. Inmenging met die inisiasie van pTC-F14 replikasie deur die IneQ prototipe plasmied is gedemonstreer deur groei vertraging van 'n replikasie-gebrekkige M13 bakteriofaag waarin die oriVpTC-F14 gekloneer is. Tweedens is die ColEl-oriVpTc-FI4 konstruk vervang deur die IneQ prototipe-afgeleide pKE462 in komplementasie proewe, en is die pKE462 plasmied op soortgelyke wyse vervang deur 'n konstruk wat slegs die pTC-F14 repBAC gene bevat. Aangesien die oriVRSF1010 area nie verenigbaar was met 'n pTC-F14 replikon nie, stel dit voor dat dit nie die oriV area is wat onverenigbaarheid uitdruk nie, maar die produkte van die IneQ prototipe plasmied se repAC gene. Dit is voorgestel dat onverenigbaarheid tussen pTC-F14 en die IneQ prototipe plasmied die gevolg is van die repAC geenprodukte wat bind aan die iterons van die verwante replikon en dat hierdie produkte nie in staat is om replikasie te inisieer nie. Die verenigbare fenotipes wat deur die lede van die IneQ plasmied familie uitgedruk word, dui aan op die ontoereikendheid van die gebruik van plasmied onverenigbaarheid as 'n klassifikasie sisteem. Vergelyking van die aminosuur volgordes van die drie replikasie proteïen ortoloë wys duidelik daarop dat die IneQ plasmied familie in twee groepe verdeel is. Om verantwoording te doen vir die replikasie proteïen verwantskap en die onverenigbare fenotipe wat uitgedruk is, word daar nou voorgestel dat die lede van die IneQ familie geklassifiseer word in subafdelings wat die verskillende IneQ-tipe replikons geïdentifiseer in hierdie studie, reflekteer. Ondersoek na die pTC-F14 replikon regulering het 'n moontlike promotor volgorde geïdentifiseer. Daar word gemeen dat hierdie promotor die inisiasie van 'n 5.l-5.7-kb polisistroniese transkrip reguleer, wat kodeer vir al die replikasie proteïene van die pTC-F14 replikon en die MobB en Mob A proteïene van die IneP-tipe mobilisasie module. Die groot polisistroniese transkrip blyk om gereguleer te word deur die RepB proteïen van die pTC-F14 replikon, en word nie gekruis-reguleer deur die IneQ plasmiede nie. Dit stel voor dat die RepB primase se funksie nie plasmiedspesifiek is nie, maar dat die reguleerbare funksie replikon-spesifiek is. 'n Tweede moontlike promotor volgorde wat stroom-op van die pTC-F14 pasAB operon geïdentifiseer is, is egter gekruis-reguleer deur die pTF-FC2 plasmied. Die pTC-F14 pas operon kodeer vir twee proteïene met hoë aminosuur volgorde verwantskappe (PasA, 81 %; PasB, 72 %) aan die plasmied-verslaafde sisteem van pTF-FC2. Dit is die tweede keer dat hierdie tipe plasmied-verslaafde sisteem in 'n IncQ-tipe plasmied gevind is.

The understanding of the phenomenon of thermophily requires investigations of both thermophilic prokaryotes and eukaryotes. In the eukaryotes, thermophily is exhibited only by a few species of fungi which can grow up to 60°C. A comparative study of homologous enzymes from thermophilic and mesophilic fungi and the analysis of the observed differences is a useful approach not only in discerning the mechanisms in thermophily but also in understanding the features of fungal growth and metabolism. Because of the availability of background information of invertase from some mesophilic sources and the convenience of assaying the enzyme, it was chosen for the projected study in a thermophilic fungus, Themomyces lanuginosus. The behaviour of invertase in the thermophilic fungus differed from invertases of mesophilic organisms in several respects, e.g., in the thermophilic fungus the enzyme was induced only in the presence of its substrate; it was intracellular and it was unstable both in mycelia and in cell-free extracts. The enzyme specific activity was maximum in 6 h-sucrose-grown mycelia following, which it progressively declined before maximal increase in biomass occurred and much of the inducer (sucrose) was still present in the growth medium. Further, invertase activity in cell-free extracts was unstable; it was completely inactivated during storage for 3 days at O°C. The enzyme activity was stabilised by the addition of thiol compounds, dithiothreitol (DTT) and glutathione (GSH) to cell-& extracts. In contrast, the addition of disulphides and thiol-modifying compounds rapidly inactivated the enzyme indicating the involvement of free sulphydryl group(s) in enzyme activity. The enzyme activity was reciprocally modulated by reduced (GSH) and oxidized (GSSG) glutathione, suggesting that invertase may be regulated by thiol/disulphide exchange reaction. Such a modulation of invert- activity has not been reported hr any other invertase. This observation suggested that the enzyme in the thermopbilic fungus is different from invert- that have been studied from mesophilic sources, notably from yeast and Neurospora. To obtain more information on this unusual behaviour of invertase of T. lanuginosus, an attempt was made to purify the enzyme and study its physico-chemical properties. Invertase was purified by ammonium sulphate fractionation of cellular proteins, ion-exchange and thiol-affinity chromatography followed by preparative electrophoresis. The final preparation of invertase after the electroelution step gave a single band on a native PAGE. However, the same preparation of invertase resolved into five bands of different molecular mass. The heterogeneity of the enzyme preparation on SDS-PAGE raised two possibilities with respect to the purity of the enzyme: (1) the final preparation contained multiple invertases of different molecular mass, or (2) the invertase preparation was associated with contaminating proteins. To distinguish between these two possibilities, proteins from induced (sucrose-grown) and non-induced (glucose-grown) mycelia were compared after identical steps of purification. The rationale of this experiment was that if heterogeneity of invertase is due to multiple forms of the same enzyme, they would most likely be absent in the non-induced mycelia. When the final preparations of proteins from both the mycelia were analysed on SDS-PAGE, it was observed that certain proteins were present in both the induced and the non-induced mycelia, suggesting that they might be the contaminating proteins present in the invertase purified by the above procedures. Some physico-chemical properties of invertase were studied. The purified enzyme was unstable during storage, losing activity completely in five days at O°C. Addition of DTT or glutathione did not prevent this loss of enzyme activity. This response of purified invertase preparations to DTT was quite opposite to that in cell-free extracts where invertase activity was stabilised by thiol compounds. To elucidate the reason for this difference in the behaviour of invertase in cell-free extracts and in pursed preparations, the approach taken was to first inactivate the enzyme in both1 type of preparations and then attempt to reactivate it. Dialysis of cell-free extracts had been found to cause an accelerated and complete inadivation oft he enzyme. The same treatment also inactivated freshly purified invertase, but to a lesser extent (60%). Whereas addition of DTT completely reatored the enzyme activity in the dialysed cell-bee extracts, it caused only a marginal revival of activity in dialysed invertase. This change in the response of purified invertase to DTT suggested that some cellular proteins were required br the reactivation of the enzyme by DTT that had been removed during the purification of invertase. A cellular protein was identified that reactivated inactive invertase in the presence of DTT. This protein was given the acronym "PRIA" for 'protein which restores i nvertase activity'. The mechanism of reactivation involved the conversion of the inactive invertase molecules into an active form. A model has been proposed to explain the requirement of UPRIA" for the reactivation of invertase. The salient features of this model are : (i) invert= requires free sulphydryl group(s) for activity, (ii) inactivation of invertase involves the formation of intramolecular disulphide bond(s) in the enzyme, (iii) the disulphide bond(s) is inaccessible to reduction by DTT, (iv) interaction of invertase and "PRIA” results in a conformational change in the enzyme that exposes the disulphide bond(s), rendering it susceptible to reduction by DTT and converting inactive invertase into active enzyme molecules. A surprising observation was the resistance of purified invertase to inactivation by the disulphides, GSSG, CoASSCoA and cystine. This was in marked contrast to their effective inhibition of invertase in the cell-& extracts. The experimental analysis of this unexpected resistance of purified invertase to disulphides revealed that following thioldnity chromatography on a Afegeldol column, invertase became resistant to disulphide inactivation. Moreover, the purified invertase was more stable during storage and to dialysis treatments in contrast to invertase activity in the cell-free extracts. These obsemtions suggested that invertase was altered- presumably it underwent a conformational change during the -el-501 chromatography step; possibly, the interaction of invertase with the gel matrix resulted in some cysteine residues in the enzyme becoming inaccessible to oxidation, thereby conferring resistance to inactivation by disulphides. The in vitro modulation of invertase activity by GSH and GSSG suggested the possibility that the enzyme may be regulated by a similar mechanism in the fungal mycelium. To substantiate this, GSH and GSSG levels in the mycelia were estimated. The GSH/GSSG ratio dememed in the mycelia between 6 and 18 h of growth and this was correlated with the decline in invertase activity. The fall in the GSHIGSSG ratio suggested that the intracellular environment waa becoming progressively oxidised during growth. Because NADPH participates in maintaining the cellular glutathione in a reduced state by the glutathione reduct- reaction, NADPH and NADPt levels were estimated. The NADPH/NADPt ratio declined by a factor of four between 6 and 36 h of growth and this decrease was positively correlated with the decrease in the flux of glucose through the pentose phosphate pathway. Incorporation of 'H-thymidine in mycelia indicated that with age of the culture, the number of growing hyphal tipslunit weight of mycelia declined. An attempt was made to integrate the changes in various biochemical parameters with the pattern of invertase development in T. lanuginosus when grown in a medium containing sucrose, i.e. invertase activity appeared rapidly as soon as perceptible growth occurred but it did not increase in parallel with the increase in biomass. Rather, the activity started to decline at approximately 6 h at which time growth was quantitatively mall. Since invertase activity in T. lanuginosus was induced by sucrose which is transported inside by a specific transporter, the development of invertase activity was linked to the uptake of sucrose by the fungal mycelia. It was considered likely that the sucrose transporter in T. lanuginosus, is localised at the hyphal tip where the entry of sucrose induces invertase. He enzyme is kept active in the hyphal tip because of a reductive environment due to a high GSHIGSSG ratio as a result of high NADPH levels. The latter serves to maintain GSH in a reduced state by the glutathione reductase reaction. In mature hyphae, lower generation of NADPH will result in lower GSHIGSSG ratio that will inactivate invertase by thiol oxidation. According to this model, the early burst of invertase activity in sucrose grown T. lanuginosus mycelia is due to the initiation of branch initials whereas the fall in enzyme activity is because of the decline in the proportion of hyphal tips per unit mass of mycelium as elongation growth and wall thickening occurs.

The understanding of the phenomenon of thermophily requires investigations of both thermophilic prokaryotes and eukaryotes. In the eukaryotes, thermophily is exhibited only by a few species of fungi which can grow up to 60°C. A comparative study of homologous enzymes from thermophilic and mesophilic fungi and the analysis of the observed differences is a useful approach not only in discerning the mechanisms in thermophily but also in understanding the features of fungal growth and metabolism. Because of the availability of background information of invertase from some mesophilic sources and the convenience of assaying the enzyme, it was chosen for the projected study in a thermophilic fungus, Themomyces lanuginosus. The behaviour of invertase in the thermophilic fungus differed from invertases of mesophilic organisms in several respects, e.g., in the thermophilic fungus the enzyme was induced only in the presence of its substrate; it was intracellular and it was unstable both in mycelia and in cell-free extracts. The enzyme specific activity was maximum in 6 h-sucrose-grown mycelia following, which it progressively declined before maximal increase in biomass occurred and much of the inducer (sucrose) was still present in the growth medium. Further, invertase activity in cell-free extracts was unstable; it was completely inactivated during storage for 3 days at O°C. The enzyme activity was stabilised by the addition of thiol compounds, dithiothreitol (DTT) and glutathione (GSH) to cell-& extracts. In contrast, the addition of disulphides and thiol-modifying compounds rapidly inactivated the enzyme indicating the involvement of free sulphydryl group(s) in enzyme activity. The enzyme activity was reciprocally modulated by reduced (GSH) and oxidized (GSSG) glutathione, suggesting that invertase may be regulated by thiol/disulphide exchange reaction. Such a modulation of invert- activity has not been reported hr any other invertase. This observation suggested that the enzyme in the thermopbilic fungus is different from invert- that have been studied from mesophilic sources, notably from yeast and Neurospora. To obtain more information on this unusual behaviour of invertase of T. lanuginosus, an attempt was made to purify the enzyme and study its physico-chemical properties. Invertase was purified by ammonium sulphate fractionation of cellular proteins, ion-exchange and thiol-affinity chromatography followed by preparative electrophoresis. The final preparation of invertase after the electroelution step gave a single band on a native PAGE. However, the same preparation of invertase resolved into five bands of different molecular mass. The heterogeneity of the enzyme preparation on SDS-PAGE raised two possibilities with respect to the purity of the enzyme: (1) the final preparation contained multiple invertases of different molecular mass, or (2) the invertase preparation was associated with contaminating proteins. To distinguish between these two possibilities, proteins from induced (sucrose-grown) and non-induced (glucose-grown) mycelia were compared after identical steps of purification. The rationale of this experiment was that if heterogeneity of invertase is due to multiple forms of the same enzyme, they would most likely be absent in the non-induced mycelia. When the final preparations of proteins from both the mycelia were analysed on SDS-PAGE, it was observed that certain proteins were present in both the induced and the non-induced mycelia, suggesting that they might be the contaminating proteins present in the invertase purified by the above procedures. Some physico-chemical properties of invertase were studied. The purified enzyme was unstable during storage, losing activity completely in five days at O°C. Addition of DTT or glutathione did not prevent this loss of enzyme activity. This response of purified invertase preparations to DTT was quite opposite to that in cell-free extracts where invertase activity was stabilised by thiol compounds. To elucidate the reason for this difference in the behaviour of invertase in cell-free extracts and in pursed preparations, the approach taken was to first inactivate the enzyme in both1 type of preparations and then attempt to reactivate it. Dialysis of cell-free extracts had been found to cause an accelerated and complete inadivation oft he enzyme. The same treatment also inactivated freshly purified invertase, but to a lesser extent (60%). Whereas addition of DTT completely reatored the enzyme activity in the dialysed cell-bee extracts, it caused only a marginal revival of activity in dialysed invertase. This change in the response of purified invertase to DTT suggested that some cellular proteins were required br the reactivation of the enzyme by DTT that had been removed during the purification of invertase. A cellular protein was identified that reactivated inactive invertase in the presence of DTT. This protein was given the acronym "PRIA" for 'protein which restores i nvertase activity'. The mechanism of reactivation involved the conversion of the inactive invertase molecules into an active form. A model has been proposed to explain the requirement of UPRIA" for the reactivation of invertase. The salient features of this model are : (i) invert= requires free sulphydryl group(s) for activity, (ii) inactivation of invertase involves the formation of intramolecular disulphide bond(s) in the enzyme, (iii) the disulphide bond(s) is inaccessible to reduction by DTT, (iv) interaction of invertase and "PRIA” results in a conformational change in the enzyme that exposes the disulphide bond(s), rendering it susceptible to reduction by DTT and converting inactive invertase into active enzyme molecules. A surprising observation was the resistance of purified invertase to inactivation by the disulphides, GSSG, CoASSCoA and cystine. This was in marked contrast to their effective inhibition of invertase in the cell-& extracts. The experimental analysis of this unexpected resistance of purified invertase to disulphides revealed that following thioldnity chromatography on a Afegeldol column, invertase became resistant to disulphide inactivation. Moreover, the purified invertase was more stable during storage and to dialysis treatments in contrast to invertase activity in the cell-free extracts. These obsemtions suggested that invertase was altered- presumably it underwent a conformational change during the -el-501 chromatography step; possibly, the interaction of invertase with the gel matrix resulted in some cysteine residues in the enzyme becoming inaccessible to oxidation, thereby conferring resistance to inactivation by disulphides. The in vitro modulation of invertase activity by GSH and GSSG suggested the possibility that the enzyme may be regulated by a similar mechanism in the fungal mycelium. To substantiate this, GSH and GSSG levels in the mycelia were estimated. The GSH/GSSG ratio dememed in the mycelia between 6 and 18 h of growth and this was correlated with the decline in invertase activity. The fall in the GSHIGSSG ratio suggested that the intracellular environment waa becoming progressively oxidised during growth. Because NADPH participates in maintaining the cellular glutathione in a reduced state by the glutathione reduct- reaction, NADPH and NADPt levels were estimated. The NADPH/NADPt ratio declined by a factor of four between 6 and 36 h of growth and this decrease was positively correlated with the decrease in the flux of glucose through the pentose phosphate pathway. Incorporation of 'H-thymidine in mycelia indicated that with age of the culture, the number of growing hyphal tipslunit weight of mycelia declined. An attempt was made to integrate the changes in various biochemical parameters with the pattern of invertase development in T. lanuginosus when grown in a medium containing sucrose, i.e. invertase activity appeared rapidly as soon as perceptible growth occurred but it did not increase in parallel with the increase in biomass. Rather, the activity started to decline at approximately 6 h at which time growth was quantitatively mall. Since invertase activity in T. lanuginosus was induced by sucrose which is transported inside by a specific transporter, the development of invertase activity was linked to the uptake of sucrose by the fungal mycelia. It was considered likely that the sucrose transporter in T. lanuginosus, is localised at the hyphal tip where the entry of sucrose induces invertase. He enzyme is kept active in the hyphal tip because of a reductive environment due to a high GSHIGSSG ratio as a result of high NADPH levels. The latter serves to maintain GSH in a reduced state by the glutathione reductase reaction. In mature hyphae, lower generation of NADPH will result in lower GSHIGSSG ratio that will inactivate invertase by thiol oxidation. According to this model, the early burst of invertase activity in sucrose grown T. lanuginosus mycelia is due to the initiation of branch initials whereas the fall in enzyme activity is because of the decline in the proportion of hyphal tips per unit mass of mycelium as elongation growth and wall thickening occurs.

This Ph. D. thesis presents results and conclusions from studies 1) investigating the interaction between metal and metalloid ions and thermophilic bacteria, and 2) characterizing microbial populations in a geothermally active habitat with relatively high concentrations of metalloid ions and compounds. In initial cadmium ion toxicity assays, the minimal inhibition concentration for 46 thermophilic bacteria of the genera Aneurinibacillus, Anoxybacillus, Bacillus, Brevibacillus, Geobacillus, and Thermus were determined. The highest tolerances to cadmium ions (Cd2+) in the range of 400 to 3200 micro;M were observed for species belonging to the genus Geobacillus. The thermophilic Gram-positive bacteria Geobacillus stearothermophilus and G. thermocatenulatus were selected to describe further biosorption reactions between cadmium ions and chemically reactive functional groups (potential ligands) within and onto the bacterial cell walls. Data obtained from electrophoretic mobility, potentiometric titration and cadmium ion adsorption experiments were used to quantify the number and concentrations of ligands and to determine the thermodynamic stability constants for the ligand-cation complexes. The first reported surface complexation models (SCMs) quantifying metal ion adsorption by thermophilic microorganisms predicted cadmium adsorption and desorption by both studied Geobacillus strains over a range of pH values and for different biomasses. The results indicated the functional group, with a deprotonation constant pK value of approximately 3.8, to be more dominant in cation biosorption accounting for 66 and 80% of all titrable groups for G. thermocatenulatus and G. stearothermophilus, respectively. The generated SCMs are different from model parameters obtained from mesophilic species that have been studied to date and might indicate a different biosorption behavior for both studied Geobacillus strains. Another objective of this thesis was to characterize microbial populations in the hot spring Champagne Pool, located in Waiotapu, New Zealand. The thermal spring is approximately 65 m in diameter and discharges water at 75eg; C and pH 5.5, which is oversaturated with arsenic and antimony compounds that precipitate and form orange deposits. Recovered nucleic acids and adenosine 5'-triphosphate (ATP) concentrations obtained for Champagne Pool water samples indicated low microbial density and were in good agreement with relatively low cell numbers of 5.6 plusmn; 0.5 x10^6 cells per ml. Denaturing gradient gel electrophoresis (DGGE) and 16S rRNA gene clone library analyses revealed the abundance of Sulfurihydrogenibium, Sulfolobus and Thermofilum-like populations in Champagne Pool. Two novel bacteria and one novel archaeon were successfully isolated with a distant phylogenetic relationship to Sulfurihydrogenibium, Thermoanaerobacter, and Thermococcus, respectively. Genotypic and metabolic characteristics differentiated isolate CP.B2 from described species of the genus Sulfurihydrogenibium. CP.B2 represents a novel genus within the Aquificales order, for which the name Venenivibrio stagnispumantis gen. nov., sp. nov. is proposed. V. stagnispumantis is a thermophilic, chemolithothrophic bacterium, that utilizes molecular hydrogen as electron donor and oxygen as electron acceptor and displayed growth in the presence of up to 8 mM NaAsO2 (As3+) and more than 20 mM Na2HAsO4.7H2O (As5+). However, growth was not observed when Na2HAsO4.7H2O and NaAsO2 were provided as the sole electron acceptor and donor pair. Arsenic resistance was conferred by the genes arsA and arsB

This study has divided the most frequently isolated types of moderate thermophiles into three groups: isolates of Sulfobacillus thermosulfidooxidans (mol% G + C 47-50), an isolate referred to as strain NAL and other closely related species (mol% G+C 54-57), and the type previously referred to as strain TH3 (mol% G+C 68). An enrichment culture was obtained that could efficiently solubilise a range of mineral sulphides at 48oC under air. Characterisation of this culture indicated the presence of two organisms essential for efficient growth under air: a typical S. thermosulfidooxidans group organism (isolate ICH), and a strain TH3 group organism (isolate ICP). Strain ICP appeared to possess an inducible, high affinity transport system for carbon dioxide during growth under air (unlike any previously studied moderate thermophiles), but extensive oxidation of ferrous iron was not achieved even at enhanced carbon dioxide levels. This lack of oxidation appeared to be the result of autotrophically-growth strain ICP having an apparent higher affinity for the end-product of iron oxidation, ferric iron (Ki 0.4 mM), than the substrate, ferrous iron (Km 0.5 mM). Only when a mixed culture of strain ICP and strain ICH was grown did extensive oxidation occur. A comparative mineral leaching study, with a mesophilic, a moderately thermophilic, and an extremely thermophilic culture indicated that the moderately thermophilic culture was the most robust during the dissolution of a range of minerals. This culture gave consistently better mineral dissolution rates than the mesophilic culture, clearly indicating their immediate commercial potential. In comparison the extremely thermophilic culture often produced faster rates of mineral dissolution than the moderately thermophilic culture, but appeared sensitive to agitation at high mineral pulp densities (10% (w/v)), limiting any present commercial applications of these organisms.

The 2-oxoacid dehydrogenase multienzyme complexes (OADHCs) catalyse the following reaction: 2-Oxoacid + NAD+ + CoASH → Acyl-SCoA + CO2 + NADH + H+ These 5-10 MDa complexes comprise multiple copies of three enzymes: E1, E2, and E3. The structural core of the OADHC is usually composed of either 24 (cubic) or 60 (dodecahedral) E2 polypeptides, around which the E1 and E3 enzymes associate non-covalently. Due to a lack of detectable activity in cell extracts, it was originally thought that Archaea did not possess any OADHCs; instead they utilised the smaller ferredoxin oxidoreductase (FOR) family of enzymes. However, the OADHC genes have since been identified in an increasing number of aerobic archaea, and given that the genomes of these organisms have been streamlined to remove any unnecessary genes, these two observations suggest that archaea may utilise both FORs and OADHC. The Thermoplasma acidophilum OADHC component enzymes have been recombinantly expressed and assembled, resulting in a thermostable and thermoactive branched-chain OADHC. This complex provides a model to investigate stability, not only of the individual enzymes but also of a multi-protein complex and its assembly. The structure of this hyper-thermostable E2 core is crucial to the thermostability of the entire complex. The Tp. acidophilum E2 structure has been solved by analytical ultra-centrifugation, X-ray crystallography and small-angle X-ray scattering; these data have shown that this E2 assembles into a novel 42-mer structure, comprising 14 trimers, with both square and pentagonal faces, rather than either of the expected cubic 24-mer or dodecahedral 60-mer structures. Analysis of the Tp. acidophilum E2 structure has identified an isoleucine ‘anchor’ residue that is key to the formation of the trimer-trimer interactions. The isoleucine side-chain extends from the C-terminal helix of one trimer into a hydrophobic pocket of the adjacent trimer and vice versa. The putative anchor residue has been removed by the insertion of a premature stop codon, resulting in E2 trimers that no longer assemble into a 42mer. This mutation has no affect on the tertiary structure of the trimers or its acyltransferase activity; moreover, the trimeric E2 retains the ability to bind the E1 and E3 enzymes, resulting in the first example of a functional trimeric OADHC.

It is widely known that fossil fuels are limited; consequently, the generation of new sources of energy in a clean and environmentally friendly manner is a research priority. Bioethanol appears to be one potential solution, especially second-generation production from renewable biomass. In order to use lignocellulosic feedstock to produce bioethanol, its polysaccharide components, cellulose and hemicellulose, must be hydrolysed into soluble sugars, which can then be converted into ethanol by fermentative microorganisms such as Geobacillus thermoglucosidasius TM242 used by the company ReBio Technologies Ltd. To date, the cost of commercial enzymes used during the hydrolysis process remains a major economic consideration in the production of second-generation bioethanol as an alternative fuel. The research project presented in this thesis aims to improve this rate-limiting step of microbial bioethanol production through an investigation of the different enzymes associated with hemicellulose hydrolysis. Firstly, the TM242 genome sequence revealed a number of genes encoding glycoside-hydrolases. Six of these genes were cloned and expressed in E. coli and the recombinant enzymes characterised; three of them, two β-xylosidases and an α arabinofuranosidase, are relevant to xylan hydrolysis, and were found to be highly active and thermostable. Crystallisation of one of the β-xylosidases permitted the determination of a high-resolution (1.7 Å) structure of the apo-enzyme along with a lower resolution (2.6 Å) structure of the enzyme-substrate complex, resulting in the first reported structure of a GH52 family member (Espina et al., 2014). Secondly, as the TM242 microorganism lacks xylanase enzymes, four genes encoding xylanases from closely-related Geobacillus strains were cloned and expressed in E. coli, with one of them being also successfully cloned and expressed in G. thermoglucosidasius TM242. This heterologous xylanase was secreted in active form representing an enhanced biomass utilisation by TM242. In conclusion, it is felt that the findings presented here have the potential to make a valuable contribution towards second-generation bioethanol production.

Kyoto University (京都大学)
0048
新制・課程博士
博士(農学)
甲第22485号
農博第2389号
新制||農||1075(附属図書館)
学位論文||R2||N5265(農学部図書室)
京都大学大学院農学研究科応用生物科学専攻
(主査)教授 吉田 天士, 教授 澤山 茂樹, 教授 菅原 達也
学位規則第4条第1項該当

Phenolic residues which accumulate in the environment as a result of agro-industrial practices has resulted in the need to find and use Eco-Friendly techniques, rather than the traditional methods of burning or burying this kind of waste. Bioremediation and bioconversion are attractive alternatives using whole cell or enzyme-based systems. The aims of this project were to isolate and uses thermophilic Actinomycetes, which produce thermo-tolerant oxidoreductase enzymes, which can be used to bioconvert a model industrial phenolic waste commonly genersated in the wine-making industry of South Africa. Current research in bioconversion and bioremediation focuses on mesophilic microbes in that their enzymes can catalyse reactions at higher temperatures without affecting its activity and lower contamination levels. Three novel Actinomycete isolates were isolated (RU-A0l , RU-A03 and RU-A06) from a compost site and characterized using a combination of conventional identification techniques and 16S rDNA methodology to identity the three isolates. All three isolates belong to the Streptomyces clade. In addition, five known Actinomycetes were selected from an internation culture collection and also screened for oxidoreductase activity in comparision to the three novel isolates. Although the five isolates were selected based on their ability to produce oxidoreductase enzymes, unexpectedly, no activity was detected. Screening assays for peroxidase, polyphenol oxidase and laccase on RU-AO 1, RU-A03 and RU-A06, showed that all three isolated produced peroxidases and peroxidases but no laccase. Substrate specificity studies revealed that the most suitable substrates to determine peroxidase and polyphenol oxidase activity on these isolates were catechol for polyphenol oxidase, 2,4-dichlorophenol for peroxidases and veratryl alcohol for lignin peroxidases. Previous studies have indicated that peroxidases and polyphenol oxidases are produced in Actinomycetes during the primary stage of growth. This was the case with RU-AOI , RU-A03 and RU-A06. Growth rates were higher that other Actinomycetes, with maxImum biomass being reached at 36 hours for the isolates RU-AOI and RU-A06 and 48 hours for isolate RUA03. pH studies showed that the three isolates were adaptable and could grow over a broad pH range. Catabolism studies of phenolic model compounds showed that the three isolates were capable of catabolizing the model phenolic compounds within a period of 24 hours. Further studies were carried out to determine the effect of these microbes and their enzymes in whole cell and enzyme-based systems on a model phenolic waste, graoe waste consisting of compressed grape skins, pips and stalks. Whole cell studies showed that the isolates were capable of bioconverting the waste at a maximum concentration of 30% grape waste (vol:vol). Peroxidase and polyphenol oxidase activity increased indicating induction of these enzymes in the presence of phenolic compounds, with a maximum increase of up to 15.9 fold increase in extracellular lignin peroxidase activity in RU-AO1. HPLC and phenolic determination assays indicated that bioconversion of the phenolic grape waste had occurred in the presence of the three isolates. Attempts were made to isolate and identify a peroxidase or phenol oxidase gene from one the isolates. As bacteria, Actinomycetes are amendable to gene manipulation making them suitable candidates for methods such as site directed evolution in comparison to fungi. Two clones were selected for sequencing based on positive activity results when assayed for peroxidase activity. However the resultant sequences did not identify a functional gene sequence. Southern Blotting was then carried out to determine the nature of the peroxidase gene. Previous studies have been focused on the catalase-peroxidase gene (CalC gene) found Actinomycetes and other bacteria. A probe was developed from the CalC gene. No hybridization occurred with any of the enzyme restricted DNA from the three isolates. The implications of these results are that the peroxidase genets in the three isolates are in fact lignin peroxidase in nature. This project has the potential in the bioconversion of phenolic wastes and is the first description of the use of thermophilic Actinomycetes in the bioconversion of an industrial phenolic waste.

The microbial oxidation of minerals is a commercially important process. Until comparatively recently only one organism capable of mineral oxidation has been extensively studied - Thiobacillus ferrooxidans. Several new, potentially commercially important isolates were studied in comparison with T.ferrooxidans. This was done with regard to their iron and sulphur oxidation systems, which are vital to the process of mineral solubilisation, and their ability to solubilise the minerals pyrite or chalcopyrite. The study of the latter was undertaken with particular reference to mixed culture leaching. The effect of growth substrate history on iron and sulphur oxidation varied between organisms. In particular, strain BC1 lost very little of its iron or sulphur oxidation capacity whether it was grown on either substrate. Conversely, the oxidation systems of T.ferrooxidans were directly influenced by growth substrate. SDS PAGE analysis and optical spectroscopy of T.ferrooxidans and strains BC1, LM2 and TH3 were used to investigate iron and sulphur oxidation systems and principally to indicate target components of these systems for further detailed study. Further study of the iron oxidation system in strain BC1 showed that there was only one major chromophore, a membrane bound cytochrome aa_. This had absorption peaks at 443 nm, 560 nm and 604.5 nm. Spectra run at 77°K indicated a shoulder on the Soret peak at approximately 450 nm. Extensive investigation into the effect on the cytochrome aa_ of various detergents culminated in the solubilisation from the membrane of the terminal oxidase in an intact form. This allowed the mid-point redox potential of this cytochrome to be determined as +524 mV. The mid-point redox potentials of T.ferrooxidans cytochrome £ and fi. were determined as +317 mV and +497 mV respectively. SDS PAGE analysis indicated two proteins that could be involved in growth on ferrous iron, at 27 kD and 31 kD. One or both of these proteins appeared to contain very high levels of iron. High levels of iron were also found in fractions from column chromatography which contained cytochrome aa_. Mineral oxidation studies showed that there was a slight increase in the rate of leaching of chalcopyrite in mixed cultures of strain BC1 and strain BC13 when compared to a pure culture of strain BC1, when the organisms where grown in shake flasks. In airlift reactor leaching of chalcopyrite the addition of strain BC13 to strain BC1 had several major effects. The pH in the mixed culture reactor was kept far more constant. Despite this, the leaching of copper from the mineral was not significantly altered in the mixed culture. However, a notable phenomenon in this mixed culture of strain BC1 and strain BC13 was the apparent reduction of the specific iron oxidation capacity of strain BC1 in comparison with its activity in pure culture.

The current research relates to a system driven by renewable energy and chemical energy contained in the feed, which will eliminate and reduce pathogens found in human excreta. A project in the form of an experiment for demonstration will be designed and built to operate in the local waste water treatment plant. Data will be analyzed and recorded, including fecal coliforms and E.coli levels, chemical oxygen demand (COD) and total solids removal (TS). The effectiveness of the system will depend upon results obtained and weather conditions. The principal objective of the research is to test and demonstrate that the "Small Decentralized ATAD" is successful in removing/eliminating enteric pathogens found in human excreta. Most importantly, the Bill and Melinda Gates Charity Foundation is providing financial support (Grants) for new sanitation ideas to help developing countries overcome diseases, specifically water borne diseases and also diseases related to hygiene and sanitation. Moreover, the "Water, Sanitation & Hygiene: Grand Challenges Explorations" granted a $100,000 Grant to Professor James Blackburn from Southern Illinois University at Carbondale in the Mechanical Engineering and Energy Processes Department. Consequently, the "Decentralized Next Generation for Diarrheal Pathogens" project will be tested using the ATAD (Autothermal Thermophilic Aerobic Digestion) to demonstrate its effectiveness in pathogen reduction and elimination.