The release of toxic metal species is of concern due to their detrimental effects on the environment and human health. Industrial effluents are a major source of mobilised metal species. Suitable technologies are needed to sequester toxic metal species at the point of source. Biosorption, which is based on the passive adsorption of contaminants onto biological materials, promises to offer an effective alternative or complementary step to existing treatment methods. However, to date there has been no widespread commercialisation of the technique. This is partly due to an insufficient understanding of the complex underlying mechanisms which makes it difficult to select suitable biomass for specific remediation problems and to predict process performance. This study characterised two gram-positive, thermophilic bacteria, Anoxybacillus flavithermus (BF) and Geobacillus stearothermophilus (BS), harvested at two different growth times, with regard to their acid-base and Cd�⁺ adsorption behaviour. The aim was to investigate the metal cation adsorption properties of thermophilic bacteria which has not been studied previously, and to gain a better understanding of the interactions responsible for bacterial metal cation adsorption. Experimental techniques employed in this study included microscopy to establish cell and cell wall morphology, batch acid-base and Cd�⁺ adsorption experiments to quantify proton active surface functional groups and Cd�⁺ adsorption, electrophoretic mobility measurements to assess the overall surface charge of the bacteria and in situ attenuated total reflection infrared (ATR-IR) spectroscopy to reveal the chemical identities of functional groups. Chemical equilibrium models based on batch acid-base titration and electrophoretic mobility data were developed to quantitatively describe proton active surface functional groups. These groups can also interact with metal cations. It was found that growth time was an important factor in all experiments with the differences between growth times often being more pronounced than the differences between the two bacterial strains. Microscopy revealed a gram-positive cell wall structure with different widths and staining behaviour for exponential phase cells of BF and BS. Stationary / death phase cells showed disintegrating cell walls. Acid-base titrations indicated that all cells possessed buffering capacity over the whole investigated pH range (pH 2 - 10). From electrophoretic mobility measurements, isoelectric points of ~ 3.2 for BF and < 1.8 and ~ 4.2 for exponential and stationary / death phase cells of BS respectively were estimated. Chemical equilibrium models including a Donnan electrostatic model were derived which described both the batch acid-base titration data and the electrophoretic mobility data reasonably well, although a comparison with IR data suggested room for further improvement. In situ ATR-IR spectroscopy of hydrated bacterial cells at various pH values revealed amide and carboxyl groups and a contribution from phosphate / polysaccharide moieties. Group specific interactions with Cd�⁺ were not detected, however, a partially reversible absorbance increase of all peaks suggested conformational changes in the presence of Cd�⁺. BF and BS adsorbed ~ 70 [mu]mol Cd�⁺ (g dry bacterial)⁻� at pH 5 in 0.01 M NaNO₃. Release of major cations occurred concomitantly with Cd�⁺ adsorption. The buffering and Cd�⁺-binding capacities of BF and BS were found to be comparable to those of mesophilic bacteria and ion exchange was identified as an important adsorption mechanism.

The periplasmic binding protein (PBP) superfamily is found throughout the genosphere of both prokaryotic and eukaryotic organisms. PBPs function as receptors in bacterial solute transport and chemotaxis systems; however the same fold is also used in transcriptional regulators, enzymes, and eukaryotic neurotransmitter receptors. This versatility has been exploited for structure-based computational protein design experiments where PBPs have been engineered to bind novel ligands and serve as biosensors for the detection of small-molecule ligands relevant to biomedical or defense-related interests. In order to further understand functional adaptation from a structural biology perspective, and to provide a set of robust starting points for engineering novel biosensors by structure-based design, I have characterized the ligand-binding properties and solved the structure of nine PBPs from various thermophilic bacteria. Analysis of these structures reveals a variety of mechanisms by which diverse function can be encoded in a common fold. It is observed that re-modeling of secondary structure elements (such as insertions, deletions, and loop movements), and re-decoration of amino acid side-chains are common diversification mechanisms in PBPs. Furthermore, the relationship between hinge-bending motion and ligand binding is critical to understanding the function of natural or engineered adaptations in PBPs. Three of these proteins were solved in both the presence and absence of ligand which allowed for the first time the observation and analysis of ligand-induced structural rearrangements in thermophilic PBPs. This work revealed that the magnitude and transduction of local and global ligand-induced motions are diverse throughout the PBP superfamily. Through the analysis of the open-to-closed transition, and the identification of natural structural adaptations in thermophilic members of the PBP superfamily, I reveal strategies which can be applied to computational protein design to significantly improve current strategies. / Dissertation

The aim of this research was the production of beta-galactosidase from thermophilic fungi for use in low lactose milk production or other possible applications. For this purpose, three thermophilic fungi Humicola insolens, Torula thermophila and Thermomyces lanuginosus were screened for lactase production. Highest lactase activity was observed in Thermomyces lanuginosus. The carbon source inducing highest extracellular lactase production in Thermomyces lanuginosus was determined as arabinose. When grown on arabinose T. lanuginosus produced two major lactase activity peaks, one being at day 4 (beta-galactosidase-A) and second starting following the initiation of biomass degradation at day 3 suggesting the existence of a cell wall-bound beta-galactosidase (beta-galactosidase-B). Maximum activity of the second enzyme was at day 10. Crude enzyme stored at 4&ordm / C and -20&ordm / C was stable over a period of one month. Optimum pH and temperature of crude enzyme were found as pH 6.8 and 65&ordm / C. For concentration of extracellular enzyme, fractional ammonium sulfate precipitation with 60-85% salt was applied. Comparisons with commercial lactase obtained from Kluyveromyces lactis revealed that partially purified lactase from Thermomyces lanuginosus was 1.3 times more efficient in hydrolysis of lactose even at 30&ordm / C which is optimum for Kluyveromyces lactis. Lactose hydrolysis was enhanced at higher temperatures and reached maximum at 50-60&ordm / C giving 4.7 fold higher hydrolysis than Kluyveromyces lactis beta-galactosidase. Molecular weight of the second enzyme was determined as 156 kDa by gel filtration. Being an extracellular enzyme with optimum pH suitable for dairy processes, high thermotolerance and stability, this enzyme has a potential for commercial use.

A thermodynamic study of five homologous HPr proteins derived from organisms inhabiting diverse environments has been undertaken. The aim of this study was to further our understanding of protein stabilization in extremes of environment. Two of the proteins were derived from moderate thermophiles (Streptococcus thermophilus and Bacillus staerothermophilus) and two from haloalkaliphilic organisms (Bacillus halodurans and Oceanobacillus iheyensis); these proteins were compared with HPr from the mesophile Bacillus subtilus. Genes for three of these homologous HPr proteins were for the first time cloned from their respective organisms into expression vectors and they were over-expressed and purified in Escherichia coli. Stability measurements were performed on these proteins under a variety of solution conditions (varying pH, salinity and temperature) by thermal and solvent induced denaturation experiments. Stability curves were determined for every homologue and these reveal very similar conformational stability for these homologues at their habitat temperatures. The BstHPr homologue is the most thermostable and also has the highest G25; the stability of other homologues was ranked as Bst>Bh>St>Bs>OiHPr. Other key thermodynamic parameters, like Cp, have been estimated for all the homologues and it was found that these values are identical within errors of estimation. Also, it was found that the values of TS are very similar for these homologues. Together these observations allow us to propose a thermodynamic mechanism toward achieving higher Tm. The crystal structures of the BstHPr and a single tryptophan-containing variant (BstF29W) of this homologue are also reported here. Also reported is a domain-swapped dimeric structure for the BstF29W variant, together with a detailed investigation into the solution oligomeric nature of this protein. The crystal structure of BstHPr is analyzed to enumerate various stabilizating interactions like hydrogen bonds and salt-bridges and these were compared with those for the mesophilic homologue BsHPr. Finally, an analysis of sequence alignments together with structural information for these homologues has allowed design of numerous variants of both Bs and BstHPr. A detailed thermodynamic study of these variants is presented in an attempt to understand the origins of the differences in stability of the HPr homologues.

No description available.

South Africa produces an average of 154 million broilers (Gallus gallus domesticus) annually, arising to an estimated 886 million kg of broiler litter. The largest population of broilers are reared in the North West province. Various applications for this largely underexploited resource have been published, including forming part of ruminant diets and direct land application. This however has several disadvantages, as it could lead to eutrophication of fresh water sources and faecal contamination of produce. Windrow composting of broiler litter has previously been studied, and found to deliver a stabilized product free of pathogenic and phytotoxic effects, therefore making it an excellent soil conditioner. This study aimed to characterize the microbial community present during the windrow composting of broiler litter. Four different formulations of substrate were tested; these being broiler litter (Windrow 1), Windrow 1 with previously composted material (Windrow 2), Windrow 2 amended with woodchips (Windrow 3) and Windrow 3 with an additional 12.5% (w/w) zeolite (Windrow 4). Broiler litter used in this experiment had a C:N ration of 10.3:1, whilst the blue gum woodchips added as an amendment had a C:N ratio of 172:1. Windrow and environmental temperatures were monitored on a regular basis. Windrow 1 largely mimicked environmental temperature, and could not sustain a true thermophilic phase during the experimental period. Windrow 2 did achieve a short lived thermophilic phase during the first few days of the composting process, however could not sustain its temperature over the whole period. In contrast Windrows 3 and 4 sustained temperature above 40°C for the largest part of the experimental period, regardless of environmental temperature. No significant difference (p < 0.05) could be observed between average moisture levels in the 4 windrows. Internal moisture profiles were however found to differ significantly, especially on the surface of the windrows. Moisture was also lost faster in Windrows 1 and 2 compared to Windrows 3 and 4. Chemical analysis showed differences between the four windrows constructed. A higher amount of nitrogen was lost in Windrows 1 and 2, mostly due to a sub-optimal initial C:N ratio in these windrows. Windrow 2 contained the highest values for plant nutrients P, Mg, Ca, Mn and Cu. Microbial population dynamics were observed using PCR-DGGE of samples collected throughout the composting of various treatments. Various commercial DNA extraction kits where tested in a previous study for their ability to remove PCR inhibitory substances, such as humic acids. The Machery-Nagel Soil DNA isolation kit was used as it gave amplifiable DNA from all samples. Samples were amplified using a nested PCR approach primer sets 27f-1492r \ 341f(GC)-907r and EF3-EF4 \ EF4(GC)-fung5 (where “GC” indicates a GC-rich clamp) for prokaryotic and eukaryotic species respectively. The PCR products were analyzed by agarose gel electrophoresis, and equal amounts of product were subjected to denaturing gradient gel electrophoresis (DGGE). Bands obtained from these polyacrylamide gels where then re-amplified using the same secondary primer sets (without the GC-clamp), and sequenced. A total of 454 prokaryotic bands in 55 distinct rf-positions were observed. Seven distinct rf-positions were observed in eukaryotic DGGE profiles. Prokaryotic profiles were aligned and the microbial diversity was analyzed by means of Ward’s clustering algorithm and the dice coefficient of similarity, as well as Simpson’s reciprocal, Shannon-Weaver and Species richness indices. Canonical correspondence analysis (CCA) was also performed on both the banding patterns as well as the bands present, together with the physico-chemical results obtained. Several bands were successfully identified as being influenced by physico-chemical parameters. Temperature, C:N ratio, ash, and moisture showed a correlation on CCA bi-plots. Sixteen bands were sequence identified. These sequences were compared to two different databases. The 16S rRNA database for Bacteria and Archaea gave identities to genus level, however maximum identity scores were low. Of the 16 sequences, 12 sequences were identified as uncultured bacteria when compared to the nucleotide collection database. In comparing the sequences with sequences collected in the nucleotide collection database, 12 were either first described in composts and soils, or animal manures. Results indicated mostly members of the genus Bacillus and Paenibacillus. The addition of a carbon source greatly affected the microbial metabolism, resulting in a thermophilic phase being achieved in amended windrows. As no thermophilic phase was observed in windrows that were not amended with woodchips, it could be concluded that the use of a carbon source is irremissible when composting broiler litter. A zeolite amendment is also strongly advised, as this further increased temperatures within the windrow. / Thesis (MSc (Environmental Sciences))--North-West University, Potchefstroom Campus, 2013.

South Africa produces an average of 154 million broilers (Gallus gallus domesticus) annually, arising to an estimated 886 million kg of broiler litter. The largest population of broilers are reared in the North West province. Various applications for this largely underexploited resource have been published, including forming part of ruminant diets and direct land application. This however has several disadvantages, as it could lead to eutrophication of fresh water sources and faecal contamination of produce. Windrow composting of broiler litter has previously been studied, and found to deliver a stabilized product free of pathogenic and phytotoxic effects, therefore making it an excellent soil conditioner. This study aimed to characterize the microbial community present during the windrow composting of broiler litter. Four different formulations of substrate were tested; these being broiler litter (Windrow 1), Windrow 1 with previously composted material (Windrow 2), Windrow 2 amended with woodchips (Windrow 3) and Windrow 3 with an additional 12.5% (w/w) zeolite (Windrow 4). Broiler litter used in this experiment had a C:N ration of 10.3:1, whilst the blue gum woodchips added as an amendment had a C:N ratio of 172:1. Windrow and environmental temperatures were monitored on a regular basis. Windrow 1 largely mimicked environmental temperature, and could not sustain a true thermophilic phase during the experimental period. Windrow 2 did achieve a short lived thermophilic phase during the first few days of the composting process, however could not sustain its temperature over the whole period. In contrast Windrows 3 and 4 sustained temperature above 40°C for the largest part of the experimental period, regardless of environmental temperature. No significant difference (p < 0.05) could be observed between average moisture levels in the 4 windrows. Internal moisture profiles were however found to differ significantly, especially on the surface of the windrows. Moisture was also lost faster in Windrows 1 and 2 compared to Windrows 3 and 4. Chemical analysis showed differences between the four windrows constructed. A higher amount of nitrogen was lost in Windrows 1 and 2, mostly due to a sub-optimal initial C:N ratio in these windrows. Windrow 2 contained the highest values for plant nutrients P, Mg, Ca, Mn and Cu. Microbial population dynamics were observed using PCR-DGGE of samples collected throughout the composting of various treatments. Various commercial DNA extraction kits where tested in a previous study for their ability to remove PCR inhibitory substances, such as humic acids. The Machery-Nagel Soil DNA isolation kit was used as it gave amplifiable DNA from all samples. Samples were amplified using a nested PCR approach primer sets 27f-1492r \ 341f(GC)-907r and EF3-EF4 \ EF4(GC)-fung5 (where “GC” indicates a GC-rich clamp) for prokaryotic and eukaryotic species respectively. The PCR products were analyzed by agarose gel electrophoresis, and equal amounts of product were subjected to denaturing gradient gel electrophoresis (DGGE). Bands obtained from these polyacrylamide gels where then re-amplified using the same secondary primer sets (without the GC-clamp), and sequenced. A total of 454 prokaryotic bands in 55 distinct rf-positions were observed. Seven distinct rf-positions were observed in eukaryotic DGGE profiles. Prokaryotic profiles were aligned and the microbial diversity was analyzed by means of Ward’s clustering algorithm and the dice coefficient of similarity, as well as Simpson’s reciprocal, Shannon-Weaver and Species richness indices. Canonical correspondence analysis (CCA) was also performed on both the banding patterns as well as the bands present, together with the physico-chemical results obtained. Several bands were successfully identified as being influenced by physico-chemical parameters. Temperature, C:N ratio, ash, and moisture showed a correlation on CCA bi-plots. Sixteen bands were sequence identified. These sequences were compared to two different databases. The 16S rRNA database for Bacteria and Archaea gave identities to genus level, however maximum identity scores were low. Of the 16 sequences, 12 sequences were identified as uncultured bacteria when compared to the nucleotide collection database. In comparing the sequences with sequences collected in the nucleotide collection database, 12 were either first described in composts and soils, or animal manures. Results indicated mostly members of the genus Bacillus and Paenibacillus. The addition of a carbon source greatly affected the microbial metabolism, resulting in a thermophilic phase being achieved in amended windrows. As no thermophilic phase was observed in windrows that were not amended with woodchips, it could be concluded that the use of a carbon source is irremissible when composting broiler litter. A zeolite amendment is also strongly advised, as this further increased temperatures within the windrow. / Thesis (MSc (Environmental Sciences))--North-West University, Potchefstroom Campus, 2013.

Polyphenol oxidases are enzymes that catalyze the oxidation of certain phenolic substrates to quinones in the presence of molecular oxygen. Polyphenol oxidases are widely used in several applications. In food industry, they are used for enhancement of flavor in coffee, tea and cocoa production, and determination of food quality. In medicine, they have several uses in treatments of Parkinson&rsquo / s disease, phenlyketonurea and leukemia. In wastewater treatment, they are used for the removal of phenolic pollutants from wastewaters. In pharmaceutical industry, differentiation of morphine from codeine is possible by means of polyphenol oxidase immobilized electrodes. In this study, a thermophilic fungus, Thermomyces lanuginosus was evaluated in terms of poyphenol oxidase production. The effect of different nutrient sources, inducers and fermentation parameters on enzyme production were investigated and maximum PPO activity of 97 U/ml was observed in bioreactor experiments at 50&deg / C, 400 rpm and pH 8.0 in a fermentation medium containing 1.4% yeast extract, 0.3% MgSO4, 1% KH2PO4, 0.003% CuSO4, 0.032% gallic acid. Type of polyphenol oxidase produced by Thermomyces lanuginosus was determined as laccase. For biochemical characterization studies, the enzyme was enriched by electrophoresis. Temperature and pH optima for the enzyme were determined as 60&deg / C and 8.0, respectively. Enzyme retained 67% activity after 1 h incubation at 80&deg / C and retained 87% of its activity after 1 hour of incubation at pH 9.0 at room temperature. The enzyme obeys Michealis-Menten kinetics with Km and Vmax values being 5 mg /ml catechol and 38 U/ml, respectively. Molecular weight of the enzyme was determined as 29 kDa and isoelectric point of enzyme was found to be approximately 6.0.

New and rapid techniques for the characterization and identification of bacteria would have an important role in clinical microbiology and in food analysis because of an increasing prevalence of infectious diseases and In this work we carried out two approaches. In the first study the characterization and differentiation of mesophilic and thermophilic bacteria were investigated by using Fourier Transform Infrared (FTIR) Spectroscopic technique. In the second study, we investigated the characterization and identification of 3 Bacillus and Micrococcus species Our results from first approach show that there was a dramatic difference between mesophilic and thermophilic bacteria. The protein concentration was high, lipid concentration, the level of triglycerides and the unsaturated acyl chains decreased in thermophilic bacteria. We found that in thermophilic bacteria PO- 2 groups become hydrogen bounded. In addition, our results suggest that the cellular DNA content was low in thermophilic bacteria. Moreover there were characteristic peaks for both mesophilic and thermophilic bacteria and these peaks can be used for the differentiation of these two bacteria group. There were also some specific peaks that can be used for the differentiation of Escherichia coli and Lactobacillus plantarum at species level. In the second approach, our results show that there were significant spectral differences between Bacillus and Micrococcus species such as the proportion of unsaturated acyl chains in triglycerides were higher in Micrococcus species. Moreover we observed different bands that may be explained by an acetate oxidation via the tricarboxylic acid cycle and an exopolymer formation in Micrococcus species. In addition to that another band similar to glycogen, may be explained by a glycogen-like storage material in Micrococcus species. Also there are characteristic peaks that can be used for identification of Micrococcus spp.

Spores of thermophilic bacilli are a common concern during the manufacture of milk powder. Spores are believed to occur in high numbers in milk powder due to their ability to survive pasteurisation, attach to stainless steel surfaces, germinate, grow as biofilms and subsequently enter the product stream and thereby contaminate the final product. In this study, thirty one thermophilic bacilli isolates were obtained from a New Zealand milk powder production line and identified as either Anoxybacillus flavithermus or Geobacillus spp. using random amplified polymorphic DNA (RAPD) and species-specific PCR. Sporulation media and a polyethylene glycol two-phase separation system were modified to produce high yields of spores free from debris. The spores of four Geobacillus spp. isolates (CGT-8, D4, E7 and E11) were characterised in terms of structure (electron microscopy), surface charge (zeta potential), hydrophobicity (contact angle and microbial adhesion to hexadecane) and attenuated total reflectance infrared spectroscopy (ATR-IR). Spores from three of the four isolates possessed an exosporium while the fourth did not. However the integrity of the exosporium varied over time. The spores were negatively charged (-10 to -20 mV) at neutral pH and high ionic strength (0.1 M KC1). Both hydrophobicity assays revealed that the spores of the four isolates were relatively hydrophilic while ATR-IR revealed the spores' surfaces consisted of protein and polysaccharides. The influence of these spore characteristics on adhesion to a variety of substrata under high flow rates was examined using the extended Derjaguin, Landau, Verwey and Overbeek (XDLVO) theory. Spores generally attached in higher numbers to hydrophobic surfaces compared to hydrophilic surfaces, however this observation was more prevalent for isolate D4. This result indicated that a single mechanism could not describe the adhesion of spores from different strains. A series of glass surfaces with modified characteristics were produced in order to test the antifouling properties on the adhesion of D4 spores. Spores suspended in a high ionic strength medium (0.1 M KC1) attached in greater numbers (1 Log₁₀ CFU cm⁻�) to positively charged and hydrophobic surfaces compared with negatively charged and hydrophilic surfaces. A clean in place (CIP) procedure, reduced spore numbers on hydrophobic and hydrophilic surfaces by 1.5 and by 2.0 Log₁₀ CFU cm⁻�, respectively. When spores were suspended in milk, there was little difference in the number of spores attaching to the different surfaces (ie. 3.5 to 3.8 Log₁₀ CFU cm⁻�), and spore removal from surfaces via a CIP regime was unchanged (1.5 to 2.0 Log₁₀ CFU cm⁻� reduction) compared with spores that attached in simple 1:1 electrolyte media. The effects of a caustic wash on spore surface characteristics and adhesion was determined. There was a significant reduction in spore viability (2 Log₁₀ CFU mL⁻�) after a 30 min caustic wash at 65 �C in the current study, however surviving spores displayed a greater propensity to attach to stainless steel. Surface characterisation results revealed an increase in hydrophobicity and a greater negative charge on the spores' surface after treatment with NaOH. Surviving spores could potentially recontaminate sections of the plant which are cleaned with this recycled caustic wash solution, thereby seeding surfaces with spores at the beginning of the next processing run. In conclusion, while surfaces that reduce spore adhesion and enhance removal can be produced, exposure to complex solutions such as milk can reduce the anti-fouling effectiveness of such surfaces to spore adhesion.