High levels of elements such as sodium (Na), potassium (K) and phosphorus (P) in iron ore minerals are known to reduce the quality and price of these minerals. South Africa, as one of the world largest exporter of iron ore, is affected by this problem. Both potassium (K) and phosphorus (P) are peculiar to South African iron ore. The present study has therefore focussed on developing an environmentally friendly biological method for lowering the levels of K and P in iron ore minerals. Short and long term experiments were set up to isolate, identify, screen and test potential bioleaching bacteria and fungi from different environmental samples. The study started by investigating the possible relationship that exists between weathering and bioleaching processes. The investigation was intended to provide relevant information on the natural role of microorganisms such as ectomycorrhizal (ECM) fungi in the mining environment. The experiments involved the use of both mycorrhizal and non-mycorrhizal Pinus patula seedlings for the weathering of iron ore minerals. Four types of ECM fungi were used, namely Pisolithus tinctorius (PT), Paxillus involutus (PI), Laccaria bicolor (LB) and Suillus tomentosus, (ST). From the results, ectomycorrhizal weathering can be said to be species-specific and significantly influenced by fungal type and particle size. In addition, it was also discovered that both mycorrhizal and non-mycorrhizal roots can participate in weathering processes. Further investigations of ECM fungi when not in symbiosis, were carried out to know how or if they can be potential candidates to mobilise K and P from iron ore minerals. The experimental set up involved in vitro pure cultures of four different ECM fungi, namely Pisolithus tinctorius (PT), Paxillus involutus (PI), Phialocephala fortini (PFR), and Suillus tomentosus (ST). In addition, the treatments involved the use of five different particle sizes of each ore type. The results obtained indicated the potential of the ECM fungi to mobilise P and K from the two iron ore types though at different levels. Factors such as ore type, particle size, organic acid production and attachment of the fungi to the iron ore were all found to influence the mobilisation of nutrients from these ores. Another experiment that addressed some of the limitations encountered with the use of pure cultures of ECM fungi was conducted. Isolated indigenous fungal pure cultures from the surfaces of iron ore minerals were screened for their abilities to solubilise minerals by lowering the levels of K and P. These isolates were identified molecularly as close relatives of three genera that included Penicillium, Alternaria (2 isolates) and Epicoccum for isolates FO, SFC2/KFC1 and SFC2B respectively. The identified Penicillium sp. turned out to be the only phosphate solubiliser among these isolates. Direct bioleaching capability of the fungus was compared to that of its metabolite. At the end, the metabolite showed better K removal than the direct use of the fungi. Interpretation of these results indicates possible relationship between K and P removal, and the organic acids production by this fungus. Other factors such as particle size and mineral type were also found to significantly influence the leaching process. Additional experiment was conducted to investigate the indigeous bacteria and their potentials in reducing the K and P contents of iron ore minerals. A total of 23 bacterial strains that belong to Proteobacteria, Firmicutes, Bacteroidetes and Actinobateria were isolated from the iron ore minerals and identified with molecular methods. All the bacterial isolates were screened for their potential as mineral solubilisers. Only eight of the isolates were selected and used in shake flask experiments that contained both KGT and SK mineral types as their sources of K and P. The experiment showed that all the eight isolates have potentials to produce organic acids especially high levels of gluconic acid but lower quantities of acetic, citric and propanoic acid. Scanning electron microscopy (SEM) and fourier transform infrared (FITR) analyses also helped to uncover the role that biofilm and extracellular polymeric substances could play in mineral solubilisation. Finally, an investigation of a new method for reduction of K and P levels of iron ore minerals was carried out, focussing on the use of cheap resources as well as septic conditions. The study involved the use of fermented spoilt grape fruits (Vitis sp.) and the solution from the product utilised in shake-flask experiments. Treatments involved two types of iron ore minerals (KGT and SK) and two different particle sizes. The result suggests the significant effect of particle size, time and organic acids on the reduction of K and P from the iron ore minerals. The important part of this finding is the discovery of a cheap microbial energy source (spoilt grape) that can be further exploited for full biobeneficiation of iron ore minerals. Another advantage of this method is the fact that the experiment can be conducted under non–sterile conditions, making it a system that can be operated outdoor. / Thesis (PhD)--University of Pretoria, 2010. / Microbiology and Plant Pathology / unrestricted

Abstract The chemical characteristics of spent wash, an effluent from the distillation of ethanol from molasses, can vary, depending on the initial feed stock and the operations of the factory. Spent wash from Australia contained large quantities of dissolved organic carbon (DOC), K, N and Cl with moderate to low concentrations of Ca, Mg, Na, and S, and small amounts of trace elements (Fe, Al, Mn, Cu, Zn). Spent wash from Thailand was obtained from whisky distillation and generally had similar chemical attributes to the spent wash from ethanol production, although the Zn concentration was much higher (399-974 mg Zn L-1). Large differences in EC and pH were found between Australian (pH: 4.08-4.49; EC: 40.4-42.2 dS m-1) and Thai spent wash (pH: 7.86-9.20; EC: 11.3-82.4 dS m-1). However, the high concentration of K in spent wash from both manufacturing processes indicates the unique chemical characteristic of wastewater produced from molasses-based distillation. While chemical characterisations of spent wash have shown its potential value as a fertilizer, its utility needs to be more practical than by direct application. This is due to its dilute nature, associated with the presence of organic carbon (OC) and nutrients in soluble form, thus making direct application susceptible to leaching losses of nutrients, especially in soils of low cation exchange capacity (CEC). It was hypothesized that capturing OC and nutrients on a high activity exchanger would overcome this problem. Since the charge on organic components in spent wash is important to the capturing process, pH manipulation is one of the major factors affecting the efficiency of this capture. The study of the influence of pH on development of charge on organic components in spent wash was undertaken using potentiometric titration. The potentiometric titration, and derived pH buffer curve, showed that the organic components in spent wash comprised a wide range of ionizable organic functional groups behaving like a mix of weak acids. While the inflection point (s) could not be obtained from the potentriometric titration, the absorbance of spent wash in the UV waveband (250 to 400 nm) confirmed the presence of phenolic groups in spent wash. The charge on organic components in spent wash was strongly pH dependent, and highest in the pH range of 4.0-5.0. The manipulation of pH and salt concentration (EC) was shown to have an effect on flocculation/dispersion and it is inferred that this was due to changes in the molecular arrangement of organic components in spent wash. A coiled configuration is suggested at acidic pH, whereas a linear flexible configuration and macromolecular structure tending to occur at neutral and alkaline pH respectively. Ca-saturated bentonite, originally sourced from Mantuan Downs, Central Queensland, was used in an investigation of the capture of OC and nutrients from Sarina spent wash at different pH values (4.5, 6.0, 8.0). This study demonstrated that manipulation of pH and electrolyte concentration had a small effect on adsorption of OC by the clay, probably the result of effects on the charge characteristics of both clay mineral surfaces and organic molecules and also on molecular sizes of the dissolved organic matter (DOM). The highest absorption of OC was only 28% for dewatered spent wash, suggesting Ca-saturated bentonite may not be a strong sorbent for DOM from a wastewater of high EC and which contains high concentration of DOC of the composition found in spent wash. The study of the adsorption of K by bentonite in a pure inorganic system (without DOM) indicated sufficiently high concentrations of K can compete with divalent Ca2+ for exchange sites on the clay surface. The adsorption of K in an organic system (the DOM component in spent wash) showed that DOM had a positive influence on adsorption of inorganic ions (e.g. K+). Characterisation of the spent wash/ clay mixture demonstrated its potential to be used as a soil ameliorant on the basis of the large quantity of OC and nutrients it contained (especially N and K). Wet and dry organic clays were produced and assessed as ameliorants in a glasshouse trial using forage oat (Avena sativa var. Coolobah) as test species. This pot study indicated a positive influence of application of either wet or dry organic clay on crop biomass at the high application rate (equivalent to 40 tonnes ha-1). The high proportional K recovery from this high application rate application suggests that organic clays can be used as K fertilizer. The crop nutrient uptake data also indicated its use as a source of S and Mn.

Poultry litter ash (PLA) is a co-product from manure-to-energy systems that originated in response to increased poultry litter (PL) volumes generated in concentrated poultry production regions. Investigating PLA as a crop fertilizer is an alternative solution to balancing poultry and crop regional nutrient cycling in the Commonwealth of Virginia. As the expanding world population places pressure on the poultry industry to meet consumption demands, increased PL production presents an obstacle to identify alternative uses for increased volumes. Currently, Virginia produces 44 million broilers with PL produced predominately in the Shenandoah Valley and Eastern Shore. Likewise, a growing world population places pressure on crop production areas and subsequently finite natural resources used for crop fertilization. Poultry litter ash is an alternative phosphorus (P) and potassium (K) source enhancing transportation logistics, repurposing PL nutrients, and offers dual purpose as a fertilizer and an energy source when compared to PL. Three PLA products [(fluidized bed bulk (FB Bulk), fluidized bed fly (FB Fly), and combustion Mix (CMix)], two manufactured co-products [(granulated poultry litter ash (GPLA), and ash coated urea (ACU)] were evaluated as P, K, and N sources for corn (Zea Mays L.) production in comparison to industry fertilizers [(PL, triple superphosphate (TSP), muriate of potash (KCL), and urea). A comprehensive examination of elemental composition, P speciation, P and K solubility, improved functionality into granulized forms, and field testing were conducted to discern PLA potential as an alternative fertilizer source. Poultry litter ash products were evaluated by total elemental analysis, backscatter-electron dispersive (BSED) microscopy, and X-ray absorption near edge structure (XANES) spectroscopy. Poultry litter ash elemental concentrations were highly variable ranging from 50.6 to 102.0 g P kg -1 and 62.6 to 120.0 g K kg -1 and were comparatively higher than PL concentrations. Phosphorus structures that provided and controlled P solubility were Ca and Ca-Mg-phosphate compounds. Spectroscopy confirmed Ca structures as predominately monetite (dicalcium phosphate anhydrous; CaHPO4; log K ̊ 0.30) and brushite (dicalcium phosphate dihydrate; CaHPO4.2H20; 0.63 log K ̊ ) species that were supported by BSED and elemental stoichiometric ratios (Ca:P; 1.12 to 1.71:1). Additionally, GPLA acidified from FB Fly had higher brushite and monetite percentages described by spectra models, translating into a more soluble Ca-phosphate species when compared to FB Fly original P species. Granulated poultry litter acidulation trials successfully identified a desired granulation point of 29% (14.5 g acid to 50 g PLA) phosphoric acid (75% H3PO4) acidulation. Acidulation dose response relationships created simple linear regression (SLR) equations that sufficiently (R2 > 0.80) described changes in total measurable P and water soluble P, pH, and exothermic reaction temperatures to increasing H3PO4 acidulation. Solubility tests included: sequential extraction, particle size effect on solubility, carbon effect on water soluble P, and Mehlich-1 extraction of PLA sources that confirmed decreased P solubility. A majority PLA P was found in bound plant unavailable fractions (87.7 to 97.7% P of total P). Granulated poultry litter ash had improved P plant available P of 36.0% P of total P. Carbon (C) effects on PLA P were examined by ashing PLA samples in a muffle furnace at 550 ̊C. Differences in total carbon content negatively impacted FB Bulk and CMix total P (1.30 and 4.56 g P kg -1); however, muffle furnace temperatures increased FB Fly total P by 6.74 g P kg -1. All fertilizer products were investigated under field conditions in separate P, K and N corn studies across Virginia coastal plain soils to determine fertilizer effects on corn plant parameters [(most mature leaf (V6), corn ear leaf (R1), and grain (R6)]. Poultry litter P treatments, averaged over rate, recorded highest yield in both years. At eight of nine field sites, FB Bulk resulted in numerically or significantly higher Mehlich-1 concentrations than other P sources post-harvest. Although Mehlich-1 P increased, yield and plant parameters did not; which leads to the conclusion that PLA sources increased soil residual P that did not translate into immediate plant availability recorded within a growing season. Across plant efficacy parameters examined, PLA K is a comparable nutrient source and improved plant parameters when compared to control. Eighteen out of twenty-one plant parameters examined found similar ACU and urea effects on N concentrations. Therefore, ACU is a comparable N source to urea. When compared to industry fertilizer sources, we concluded that PLA is a slowly available P source, decreased P availability negatively affected early plant growth, K is a comparable nutrient source and improved plant parameters compared to control, and ACU effectively provided N to maintain sufficient corn growth. In conclusion, PLA co-products serve as a densified nutrient source that may provide plant available nutrients if processed to aid in nutrient distribution to grain producing areas. / Doctor of Philosophy / Poultry litter ash (PLA) is a co-product from manure-to-energy systems that originated in response to increased poultry litter (PL) volumes generated in concentrated poultry production regions. Investigating PLA as an alternative crop fertilizer is essential to balancing poultry and crop regional nutrient cycling in the Commonwealth of Virginia. As the expanding world population places pressure on the poultry industry to meet consumption demands, heightened PL production presents an obstacle to identify alternative uses for increased volumes. Currently, Virginia produces 44,683,904 broilers with PL produced predominately in the Shenandoah Valley and Eastern Shore. Likewise, a growing world population places pressure on crop production areas and subsequently finite natural resources used for fertilization vital to maintaining crop yields. Poultry litter ash, a co-product from manure-to-energy systems, is an alternative phosphorus (P) and potassium (K) source enhancing transportation logistics, repurposing PL nutrients, and offers dual purpose as a fertilizer and an energy source when compared to PL. In this dissertation, three PLA products [(fluidized bed bulk (FB Bulk), fluidized bed fly (FB Fly), and combustion Mix (CMix)], two manufactured co-products [(granulated poultry litter ash (GPLA), and ash coated urea (ACU)] were evaluated as P, K, and N source for corn (Zea Mays L.) production in comparison to industry fertilizers (PL, triple superphosphate (TSP), muriate of potash (KCL), and urea). Each of the following chapters provides a comprehensive examination of the following topics: elemental composition, P speciation, P and K solubility, improved functionality into granulized forms, and field testing designed to provide parameters to conclude PLA potential as an alternative P, K and N source. In the second chapter, PLA products were evaluated by total elemental analysis, backscatter-electron dispersive (BSED) microscopy, and X-ray absorption near edge structure (XANES) spectroscopy. Poultry litter ash elemental concentrations are highly variable and are comparatively higher than PL concentrations. Phosphorus structure and species identified Ca as the primary element controlling P structure and subsequent solubility. The third component of this dissertation is granulation trials investigating phosphoric acid effects on granulizing and increasing total and water soluble P. Our results identified 29% (14.5 g acid to 50 g PLA) phosphoric acid acidulation for desired granule size. The third dissertation component examines PLA solubility. The results demonstrated PLA decreased P water solubility when compared to industry fertilizer sources. Granulated poultry litter ash demonstrated improved P plant availability due to the granulation process. The final and fourth dissertation components investigated PLA sources under field conditions in separate P, K and N corn studies across Virginia coastal plain soils to determine fertilizer effects on corn plant parameters. Minority of plant parameters tested revealed P control yielded numerically higher P concentrations than PLA P sources tested. Poultry litter P treatments, averaged over rate, recorded highest yield in both years. At eight of nine field sites, FB Bulk resulted in numerically or significantly higher Mehlich-1 concentrations than other P sources post-harvest. Although Mehlich-1 P concentrations increased, yield and plant parameters did not; which leads to the conclusion that PLA sources increased soil residual P that did not translate into immediate plant availability recorded within a growing season. Across plant efficacy parameters examined, PLA K is a comparable nutrient source and improved plant parameters when compared to controls. The majority of plant parameters examined found similar ACU and urea effects on N concentrations. Therefore, ACU is a comparable N source to urea. When compared to industry fertilizer sources, field results concluded that PLA is a slowly available P source, decreased P availability negatively affected early plant growth, K is a comparable nutrient source and improve plant parameters compared to control, ACU effectively provides N to maintain sufficient corn growth. In conclusion, PLA co-products serve as a densified nutrient source that may provide plant available nutrients if processed to aid in nutrient distribution to grain producing areas.

El trabajo realizado en la Tesis Doctoral llega a las siguientes conclusiones:1.- T. halophila muestra una relación en peso raíz/parte aérea mayor que A. thaliana, y esta diferencia se ve incrementada en condiciones de estrés salino, lo que podría suponer una ventaja para afrontarlo.2.- El estrés salino produce en A. thaliana mayores reducciones en la absorción y en las concentraciones internas de K+ que en T. halophila, a la vez que T. halophila presenta menor absorción de Na+ y transporte a la parte aérea que A. thaliana. Ambas circunstancias resultan en una mayor relación K+/Na+ en T. halophila, lo que puede suponer una mayor tolerancia a la salinidad.3.- El gen ThHAK5 codifica para un transportador que media un transporte de K+ de alta afinidad en levaduras similar al observado en las plantas de T. halophila lo que sugiere que este transportador juega un papel fundamental en la absorción de K+ en el rango de la alta afinidad en esta especie vegetal.4.- Aunque AtHAK5 y ThHAK5 presentan una gran homología de secuencia y unas características funcionales similares, la regulación de los genes que los codifican difieren en condiciones salinas. Así, la salinidad reduce en menor medida la inducción de ThHAK5 por ayuno de K+. En consecuencia, la absorción de K+ de alta afinidad está menos afectada por la presencia de NaCl en el medio externo en T. halophila.5.- La mutagénesis al azar permite encontrar aminoácidos importantes para la función de las proteínas y ésta ha permitido identificar dos versiones mutantes del transportador de K+ de alta afinidad AtHAK5 más eficientes, capaces de transportar K+ a concentraciones externas de Na+ muy elevadas (0.1 mM K+ y 800 mM Na+). / The work done in this Thesis provides some interesting conclusions:1.- Thellungiella halophila show a weight ratio root/shoot bigger than Arabidopsis thaliana, and this difference arise under salt stress, what might provide an effective mechanism of salt tolerance to T. halophila.2.- In A. thaliana, salt stress induces a bigger reduction of K+ uptake and tissue concentrations than in T. halophila, and at the same time T. halophila shows a reduced Na+ uptake and Na+ transport to the shoot. Both properties enable a higher ratio K+/Na+ in T. halophila which might be another mechanism of salt tolerance. 3.- The ThHAK5 gene isolated in this Thesis, encode a K+ transporter that mediates high affinity K+ transport in Saccharomyces cerevisiae similar to the observed in intact plants of T. halophila, which suggest a key role of this transporter in the high affinity range of concentrations.4.- Although AtHAK5 and ThHA5 shows high sequence homology and similar functional properties, gene regulation is different under salt stress. Thus, salinity reduces to a lesser extent the K+-starvation ThHAK5 induction. As a consequence, high affinity K+ uptake is less affected by NaCl in T. halophila. 5.- Random mutagenesis allows the identification of important aminoacids for protein function, and with this technique two more efficient mutant versions of AtHAK5 have been isolated. The evolved AtHAK5 mutant versions are able to transport K+ at high Na+ external concentrations (0.1 mM K+ and 800 mM Na+) in yeast.