Thesis (Ph. D.)--University of Hong Kong, 1989.

In the modern ocean, manganese is oxidized over a timescale of days. To better understand the mechanisms and driving factors for manganese oxidation in the natural environment, experiments were performed with surface water samples collected from the San Antonio Bay. In this study area, the formaldoxime assay was utilized to determine that manganese oxidation is catalyzed via multiple pathways utilizing various catalysts and proximal oxidants. The contribution of catalysts such as colloidal matter, microorganisms and the proximal oxidant superoxide were investigated in the San Antonio Bay. The study suggests that superoxide contributed about 30% of Mn oxidation. The microorganisms and colloids were equal in terms of catalysis and accounted for approximately 100% of Mn oxidation. This study is important because gaining more understanding on the mechanisms by which Mn is oxidized will contribute to its use as a geochemical redox indicator.

Take-all is the most devastating root disease of wheat worldwide. The causal agent is <I>Gaeumannomyces</I> <I>graminis</I> (Sacc.) Arx & Olivier. Based on morphological characteristics and host ranges, three varieties of <I>G</I>. <I>graminis</I> have been recognized. <I>G</I>. <I>graminis</I> var. <I>tritici</I> Walker (Ggt) is the major causal agent of take-all of wheat and barley and the most economically important take-all pathogen. <I>G</I>. <I>graminis</I> var. <I>avenae</I> (Turner) Dennis (Gga) attack oats and causes take-all patch of turf grasses while <I>G</I>. <I>graminis</I> var. <I>graminis</I> (Ggg) is pathogenic on turf grasses but is non-pathogenic on wheat. Conventional diagnosis of take-all pathogens is based on field symptoms such as blackened roots, stunted growth, and white-heads and morphological characteristics such as hyphopodia type, size of perithecia, asci, and ascospores. These procedures are time-consuming, laborious, and often inconclusive. The objective of this study was to develop a rapid, simple, and specific method for differentiation of <I>G</I>. <I>graminis</I> varieties using PCR and molecular-based technology. Exploitation of genes associated with pathogenicity of <I>G</I>. <I>graminis</I> as markers for the test was proposed. Metabolic activities of <I>G</I>. <I>graminis</I> associated with pathogenesis were investigated, namely, the abilities to produce avenacinase and to oxidize manganese. Avenacinase, an avenacin detoxifying enzyme, was associated with Gga pathogenicity for oats but this enzyme is not important in Ggt pathogenicity for wheat. Manganese oxidation was also correlated with Ggt virulence. In this study, avenacinase-like genes were discovered in Ggt and Ggg and manganese oxidation was confirmed for Ggt, Gga, and Ggg. All isolates of Ggt except isolate ATCC 28230 were manganese oxidizers. Ggg and Gga isolates could oxidize manganese but their precipitation patterns were not as intense or closely correlated with mycelial growth as for Ggt. Pathogenicity assays on oats for Ggt, Gga, and Ggg isolates confirmed that Ggt isolates could not cause disease on oats aside from occasional slight root damage. Root weight was reduced for oat seedlings inoculated with Gga isolates. Comparison of partial sequences of avenacinase-like genes from Ggt and Ggg showed strong homology to that of Gga (94.8% identity to Ggt and 94.6% identity to Ggg). However, the Ggt gene was more closely related to that of Ggg (99.2% identity) than to Gga. DNA restriction endonuclease polymorphisms of the genes supported DNA sequencing information and revealed that there were variations within the genes among Ggt, Gga, and Ggg. Variety-specific electrophoretic patterns were obtained when the genes were digested with <I>Hae</I>III. Ggt, Gga, and Ggg upstream (5') variety-specific primers and a downstream (3') universal primer were designed from the avenacinase and avenacinase-like DNA sequences. PCR amplification with Ggt-, Gga-, and Ggg-specific primers generated fragments of 870, 617, and 1,086 bp, respectively. Each 5'-specific primer showed high specificity for its own DNA template in mixed populations of DNA templates. The optimized PCR procedure was sensitive to DNA template concentration as low as 100 pg. Genomic DNA of sixteen Ggt isolates, seven Gga isolates, and five Ggg isolates were tested. Although all Ggt isolates were originally isolated from wheat, seven isolates produced Ggg-specific fragments. This result corresponded well with <I>Hae</I>III DNA polymorphisms, pathogenicity assay, and manganese oxidizing ability. All but one Gga isolates produced the variety-specific fragment. Ggt- and Gga- specific products were generated from Gga isolate RB-W. Although Ggg-specific fragments were produced from all Ggg isolates, non-specific products were also observed from isolates that were not from wheat origin suggesting some genetic variations due to host ranges. Additionally, no non-specific amplification was obtained from any closely related fungi such as <I>Gaeumannomyces</I> <I>cylindrosporus</I> or <I>Phialophora</I> spp. The test developed in this study is the first test capable of identification of Ggt, Gga, and Ggg in a single PCR tube with a basic PCR protocol. The test is rapid and specific. Interpretation of results is simple and conclusive based on differences in size of each variety-specific fragment. / Ph. D.

The possible effects of pipe materials on drinking water quality have been analyzed in the distribution system of the water treatment plant of "La La Concepciónâ " in Tegucigalpa, Honduras. "La La Concepciónâ " is a surface water reservoir experiencing biogeochemical cycling of manganese. Black water problems have been reported in the distribution system since 1998. An evaluation of the potential influence that PVC and iron pipes could have on the concentration of iron and manganese in drinking water, the effects caused by the presence of manganese in PVC and iron pipe surfaces, and residual chlorine and Pb concentrations in the distribution system was performed. The sampled neighborhoods received an intermittent service. Water was suspended for 8 hours every day due to water quantity problems in the city. Water and pipe samples were obtained for PVC and galvanized iron pipes because these constitute the majority of the infrastructure used for distribution systems in Honduras. Thermodynamic and kinetic conditions for possible manganese oxidation by chlorine and dissolved oxygen in the distribution system were also evaluated. As expected, total Fe concentrations were greater for first flush conditions from the iron pipe. Water samples obtained from the PVC pipe showed higher total Mn concentrations and more black color than those obtained from the iron pipe for both first flush and continuous flow conditions. Residual chlorine decayed relatively fast along the sampled section of the distribution system. Pb concentrations were detected on water samples obtained from PVC for first flush and continuous flow and on iron pipe for first flush. Preliminary experiments showed that manganese-oxidizing and -reducing bacteria were present in the walls of both PVC and iron pipes. Higher numbers of colony-forming microorganisms were recovered from iron (30-fold more) compared to PVC pipe sections. However, the majority of isolates from the PVC biofilm (8 of 10, 80 %) were capable of Mn-oxidation while only 35 % (11 of 31) of isolates from the iron biofilm sample demonstrated Mn-oxidation. This research demonstrates the importance of the different interactions between water and the infrastructure used for its supply in producing safe drinking water. / Master of Science

No description available.

In the following study, the potential role that microorganisms play in the removal of Mn (II) and Fe (II) was assessed using biofilter sand and water samples collected from a biofiltration system (operated by Umgeni Water in KwaZulu-Natal, Nottingham Road, at the Nottingham combined school, South Africa) treating borehole water containing manganese and iron. Initially the presence of Mn (II) and Fe (II) oxidizing bacteria was demonstrated in the biofiltration system. Thereafter, the contribution of individual microorganisms to the overall removal of manganese and iron was assessed in the laboratory by determining the difference in metal oxidation in the presence and absence of active bacteria at neutral pH, simulating conditions in the biofilter. Controls were run to verify the elimination via physiochemical reactions occurring within the biofiltration system. Finally a diversity snapshot of the bacteria present within the biofilter matrix was established via analysis of a clone library. Viable bacterial counts for the biofiltration system were established using MSVP (minimal salts vitamins pyruvate) medium - plus added manganese sulfate or iron sulfate targeting Mn (II) and Fe (II) oxidizing bacteria - and R2A for heterotrophic bacteria. In the first experimental chapter, batch tests using MSVP were employed to determine manganese oxidation, by measuring the pH and ORP (oxidation reduction potential) in experimental flasks and controls over time. There was a clear drop in pH and a concomitant increase in ORP when an isolated manganese oxidizing strain (designated LB1) was grown in MSVP plus added manganese sulfate, indicating manganese oxidation. Based on physiological characteristics established by the VITEK-2 system as well as by 16S rRNA gene sequence analysis and MALDI-TOF (Matrix assisted laser desorption ionization-time of flight mass spectrometry) mass spectrometry of cell extracts, the isolate was identified as a member of the genus Acinetobacter. EDX (energy dispersive X-ray analysis) analysis of crystals formed in batch culture tests, containing MSVP plus either added manganese or iron sulfate, confirmed the ability of the isolate to oxidize both Mn (II) and Fe (II). The leucoberbelin blue colorimetric assay and batch tests using MSVP both demonstrated that in the presence of the isolated strain, Acinetobacter sp. LB1, the rate of Mn (II) oxidation at neutral pH was enhanced as compared to abiotic controls. In the second experimental chapter the difference in Fe (II) oxidation between biological and abiological systems at neutral pH was determined using batch tests run with Acinetobacter sp. LB1 and Fe (II) in saline. In addition, the rate of Fe (II) oxidation was also determined at acidic pH and at alkaline pH in experimental and control flasks. To determine Fe (II) removal under conditions simulating those in the biofiltration system, batch tests were set up using borehole water freshly collected from the biofiltration system. In order to verify the contribution of native microorganisms in the borehole water to Fe (II) oxidation, these flasks were spiked with bacterial strains isolated from the biofiltration system - Acinetobacter sp. LB1 and Burkholderia sp. strain LB2 - and two known iron oxidizing strains Leptothrix mobilis (DSM 10617) and Sphaerotilus natans (DSM 565) were used to determine the contribution of reference iron oxidizers to Fe (II) oxidation. A separate set of the same flasks with the addition of filter sand was used to qualitatively demonstrate iron oxidation as it would occur within the biofiltration system. The ferrozine assay was employed to quantify the amount of Fe (II) in batch tests employing saline medium and in batch tests employing borehole water. EDX analysis was employed to confirm the presence of Fe (II) in oxidation products in the batch test flask with filter sand spiked with Acinetobacter sp. LB1. In the presence of Acinetobacter sp. LB1 at neutral pH in saline medium, the rate of Fe (II) oxidation was very similar to that in the abiological controls thus demonstrating that the presence of metabolically active microorganisms does not per se enhance the oxidation of Fe (II) like in the case of Mn (II) at neutral pH. Surprisingly, in the heat inactivated control, apparently the highest amount of Fe (II) was oxidized. As expected, at acidic pH very little oxidation of Fe (II) took place and at alkaline pH almost all Fe (II) in the flasks was removed and small amounts oxidized as determined by the amount of Fe (III) produced. Batch tests using borehole water proved that native microorganisms within the biofiltration system were more efficient in the oxidative removal of Fe (II) from the system, in comparison to the reference iron oxidizing strains. In the final experimental chapter, the presence of biofilms with actively metabolizing cells was examined on a pooled sample of biofilter matrix from the manganese and iron filter using CLSM (confocal laser scanning microscopy) image analysis. DNA was extracted from the biofilm material associated with biofilter matrix to establish a diversity snapshot of the bacteria present within the biofilter matrix. ARDRA (amplified “rDNA” restriction analysis) analysis of the clone library revealed the presence of 15 unique OTU’s (operational taxonomic unit) based upon restriction patterns of amplified 16S rRNA genes of a total of 100 randomly selected clones. The majority of the clones were closely related to the genera Nitrospira and Lactococcus. Overall, 42% of the clones were assigned to the phylum Proteobacteria, 13% to the phylum Actinobacteria, 24% to the phylum Firmicutes and 21% to the phylum Nitrospirae. Overall, the results demonstrate that bacteria present within an established biofiltration system at neutral pH can contribute to the oxidative removal of Mn (II) and, apparently only to a smaller degree, to that of Fe (II) present in borehole water and that species within the proteobacterial genus Acinetobacter are potentially involved in the geochemical cycling of these two metals. Keywords: Biofiltration, iron and manganese oxidation, Acinetobacter sp. LB1, batch tests, 16S rRNA, MALDI-TOF MS analysis, Mn (II) and Fe (II) colorimetric assays, EDX analysis, biofilm formation, CLSM image analysis, 16S rRNA clone library Abbreviations: MSVP (minimal salts vitamins pyruvate), ORP (oxidation reduction potential), EDX (energy dispersive X-ray analysis), MALDI-TOF MS (Matrix assisted laser desorption ionization-time of flight mass spectrometry), rRNA (ribosomal RNA), ARDRA (amplified “rDNA” restriction analysis), CLSM (confocal laser scanning microscopy), OTU (operational taxonomic unit) / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2013.