Nitrification is a central part of the nitrogen cycle, whereby the most reduced form, ammonia, is converted to the most oxidised form, nitrate via nitrite. The first step is performed by ammonia oxidising bacteria (AOB) and archaea (AOA), with the second step performed by nitrite oxidising bacteria (NOB). Although both groups are closely associated in nature, ammonia oxidisers have received more attention compared to NOB as ammonia oxidation is considered the rate-limiting step. Nitrobacter and Nitrospira are two important groups of soil NOB. To determine whether there are specific associations of AOA or AOB with certain NOB, the effect of organic and inorganic ammonia sources was tested by adding glutamate or ammonium sulphate to soil together with either 5% 12CO2 or 13CO2 to determined autotrophic growth by DNA-SIP. The results demonstrated that while the various ammonia and nitrite oxidisers responded differently, there was no direct evidence of specific coupled interactions. The effects of soil pH on Nitrobacter and Nitrospira was then investigated in a long-term pH gradient in an agricultural field. The results demonstrated that Nitrospira abundance was lower in acidic soils, whereas Nitrobacter abundance remained equally or more abundant. pH also influenced the relative distribution of Nitrobacter and Nitrospira populations, with distinct community structures at both high and low pH. The interaction of AOA and NOB was further investigated in a co-culture experiment, and demonstrated that the removal of nitrite and free nitrous acid NOB enhanced both rates and amounts of ammonia oxidised, indicating that in acidic environments these relationships may be particularly critical. Finally, the use of the compound PTIO was investigated for potential use in elucidating specific relationships between AOA and NOB. Results demonstrated a lack of specificity for the target group, and was unstable in soil, and therefore its use in soil should proceed with caution.

A total of 200 Rhizobium meliloti isolates were sampled from alfalfa (Medicago sativa L.) nodules in five uninoculated fields throughout Arizona. Dominant strains (≥ 20% nodule occupancy at each site) were identified using plasmid profile analysis and intrinsic antibiotic resistance patterns. The major dominant strains and a commercial strain (102F77b) were evaluated for their N fixing effectiveness in a Leonard jar study. All strains were highly effective, and no significant differences were found (p ≥ 0.05) in shoot weight, root weight, nodule weight, acetylene reduction and total N content among the strain treatments. These effective dominant R. meliloti strains indigenous to Arizona soil probably contribute to the state's high alfalfa yield. Furthermore, indigenous strains AZTCYJ, AZSC, and AZY have potential as inoculants for arid lands due to their high effectiveness and unique resistances to extreme abiotic stresses present in arid land soils.

Wastewater treatment plants (WWTPs) are required to remove ammonium (NH4+) from wastewater due to its oxygen demand and toxicity to the aquatic organisms. Ammonium is removed in the activated sludge treatment system by nitrification and denitrification processes. Nitrification is the oxidation of NH4+ to nitrate (NO3-) by autotrophic nitrifying bacteria which use carbon dioxide (CO2) as a carbon source for growth. These bacteria grow slowly with low nitrification rates limiting WWTPs capacity. In this research it was hypothesized that supplying higher concentrations of CO2 during aeration increases nitrification rates, resulting in a reduction of the solids retention time (SRT). This hypothesis was tested with two lab-scale sequencing batch reactors seeded with sludge from a full-scale activated sludge WWTP and fed synthetic wastewater. The control reactor was aerated with regular air (0.03% CO2) and the experimental reactor was aerated with air containing 1% CO2. Ammonium and NO3- were measured online to determine the nitrification rates. Samples for solids and chemical oxygen demand (COD) determination were collected to evaluate the system performance. Supplying CO2 to the experimental reactor throughout the entire react cycle resulted in proliferation of filamentous bacteria, poor settling, and washout of the biomass. However, nitrate formation rates in the experimental reactor were 3 times higher than the control before washout occurred. In a subsequent experiment, CO2 was supplied to the experimental reactor only during the last 5 hours of the cycle, resulting in excellent settling and nitrification rates 6 times higher than in the control. A confirmatory experiment was conducted that lowered the SRT from 8 days to 6, 4, and 2 days. Nitrate formation rates were up to 12 times higher in the experimental reactor compared to the control, with an average of 4 times higher. Additionally, the sludge volume index (SVI) suggested a positive impact of CO2 on settling performance. No impact of CO2 on COD removal was observed. The results obtained suggest a positive effect of CO2 on the nitrate formation and settling performance in the activated sludge system, indicating that nitrification can be achieved at low SRTs which might optimize WWTPs capacity.

Graduation date: 1996

M.S.

Metalaxyl, [N—(2,6-Dimethylphenyl)-N-(Methoxyacetyl)-alanine methyl ester], is used extensively in tobacco (Nicotiana tabacum L.) production for prevention of black shank (Phytophthora parasitica Dast. var. nicotianae), blue mold (Peronospora tabacina Adam), and damping-off (Pythigm spp.). Metalaxyl is also patented as a nitrification inhibitor, although not marketed for that purpose. Proper maturity and ripening of flue-cured tobacco depends on an adequate supply of N through the time of removal of the inflorescence, with a declining supply of N from that point. Use of a chemical which might prolong the availability of N in tobacco could delay maturity and reduce the quality of the cured leaf. These studies were conducted to determine whether metalaxyl might inhibit nitrification under a broad range of soil physical and environmental conditions prevalent in the tobacco producing areas of Virginia. The influence of soil type, soil pH, soil temperature, and soil moisture on inhibition of nitrification by metalaxyl (1 mg kg⁻¹) were investigated in three soils used extensively for tobacco production. Soils used in the study were Cecil sandy loam (clayey, kaolinitic, thermic Typic Hapludult), Appomattox fine sandy loam (clayey, mixed, thermic Typic Kandhapludult), and Mattoponi sandy loam (clayey, mixed, thermic Typic Hapludult). Metalaxyl did not inhibit nitrification under any of the conditions studied. However, NO₂⁻ accumulation with metalaxyl was sometimes greater than the control, especially at high pH (7.0) in the Cecil and Appomattox soils, and at 10 and 20°C. Nitrite and NO₃⁻ accumulations from four rates of metalaxyl (1, 5, 25, and 125 mg kg⁻¹) were compared with those of an untreated control and a nitrapyrin standard over a seven week soil incubation period in further studies using the same soils and adjusted pH levels. Significant NO₂⁻ accumulation occurred during the first week after treatment at high pH in all soil types, with 5, 25, and 125 mg kg⁻¹ metalaxyl. Only the 125 mg kg⁻¹ metalaxyl treatment caused NO₂⁻ accumulation at the high pH in all soils beyond the second week after treatment, with the peak occurring in most cases between weeks three and four. Nitrate accumulation proceeded normally in all soil types and pH levels except with treatments of 25 and 125 mg kg". Nitrate accumulations with 25 mg kg⁻¹ were similar to those for nitrapyrin. The 125 mg kg⁻¹ rate was consistent in causing near total inhibition of NO₃⁻ accumulation at all pH levels in all soils. Nitrate accumulation tended to be lower at lower soil pH levels compared to the highest pH for all soils. Little difference in nitrification due to soil appears to be evident. Use of metalaxyl at recommended rates of 0.25 to 1.5 mg kg⁻¹ would not be expected to inhibit nitrification. / Ph. D.

The newly recognized ammonia-oxidizing archaea (AOA) makes re-evaluation of the contribution to ammonia oxidization by both AOA and ammonia-oxidizing bacteria (AOB) necessary and meaningful. The growing population and increasing anthropogenic activities around coastlines have affected wetland and coastal marine ecosystems through discharging polluted water containing large amounts of reactive inorganic nitrogen. The objectives of this study were to detect the phylogenetic diversity and abundance of ammonia oxidizers including AOA and AOB on different scales and to elucidate the distribution patterns along an anthropogenic pollution gradient from the coastal wetland of the Mai Po Nature Reserve in Hong Kong to the South China Sea (SCS). Generally, besides lineages shared by similar environments, various endemic lineages were also observed in the polluted mangrove sediments of Hong Kong, and in the coastal, and deep-sea surface and subsurface sediments from the SCS indicating their geographical distance should be responsible for these phylogenetic distinctions. The community structures of AOA and AOB observed were proposed to be associated with environmental parameters including metals and total phosphorus (TP) separately in the sediments while their abundance was correlated with the pH value and temperature. On the other hand, along a profile of surface sediments with stable salinity from the coastal margin to the slope in the SCS, a clear community structure transition was detected for both AOA and AOB, showing major differences in each of their responses. Although the abundance of AOA was lower than that of AOB in the subsurface sediment samples from the SCS, the statistical support for relationships between AOA and nitrite concentration shed new light on the active contributor to the subsurface nitrogen cycle in the oxygen minimum zone from the deep-sea sediments. On a large scale, along the anthropogenic pollution gradient from the Pearl River Delta to the coastal margin and then the SCS, the dominant genus transition from Nitrosomonas to Nitrosospira was detected in response to the salinity and anthropogenic influences. Among a wide spectrum of environmental conditions in the western Pacific, a suite of statistical analyses clearly delineated the shallow and deep-sea sediments clusters suggesting that the depth and other contributing environmental factors involved shape the current distribution pattern of AOA. On a global scale, our understanding about the systematics and evolution of AOA was advanced through phylogenetic analyses. Salinity, lifestyle and temperature were proposed to be responsible for the global distribution patterns of AOA. On the basis of studies in the anthropogenic influence areas, the methods to detect specific responses of ammonia oxidizers to known anthropogenic pollution were concluded. Highlights of this study advance not only our understandings about phylogenetic diversity of ammonia oxidizers and the driving forces shaping their community structure and distribution patterns, but also a revised comprehensive view about them on the larger scale. / published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

No description available.

Legumes play an important role in agriculture as major food sources for humans and as feed for animals. Bioavailable nitrogen is a limiting nutrient for crop growth. Legumes are important because they can form a symbiotic relationship with soil bacteria called rhizobia that results in nitrogen-fixing root nodules. In this symbiosis, rhizobia provide nitrogen to the legumes and the legumes provide carbon sources to the rhizobia. The Medicago truncatula NPF1.7/NIP/LATD gene is essential for root nodule development and also for proper development of root architecture. Work in our lab on the MtNPF1.7/MtNIP/LATD gene has established that it encodes a nitrate transporter and strongly suggests it has another function. Mtnip-1/latd mutants have pleiotropic defects, which are only partially explained by defects in nitrate transport. MtNPF1.7/NIP/LATD is a member of the large and diverse NPF/NRT1(PTR) transporter family. NPF/NRT1(PTR) members have been shown to transport other compounds in addition to nitrate: nitrite, amino acids, di- and tri-peptides, dicarboxylates, auxin, abscisic acid and glucosinolates. In Arabidopsis thaliana, the AtNPF6.3/NRT1.1( CHL1) transporter was shown to transport auxin as well as nitrate. Atchl1 mutants have defects in root architecture, which may be explained by defects in auxin transport and/or nitrate sensing. Considering the pleiotropic phenotypes observed in Mtnip-1/latd mutant plants, it is possible that MtNPF1.7/NIP/LATD could have similar activity as AtNPF6.3/NRT1.1(CHL1). Experimental evidence shows that the MtNPF1.7/NIP/LATD gene is able to restore nitrate-absent responsiveness defects of the Atchl1-5 mutant. The constitutive expression of MtNPF1.7/NIP/LATD gene was able to partially, but not fully restore the wild-type phenotype in the Atchl1-5 mutant line in response to auxin and cytokinin. The constitutive expression of MtNPF1.7/NIP/LATD gene affects the lateral root density of wild-type Col-0 plants differently in response to IAA in the presence of high (1mM) or low (0.1 mM) nitrate. MtNPF1.7/NIP/LATD gene expression is not regulated by nitrate at the concentrations tested and MtNPF1.7/NIP/LATD does not regulate the nitrate-responsive MtNRT2.1 gene. Mtnip-1 plants have an abnormal gravitropic root response implicating an auxin defect. Together with these results, MtNPF1.7/NIP/LATD is associated with nitrate and auxin; however, it does not act in a homologous fashion as AtNPF6.3/NRT1.1(CHL1) does in A. thaliana.

Submitted in fulfillment for the requirements for the Degree of Doctor of Technology: Biotechnology, Durban University of Technology, 2012. / Nitrification is a crucial step in biological nutrient removal (BNR) processes, mostly carried out by a group of nitrifying bacteria which includes ammonia-oxidising bacteria (AOB) and nitrite-oxidising bacteria (NOB). Nitrification failure has proven to be a common operational problem in full-scale wastewater treatment plants (WWTP) since nitrifying bacteria are very sensitive to sudden changes in environmental or plant operating conditions. The current investigation was carried out to advance our understanding of the distribution of nitrifying bacterial populations and their performance at three different BNR plants in KwaZulu-Natal, South Africa. The latest molecular techniques such as fluorescent in situ hybridisation (FISH)-confocal scanning laser microscopy (CSLM), polymerase chain reaction (PCR) and real-time quantitative PCR (Q-PCR) were applied to detect and quantify nitrifying bacteria. When using FISH to target the nitrifying population, it necessitated optimising pre-treatment protocols of the samples to improve accuracy during quantification. Sonication was found to be the superior method of dispersion based on the least disruption of nitrifier cell integrity, irrespective of the sludge type. The effect of plant configurations and wastewater characteristics on the distribution of the nitrifying bacterial population and subsequently on the nitrification performance was evaluated using FISH and PCR. FISH results revealed the dominance of Nitrosomonas (AOB), Nitrobacter (NOB) and Nitrospira (NOB) for all BNR plants. The 16S rRNA analysis of PCR products using genus-specific primers, revealed the presence of more than one species of the same group at these plants. Nitrosomonas spp. including Nitrosomonas halophila, Nitrosomonas eutropha, Nitrosomonas europaea, Nitrosomonas aestuarii and an unidentified Nitrosomonas spp. were found to dominate among the AOB and Nitrobacter vulgaris, Nitrobacter alkalicus, Nitrobacter hamburgensis and an unidentified Nitrobacter spp. were the dominant species for NOB. Among these species, Nitrosomonas aestuarii, Nitrosomonas europaea, Nitrobacter hamburgensis were detected only from the industrial wastewater samples. The efficiency of two commonly used techniques viz., FISH and Q-PCR for the detection of nitrifiers from WWTP were also studied and compared, specifically targeting Nitrobacter sp. Even though there were slight variations in the quantification results, changes in the Nitrobacter community at these plants were consistent for both FISH and Q-PCR results. Both techniques have their own limitations and advantages. This study has helped to add to the platform of understanding the distribution and activity of nitrifying bacteria by correlating population dynamics with the operational parameters at full-scale level. The observations made in this study will assist researchers and engineers to minimise future nitrification failure at full-scale BNR plants. This study also confirmed the highly complex activities of wastewater treatment processes, which is dependant on a number of factors. Specific AOB or NOB predominant in wastewater rather suggests that the wastewater type and characteristics may contribute to significantly different microbial environments. Among the AOB, Nitrosomonas dominated at all BNR plants throughout the study period and for NOB both Nitrobacter and Nitrospira were found in significant numbers but their dominance varied across the plants. These dissimilar, distinct distribution patterns could be attributed to their environment which in turn impacted on the nitrification performance of the system. It was also noted that the co-existence of more than one group of these communities at the same plant could help the plant escape complete functional failures such as nitrification, due to sudden changes in temperature and substrate concentrations, as this function can be performed by different groups. Although it would have been meritorious to conduct a nitrogen balance in this study, this was not possible since the research focused on full-scale systems. / National Research Foundation / D