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European legislation has increased pressure on the use of land to represent the major disposal option for sewage sludge. Owing to their importance in regulating soil fertility, much research has been conducted into the effects of sewage sludge application on soil microorganisms. However, little knowledge is known about its effects on community dynamics of the beta-subgroup ammonia oxidising bacteria. This is despite the fact that nitrification activity of these bacteria plays an important role in soil nitrogen cycling. This investigation aimed to examine community dynamics of the beta-subgroup ammonia oxidising bacteria in sewage sludge amended soil by employing recently developed molecular based techniques. Firstly, a soil DNA extraction protocol was identified that allowed routine nested PCR amplification of 16S rDNA using beta-subgroup ammonia oxidiser directed primers. Reproducibility observed in denaturant gradient gel electrophoresis (DGGE) profiles suggested that 0.5 g samples used for DNA extraction allowed consistent detection of dominant beta-subgroup ammonia oxidiser community members. The effects of applying primary treatment sewage sludge to a variety of contrasting soils on (3-subgroup ammonia oxidiser community structure was unclear. This was partially hampered by lack of specificity of PCR primers for non ammonia oxidiser 16S rDNA sequences. Further, through measurement of net nitrification, there was no indication that sewage sludge addition stimulated the activity of nitrifier populations. Nevertheless, this study indicated the usefulness of DGGE for screening multiple environmental samples. A set of hierarchical oligonucleotide probes exhibiting specificity at the group, genus and cluster level were optimised using a non-radioactive system. These probes were used to assess the effects of application of aerobically digested sewage sludge to soil for 4 y on beta-subgroup ammonia oxidiser community structure. Despite significant changes in measurable soil parameters including net nitrification activity no changes were observed in beta-subgroup ammonia oxidiser community structure. This indicated resilience of these communities to change. A final study was conducted examining the effects of application of anaerobically digested sewage sludge to soil on beta-subgroup ammonia oxidiser community structure. Inhibition of nitrification by acetylene indicated the presence of a viable population of ammonia oxidising bacteria in continuously shaken samples of sludge. DGGE and oligonucleotide probing analysis provided evidence that sewage populations had the potential to outcompete indigenous soil populations of beta-subgroup ammonia oxidising bacteria. This was despite the fact that MPN enumeration suggested that soil populations of these bacteria were in some cases 10-fold greater than sludge populations. Evidence was also provided that suggested net nitrification to be an unreliable indicator of ammonia oxidiser activity in soil. It is possible to conclude that community structure of the beta-subgroup ammonia oxidising bacteria may be altered by application of sewage sludge to soil. The effects on community structure are likely to be influenced by both the type and level of sludge applied to soil.

The spring application of fertiliser N often fails to stimulate grass growth in improved grass pastures on peaty soils. Fertiliser utilisation efficiencies under these conditions have been found to be low, suggesting that available N is not taken up by the plant. Previous work has suggested that in this type of system, the soil microbial biomass may function as a strong sink for fertiliser N and therefore limit plant growth in the Spring. A series of laboratory based experiments utilising reconstituted and intact cores, and homogenised peat, was set up to identify the factors controlling the competition between N uptake by plants and N immobilisation by soil microorganisms following the addition of fertiliser N to peat. Microbial biomass N concentrations were determined in order to quantify the amount of N present in the microbial pool. The use of 15N labelled fertilisers and selective biocides provides a powerful tool with which to characterise the microbial population responsible for the immobilisation of N under these conditions. Improvement of a grass pasture at Sletill Hill has resulted in the formation of a distinct layer comprised of partially decomposed roots, underneath the surface vegetation and it was within this layer, that microbial immobilisation of fertiliser N was found to occur. Approximately 30% of applied N (equivalent to ca 50 kgN ha-1) was found within the microbial biomass in this layer, 30 days after the addition of fertiliser N. Intact cores were removed from Sletill Hill and maintained under controlled abiotic conditions. Water table level and temperature were found to be important in controlling the extent of microbial immobilisation of applied N. Lowering the water table level increased the quantity of N present in plant and microbial N pools, particularly at lower temperatures (8°C). At higher temperatures (20°C), plant uptake of N tended to be less due to a restriction on plant growth caused by 'droughty' soil conditions.

Field experiments were conducted on an Alfisol in the semi-arid tropics of northern Australia to investigate nitrogen (N) cycling in the leucaena (Leucaena leucocephala) alley cropping system. This is a farming system in which maize (Zea mays L.) is grown in alleys formed by leucaena hedgerows spaced 4.5 metres apart. Mineralization of N from Ieucaena (prunings) and maize residues was studied under field conditions. Response of maize growth to addition of N fertilizer and plant residues was evaluated both in field plot and microplot experiments. The fate of fertilizer N and leucaena N was examined over four consecutive seasons. The decomposition (loss of mass) of dry, cut 15N-labelled leucaena residues differed from that of intact fresh leucaena prunings in the first cropping season although no difference was detected after one year. At the end of one cropping season, 3 months after application, 58-72% of 15N-labelled leucaena had decomposed compared to only 34-36% of fresh leucaena prunings. Similar trends occurred at 20 and 52 days after application. The extent of decomposition of fresh leucaena prunings (28-33%) was similar at two loading rates (2.4 and 4.7 t DM ha -1) by 3 months after addition. About 72% of young 15N labelled maize residues was decomposed by 3 months after addition in the presence of fresh leucaena prunings. Decomposition of 15N-labelled leucaena residues and unlabelled fresh prunings was 91% and 88% respectively 14 months after addition. After 2 years the corresponding values were 96% and 94%. When N content of the recovered residues was taken into account, the values were 95% and 94% after 14 months, and the same (97%) after 2 years. Maize yield and N uptake were significantly increased following addition of either unlabelled fresh leucaena residues or 15N-labelled thy Ieucaena residues. Application of N ferilizer produced a thither increase in the presence of the residues. The maize yield and N uptake with the 15N-labelled leucaena were not different from those with the unlabelled residues. There was a significant positive interaction between N fertilizer and leucaena prunings which increased maize production. Addition of maize residues decreased the yield and N uptake of maize compared with that obtained in the presence of N fertilizer at 40 kg N ha~1 and leucaena residues (2.4 t DM ha-1). There was a marked residual benefit of N fertilizer applied in the first season at 36 kgN hat in the presence of leucaena prunings on the second maize crop yield and N uptake, but not on the third crop. However, a significant residual benefit of leucaena prunings added in the first season was found in DM yield and N uptake of the second and third maize crop. The short-term fate of 15N applied in plant residues was examined during two separate cropping seasons. By 20 days after application of separate 15N-labelled leucaena leaves, stems and petioles, 3-9% of the added 15N could be found in maize plants, 33-49% was in surface residues, 36-48% in the 2 m soil proffle and 0.3-22% unaccounted for. In a separate experiment when leucaena components were not separated, 5% of 15N applied in leucaena residues was taken up by maize 52 days after addition, 45% was in residues, 25% was in soil and 25% was unaccounted for. Jn another experiment, maize recovered 6% of added leucaena 15N after 2 months, 39% remained in residues, 28% was in soil and 27% was not recovered. Incorporation of 15N-labelled leucaena residues in the soil did not increase recoveiy of leucaena 15N by maize compared with placement of the residues on the soil surface. By the end of one cropping season (3 months after application), 9% of added 15N was recovered by maize from 15N-labelled leucaena. There was a similar 15N recoveiy from 15N-labelled maize residues applied as mulch at 1.7 t DM ha1 together with unlabelled leucaena prunings at 2.4 t DM ha ~. In both cases, 30-32% of added 15N was detected in soil, 28% in residues, and 31-34% apparently lost. The short-term fate of fertilizer 15N was different from that of 15N added in plant residues. In a 52-day experiment, maize recovered 65-79% of fertilizer 15N applied at low rates (6.1 and 12.2 kg N ha -1) in the presence of leucaena prunings, 21-34% was present in soil, and less than 1% was not recovered. By 2 months after application, recoveiy of fertilizer 15N by maize was 41% from N fertilizer added at 80 kg N ha -1, 35% from N fertilizer at 40 kg N ha -1 in the presence of leucaena prunings, and 24% from N fertilizer at 40 kg N ha -1 in the presence of maize residues and leucaena prunings. The corresponding deficits (unaccounted-for 15N) were 37%, 38% and 47% respectively. A small but significant amount of the fertilizer 15N was present in the unlabelled leucaena residues (3%) and in the mixture of unlabelled leucaena and maize residues (7%) present on the soil surface. However, application of the plant residues did not affect recoveiy of the fertilizer 15N in soil (21-24%). When N fertilizer was applied at 40 kg N hi1 in the presence of leucaena prunings, 43% of fertilizer 15N was recovered by maize at the end of cropping season, 20% in soil, 2% in residues, and 35% unaccounted for. The long-term fate of fertilizer 15N was compared with that of leucaena 15N in an experiment over four cropping seasons. In the first season, maize tops recovered 50% of the fertilizer 15N but only 4% of the leucaena 15N. In the second, third and fourth seasons, maize (tops + roots) recovered 0.7%, 0.4% and 0.3% of the initial fertilizer 15N compared with 2.6%, 1.8% and 1.4% of the initial leucaena 15N. In the second, third and fourth seasons, recovery of the initial fertilizer 15N (12-14%) in soil was much lower than that of the initial leucaena 15N (38-40%). There was no further loss of the fertilizer 15N after the first season. However, the cumulative 15N deficit for the leucaena 1N in the first two seasons was 50%--thissuggested an additional loss of 23% since the end of the first season. There was no further loss of 15N from either residual fertilizer 15N or residual leucaena 15N in the third and fourth seasons. In conclusion, application of leucaena prunings could substantially increase maize yield and N uptake although some supplementary N fertilizer may be required to achieve maximum crop yield. Maize recovered only a small amount of added leucaena N in the first year. Most of the leucaena residue N was present in the soil and remaining residues after one season. This residue N would be gradually available for plant uptake by subsequent crops. Of course, annual additions of leucaena prunings would appreciably increase the pool of available N over time. Thus, application of leucaena prunings could substantially improve soil fertility in the long term.

Exotic plant invasions represent a significant threat to the integrity of native grasslands. Across the Northern Great Plains, grasslands invaded by smooth brome (Bromus inermis Leyss) support lower plant diversity, potentially resulting in important consequences for ecosystem function. Previous research on smooth brome has primarily focused on aboveground changes in plant communities, but there is growing evidence that the soil ecosystem can be significantly altered with invasion. The two objectives of this thesis were to examine whether smooth brome invasion alters soil nitrogen cycling, and to determine if changes in plant community diversity or productivity influence soil bacterial communities. Relationships between smooth brome and the soil ecosystem were assessed using data collected from a Festuca hallii Vasey (Piper) (plains rough fescue) grassland located near Macrorie, SK. Gross rates of nitrogen cycling and community productivity from smooth brome invaded and native grassland sites were compared to determine the potential influence of smooth brome invasion on the soil nitrogen cycle. The relationship between increasing smooth brome abundance and soil bacterial structure and composition was also studied. Gross mineralization rates and total soil nitrogen were significantly higher in smooth brome-invaded areas relative to native grassland. Bacterial and archaeal amoA, used as indicators of ammonia-oxidizer population sizes, were altered by smooth brome cover. Higher gross mineralization rates were likely due to stimulated microbial activity caused by increased litter and root production in areas invaded by smooth brome. Smooth brome decreased plant species richness through increased litter production, but had the opposite effect on bacterial communities. Bacterial communities had higher species richness and evenness in soils invaded by smooth brome, and smooth brome invasion was also associated with bacteria important for soil nitrogen cycling. As bacteria dominate microbial biomass and are important for decomposition processes, a more even bacterial community may have supported increased mineralization rates in smooth brome-invaded soils. Specifically, a more even bacterial community may have increased mineralization rates through greater resource utilization and niche partitioning. The responses observed in these studies suggest that belowground changes with smooth brome invasion have the potential to have important consequences for ecosystem processes.

The study of biogeochemical cycles and their role in ecosystem function has helped to highlight the impacts of human activities on natural processes. However, our understanding of the effects of nitrogen (N) deposition on forested ecosystems remains limited due to the variable controls on N cycling. Soils, microclimate, and vegetation can influence rates and processes of N cycling, singly or in concert at multiple scales. Understanding how these factors influence N cycling across the landscape will help to elucidate the impacts of N deposition. The objectives of this study were to characterize variation in soils, microclimate and vegetation characteristics, and N cycling and decomposition dynamics across the landscape in a region impacted by N deposition. Relationships among these factors were explored to determine the main factors influencing N cycling and decomposition. Strong differences in net N mineralization and nitrification were found between forest stands with contrasting species composition and moisture availability. Nitrate production and leaching were related to sugar maple abundance, and base cation leaching was correlated with nitrate concentrations in soil solutions. Decomposition experiments were installed to examine the effects of substrate quality, microclimate and N availability on decay rates. Nitrogen amendments for the most part did not affect decomposition rates of wood and cellulose, and mass loss rates were correlated with microclimate and forest floor characteristics. In contrast, microclimate did not seem to affect leaf litter decay rates, and the results suggest that the presence of invertebrates may influence mass loss to a greater degree than moisture or litter quality. This work highlights the large degree of variability in N processing across the landscape and suggests that differences in microclimate and species composition may help to predict the impacts of chronic N deposition on N cycling and retention.

Biogeochemical processes in the marine biosphere are important in global element cycling and greatly influence the gas composition of the Earth’s atmosphere. The nitrogen cycle is a key component of marine biogeochemical cycles. Nitrogen is an essential constituent of living organisms, but bioavailable nitrogen is often short in supply thus limiting primary production. The largest input of nitrogen to the marine environment is by N2-fixation, the transformation of inert N2 gas into bioavailable ammonium by a distinct group of microbes. Hence, N2-fixation bypasses nitrogen limitation and stimulates productivity in oligotrophic regions of the marine biosphere. Extensive blooms of N2-fixing cyanobacteria occur regularly during summer in the Baltic Sea. N2-fixation during these blooms adds several hundred kilotons of new nitrogen into the Baltic Proper, which is similar in magnitude to the annual nitrogen load by riverine discharge and more than twice the atmospheric nitrogen deposition in this area. N2-fixing cyanobacteria are therefore a critical constituent of nitrogen cycling in the Baltic Sea. In this thesis N2 fixation of common cyanobacteria in the Baltic Sea and the direct fate of newly fixed nitrogen in otherwise nitrogen-impoverished waters were investigated. Initially, the commonly used 15N-stable isotope assay for N2-fixation measurements was evaluated and optimized in terms of reliability and practicality (Paper I), and later applied for N2-fixation assessments (Paper II–IV). N2 fixation in surface waters of the Baltic Sea was restricted to large filamentous heterocystous cyanobacteria (Aphanizomenon sp., Nodularia spumigena, Dolichospermum spp.) and absent in smaller filamentous cyanobacteria such as Pseudanabaena sp., and unicellular and colonial picocyanobacteria (Paper II-III). Most of the N2-fixation in the Northern Baltic Proper was contributed by Aphanizomenon sp. due to its high abundance throughout the summer and similar rates of specific N2-fixation as Dolichospermum spp. and N. spumigena. Specific N2 fixation was substantially higher near the coast than in an offshore region (Paper II). Half of the fixed nitrogen was released as ammonium at the site near the coast and taken up by non-N2-fixing organisms including phototrophic and heterotrophic, prokaryotic and eukaryotic planktonic organisms. Newly fixed nitrogen was thereby rapidly turned-over in the nitrogen-depleted waters (Paper III). In colonies of N. spumigena even the potential for a complete nitrogen cycle condensed to a microcosm of a few millimeters could be demonstrated (Paper IV). Cyanobacterial colonies can therefore be hot-spots of nitrogen transformation processes potentially including nitrogen gain, recycling and loss processes. In conclusion, blooms of cyanobacteria are instrumental for productivity and CO2 sequestration in the Baltic Sea. These findings advance our understanding of biogeochemical cycles and ecosystem functioning in relation to cyanobacterial blooms in the Baltic Sea with relevance for both ecosystem-based management in the Baltic Sea, and N2-fixation and nitrogen cycling in the global ocean. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 2: Manuscript.</p>

Limitations in the quality of transformation rates used with water quality models such as QUAL2K have led to the need to identify methods that can effectively and accurately determine these rates. During the early stages of method development of a Utah Department of Environmental Quality (Utah DEQ) funded project seeking to identify such methods, it was determined that under certain situations external carbon amendment may be required to successfully determine denitrification rates. The focus of this study was to evaluate the potential impacts that introducing external carbon sources may have on estimating the laboratory denitrification rates resulting from these methods. Ultimately it was shown that, carbon amendment does not negatively impact the comparability of laboratory-generated rates to those that could be measured using in-situ methods. Additionally, it was determined that the Substrate Chemical Product method discussed herein should be used along with a no carbon, glucose, and sodium acetate amended treatments. After analysis of the data, the highest rate should be used if differences caused by carbon amendment can be identified.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第20416号 / 農博第2201号 / 新制||農||1047(附属図書館) / 学位論文||H29||N5037(農学部図書室) / 京都大学大学院農学研究科農学専攻 / (主査)教授 稲村 達也, 教授 白岩 立彦, 教授 廣岡 博之 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

Overapplication of manure in sensitive watersheds is an issue of increasing environmental concern due to increased nutrient loading and antibiotic release into aquatic environments. Manure is typically surface applied, leaving nutrients and antibiotics vulnerable to loss at the soil surface. Elevated nutrient and antibiotic loading into water bodies can increase the rate of eutrophication and occurrence of antibiotic resistance genes in areas of high animal agriculture production, such as the Chesapeake Bay watershed. Manure injection is a new technology that incorporates manure into the soil with minimal disturbance, and management strategies that reduce manure loss from agricultural fields could prevent the transport of nutrients and antibiotics to sensitive waterways. However, little is known about the efficacy of dry litter injection to decrease nitrogen (N) loss when compared to surface application. Also, there are no studies that determine the effects of injection on antibiotic transport and transformation after manure application. Therefore, this project focused on changes in N cycling, orchardgrass hay yield and quality, and transport and transformation of pirlimycin and cephapirin, two common antibiotics in dairy production, when manure is injected. Subsurface injection eliminated ammonia volatilization and N loss in runoff and increased soil inorganic N when compared to surface application after volatilization, incubation, and rainfall simulation studies. Although these benefits did not translate to higher yields in orchardgrass hay, protein increased when poultry litter was injected, indicating greater N uptake. Injection of dairy manure decreased losses of pirlimycin to levels of the control when compared to surface application. Although, pirlimycin had a slower degradation rate within the injection slit compared to surface application, potentially increasing the amount of time soil microbes are exposed to antibiotics. In an incubation study, pirlimycin concentrations decreased after 7 days, but concentrations increased sharply after 14 days. This indicates that conjugates formed in the liver or digestive tract of dairy cows may revert back to the parent compound after manure application. With increased retention of nutrients and antibiotics, injection could be a best management practice used to reduce the loss of these compounds to the environment while increasing the quality of crops produced. / Ph. D.