Grandes esforços têm sido empreendidos para preservar recursos naturais e desenvolver sistemas de produção agrícola mais sustentáveis, reduzindo o uso de fertilizantes. Uma das alternativas ao uso de fertilizantes nitrogenados na agricultura são os inoculantes com bactérias diazotróficas. Os micro-organismos diazotróficos mais estudados para produção de inoculantes são os simbiontes noduladores de leguminosas. Para outros grupos de plantas, os diazotróficos mais utilizados são assimbióticos. Estudos conduzidos na Mata Atlântica e na Amazônia tem mostrado que a fixação biológica de nitrogêncio (FBN) assimbiótica associada a filosfera pode contribuir com aportes de N significativos, e que esses biomas podem abrigar uma grande diversidade de bactérias diazotróficas, sendo ambientes apropriados para prospectar diazotróficos eficientes para o uso na agricultura. O objetivo deste trabalho foi isolar novos genótipos de bactérias diazotróficas assimbióticas da filosfera e serapilheira de espécies arbóreas da floresta Amazônica, e estimar suas taxas de FBN e de síntese de ácido indolacético in vitro, bem como identificar taxonomicamente os isolados considerados mais promissores para uso biotecnológico. Bactérias diazotróficas de vida-livre foram isoladas da filosfera e serapilheira de três espécies arbóreas (Rinorea pubiflora, Amphirrhox longifolia e Chamaecrista xinguensis), e cultivadas em meio isento de N. Um total de 86 isolados foram obtidos. A taxa de fixação de nitrogênio foi estimada para todos os isolados, através do ensaio de redução de acetileno (ARA), pelo complexo nitrogenase. Para todos os isolados, também foi determinada a taxa de produção de ácido indolacético (AIA), na presença de L-triptofano. Os dados obtidos foram submetidos à análise de variância e as médias comparadas pelo teste de Scott-Knott e pelo teste de Duncan (p<0,05), através do software estatístico R. Os isolados que apresentaram as maiores taxas de redução de acetileno e de síntese de AIA foram identificados através do sequenciamento parcial do gene rRNA 16S. Cerca de 84,8% dos isolados apresentaram atividade de nitrogenase in vitro, e o teste estatístico desmonstrou não haver diferenças significativas entre as taxas de redução de acetileno entre os isolados, exceto o isolado 10RF, o qual apresentou uma taxa de redução de acetileno maior do que a dos demais isolados (p<0,05). Todos os isolados produziram AIA in vitro, e houve diferenças significativas na síntese de AIA entre os isolados, e quanto origem dos mesmos. Isolados bacterianos de Amphirrox longifolia sintetizaram mais AIA in vitro, em comparação aos isolados das outras duas espécies arbóreas. Cerca de 22% dos isolados apresentaram taxas de fixação de nitrogêncio maiores que 0.025ng N &mu;g-1 h-1, e 20% dos isolados produziram mais de 21,87 &mu;g/mL-1 de AIA. 21 isolados, considerados promissores para uso agrícola (fixadores de N e produtores de AIA), foram identificados através do sequenciamento do gene rRNA 16S, e afiliados aos gêneros Pseudomonas, Bacillus, Burkholderia, Citrobacter, Rhizobium e Stenotrophomonas. Aproximadamente 71 % dos isolados considerados promissores para promoverem o crescimento de plantas foram isolados da filosfera, indicando ser este um ambiente propício à prospecção de bactérias diazotróficas promotoras de crescimento de plantas. / Great efforts have been made to preserve natural resources and develop more sustainable agricultural production systems, reducing the use of fertilizers. One of the alternatives to the use of nitrogen fertilizers in agriculture is the use of inoculants with diazotrophic bacteria. The most studied diazotrophic microorganisms for the production of inoculants are nodule-forming symbionts of legumes. For other groups of plants, the most used diazotrophs are asymbiotic. Several studies in the Atlantic and Amazon forests have shown that the biological nitrogen fixation (BNF) associated with the phyllosphere may contribute with significant N inputs, and that these biomes can harbor a great diversity of diazotrophic bacteria, making them important environments for the prospection of efficient diazotrophs for agricultural use. The objective of this work was to isolate new genotypes of asymbiotic diazotrophic bacteria from the phyllosphere and litter of tree species of the Amazon forest, and to estimate their rates of BNF and indoleacetic acid biosynthesis in vitro, as well as to identify taxonomically the most promising isolates for biotechnological use. Asymbiotic diazotrophic bacteria were isolated from the phyllosphere and litter of three tree species (Rinorea pubiflora, Amphirrhox longifolia and Chamaecrista xinguensis), and cultivated in N-free medium. A total of 86 isolates were obtained. The nitrogen fixation rate was estimated for all isolates measuring the nitrogenase activity by the acetylene reduction assay (ARA). The rates of indoleacetic acid (IAA) production in the presence of L-tryptophan was also determined for all isolates. The data obtained were submitted to analysis of variance and the means were compared by the Scott-Knott test and the Duncan test (p<0.05) using the statistical software R. The isolates showing the highest rates of acetylene reduction and IAA biosynthesis were identified through the partial sequencing of the 16S rRNA gene. Approximately 84.8% of the isolates were positive for nitrogenase activity in vitro, and the statistical analyses showed that there were no significant differences between the acetylene reduction rates among the isolates, except the isolate 10RF, which showed a higher acetylene reduction rate than the other isolates (p<0.05). All isolates produced IAA in vitro, and there were significant differences in the biosynthesis rates among the isolates, as well as regarding the origin of the isolate. Bacterial isolates from Amphirrox longifolia synthesized higher amounts of IAA in vitro as compared to the isolates from the other two tree species. Approximately 22% of the isolates showed nitrogen fixation rates greater than 0.025 ng N &mu;g-1 h-1, and 20% of the isolates produced more than 21.87 &mu;g mL-1 of IAA. 21 isolates, considered promising for agricultural use (N-fixers and IAA producers), were identified by sequencing the 16S rRNA gene, and affiliated to the genera Pseudomonas, Bacillus, Burkholderia, Citrobacter, Rhizobium and Stenotrophomonas. Approximately 71% of the isolates considered promising to promote plant growth were isolated from the phyllosphere, suggesting that this environment is propitious to the prospection of diazotrophic plant growth promoting bacteria.

Submitted by Jean Medeiros (jeanletras@uepb.edu.br) on 2016-08-29T12:34:04Z No. of bitstreams: 1 PDF - Dalila Ribeiro Rodrigues.pdf: 1923244 bytes, checksum: f45088c1a8a811273f1ac7c37c1e3aec (MD5) / Approved for entry into archive by Secta BC (secta.csu.bc@uepb.edu.br) on 2016-08-29T17:02:09Z (GMT) No. of bitstreams: 1 PDF - Dalila Ribeiro Rodrigues.pdf: 1923244 bytes, checksum: f45088c1a8a811273f1ac7c37c1e3aec (MD5) / Made available in DSpace on 2016-08-29T17:02:09Z (GMT). No. of bitstreams: 1 PDF - Dalila Ribeiro Rodrigues.pdf: 1923244 bytes, checksum: f45088c1a8a811273f1ac7c37c1e3aec (MD5) Previous issue date: 2016-04-01 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The use of legumes inoculated with competitive and efficient nitrogen-fixing bacteria is a strategy to enhance the practices in the regeneration of degraded areas. In addition to encouraging the establishment of plants, the use of efficient strains reduces the cost of deployment and maintenance of agricultural systems, eliminating the use of nitrogenous fertilizers. But for the success of these practices is necessary to know the diversity of bacteria in the legumes nodules and evaluate their proper efficiency. The objective of this study was to evaluate the diversity and efficiency of bacterial isolates from three native species nodules Caatinga, grown in the State of Pernambuco soils deposited in the Agricultural Interest Collection of Microorganisms of Embrapa Semi -Arid. After certification purity of the stock of bacteria, all 308 isolates passed through a selection of amplicons nodC and nifH genes using a duplex-PCR approach. Among the bacteria evaluated, positive amplification were obtained for 18 isolated from mulungu, 40 to jurema-preta and 44 to angico. All these isolates were evaluated for NaCl tolerance and high temperatures "in vitro", ability to metabolize different carbon sources and enzymatic activity (only bacteria from mulungu), their “in vitro” plant growth promotion mechanisms by means of the production of indole acetic aci d (IAA) and calcium phosphate solubilization and finally tested in the greenhouse with the original 2 host plants, to determine the symbiotic efficiency of the isolates. Isolates of mulungu showed variability for enzyme activity and metabolism of carbon sources. Five isolated mulungu and jurema-preta and 6 isolates from angico were able to produce IAA in L-tryptophan supplemented medium, reaching rates above those achieved by the reference strain. As the potential of calcium phosphate solubilization for mulungu isolates, only one bacteria was able to achieve high solubilization equal to the reference strain, seven and two isolates from jurema-preta and angico, solubilized calcium phosphate above the reference strains value. For each species there bacteria were able to show tolerance to high temperatures and salinity. Isolates of angico and jurema were submitted to ARDRA using three endonucleases: MspI, HinfI and HhaI. The genetic diversity evaluation indicated that to angico a large cluster with around 40% of similarity was formed. To jurema-preta a major cluster with 65% of similarity among the isolates were achieved. In addition to high diversity of bacteria, patterns regarding its geographical occurrence were also observed. Bacteria from mulungu had the sequences of the 16S rRNA gene partially determinated. Among the 13 bacteria with good sequences, six belong to Rhizobium, six Bradyrhizobium and one belongs to Burkholderia. The Efficiency of tests, 22 isolates of mimosa and 10 mulungu were able to renodulate their original host strains and A27 from Angico and M14 and M31 can be highlighted to mulungu. / A utilização de leguminosas inoculadas com bactérias fixadoras de nitrogênio nativas eficientes e competitivas representa uma estratégia para potencializar as práticas na regeneração de áreas degradadas. Além de favorecer o estabelecimento das plantas, a utilização de estirpes eficientes reduz os custos da implantação e manutenção dos sistemas agrícolas, dispensando a utilização de fertilizantes químicos nitrogenados. Mas, para o sucesso destas práticas é preciso conhecer a diversidade das bactérias presentes nos nódulos de tais espécies de leguminosas e sua devida eficiência. O objetivo deste trabalho foi avaliar a diversidade e eficiência de bactérias oriundas de solos do Estado de Pernambuco e isoladas de nódulos de três espécies leguminosas arbóreas nativas da Caatinga, depositadas na Coleção de Microorganismos de Interesse Agrícola da Embrapa Semiárido. Após certificação da pureza do estoque de bactérias, os isolados passaram por uma seleção através da amplificação de fragmentos dos genes nodC e nifH utilizando a abordagem de duplex PCR. Apresentaram amplificação positiva 18 isolados de mulungu, 40 de jurema-preta e 44 de angico, esses isolados foram avaliados quanto a tolerância de NaCl e altas temperaturas “in vitro”, habilidade de metabolizar diferentes fontes de carbono e atividade enzimática (apenas isolados de mulungu), sua capacidade de promoção do crescimento vegetal, através da produção do Ácido indol-acético (AIA) e solubilização de fosfato de cálcio e, por fim, foram testados em casa de vegetação com as plantas hospedeiras originais, para determinação da eficiência simbiótica. Os isolados do mulungu mostraram variabilidade para atividade enzimática e para metabolização das fontes de carbono. Cinco isolados de mulungu e jurema-preta e 6 isolados de angico foram capazes de produzir AIA, na presença do L-triptofano, acima da estirpe de referência. Quanto ao potencial de solubilização de fosfato de cálcio apenas um isolado de mulungu apresentou valor igual a estirpe de referência, sete isolados de jurema-preta e dois de angico, solubilizaram fosfato de cálcio acima do valor da referência. Para cada espécie existem representantes potenciais na tolerância de altas temperaturas e altos teores salinos. Os isolados de angico e jurema-preta com características promíscuas foram submetidos ao ARDRA, utilizando três enzimas de restrição MspI, HinfI e HhaI, gerando para o angico um grande grupo com aproximadamente 40% de similaridade e para juremapreta um principal grupo com 65% de similaridade entre os isolados, constatando então a presença da diversidade dentre os isolados. Nos testes de autenticidade, 22 isolados de angico e 10 de mulungu foram capazes de renodular a estirpe hospedeira e o isolado A27, é eficiente na fixação biológica de nitrogênio, quando comparado ao controle nitrogenado. As bactérias do mulungu que se destacaram tiveram o gene 16S do rRNA parcialmente sequenciado para determinar o posicionamento taxonômico.

A adoção da rotação de culturas com leguminosas em áreas de implantação e reforma de canaviais é uma estratégia promissora para recuperação e manutenção da fertilidade do solo, e tem potencial no aproveitamento do N para as soqueiras. A análise mais detalhada do comportamento do N no solo durante o ano agrícola e dos parâmetros de produção em locais que possuem solo e clima contrastantes, permitem aprimorar o manejo da adubação nitrogenada da cana-de-açúcar. As hipóteses deste trabalho são (1) a rotação de culturas irá aumentar a disponibilidade de N no solo, a qual suprirá parte da demanda do N das soqueiras subsequentes e (2) a rotação de culturas promoverá redução da resposta das soqueiras à adubação nitrogenada. Objetivou-se avaliar o efeito da rotação de culturas nas transformações do N no solo (N mineral, N-ISNT e N e C da biomassa microbiana) e na produtividade da primeira soqueira. Os experimentos foram instalados em quatro regiões representativas de cultivo de cana-de-açúcar na região Centro Sul do Brasil: Quatá/SP (Local A); Iracemápolis/SP (Local B); Chapadão do Céu/GO (Local C) e Quirinópolis/GO (Local D). Em cada local, foi instalado um experimento em esquema de parcelas subdivididas no delineamento blocos ao acaso com quatro repetições, em áreas submetidas (com rotação) ou não (sem rotação) ao plantio de leguminosa do gênero crotalária. Após a colheita da cana planta, foram implantados os seguintes tratamentos: 60, 120 e 180 kg ha-1 de N e um controle (sem N), aplicados superficialmente sobre a palhada. Foram realizadas três amostragens para determinação do conteúdo de N mineral, N-ISNT e C e N da biomassa microbiana. Entre 150 a 180 dias após a colheita da cana-planta, foram realizadas avaliações de perfilhamento e índice SPAD. Na colheita, avaliou-se matéria seca da parte aérea, produtividade e atributos tecnológicos. Os resultados demonstraram que a mineralização dos resíduos da crotalária influenciaram a dinâmica do C e N e os reservatórios de N no perfil do solo ao longo do ciclo da primeira soqueira, com destaque para o solo de textura média (Local A). Apesar da rotação de culturas ter modificado o conteúdo de N mineral, N-ISNT, e aumentado o perfilhamento e índice SPAD no local A, somente nos solos argilosos (Locais C e D), a rotação aumentou a produtividade da soqueira. A hipótese de que a rotação de culturas reduz a demanda de fertilizantes nitrogenados não foi aceita; houve resposta à adubação nitrogenada nos quatro locais, independentemente da rotação de culturas. Porém, a rotação demonstrou potencial em aumentar a produção de biomassa com manutenção da dose de N. Esta pesquisa demonstrou a viabilidade de rotação de culturas em solos argilosos com elevado conteúdo de C e N total, com potencial de aumentar a produtividade do canavial, porém sem reduzir a demanda de adubação nitrogenada. / The adoption of crop rotation with sugarcane is a promising strategy for the recovery and maintenance of soil fertility, and it shows potential in the use of N to the sugarcane ratoon. A more detailed analysis of the transformations N in soil during the agricultural year and production parameters in sites that have contrasting soil texture and climate, allows to improve management of N fertilization for sugarcane. The hypothesis of this work are (1) crop rotation will increase soil N availability, which will satisfy part of N requirements of sugarcane ratoon and (2) crop rotation will reduce the sugarcane ratoon response to N fertilization. The objective was to evaluate the effects of sunn hemp rotation in the transformations of N in soil (mineral N content, ISNT-N and microbial biomass C and N) and sugarcane ratoon yield. The field trials were installed in four representative regions of cultivation of sugarcane in Brazil: Quatá/SP (Site A); Iracemápolis/SP (Site B); Chapadão do Céu/GO (Site C) e Quirinópolis/GO (Site D). At each site, a field trial was installed in split plot in a randomized block experimental design with four replications, in areas sowed (rotation) and not sowed (no rotation) with sunn hemp. After the harvest of cane-plant, the following treatments were applied: 60, 120 and 180 kg ha-1 of N and a control (without N) over the straw. Three sampling were performed to determine the soil inorganic N content, ISNT-N, C and N of microbial biomass. Between 150 to 180 days after the harvest of the cane-plant, tillering was counted and SPAD index was measured. At harvest of the sugarcane ratoon, biometric parameters, yield and quality parameters were evaluated. The results showed that mineralization of residues of sunn hemp influenced the dynamics of C and N and N content in the soil profile throughout the cycle of the sugarcane ratoon, with emphasis on medium-textured soil (Site A). Despite crop rotation has modified mineral N stocks, ISNT-N, and increased tillering and SPAD index in site A, crop rotation only increased the sugarcane ratoon yield on clay soils (Sites C and D). The hypothesis that crop rotation reduces N-fertilizer requirement was not accepted; there was response to N fertilization in four sites, regardless of crop rotation. However, the rotation showed potential in increase biomass production with maintenance of N rate. This research demonstrated the viability of crop rotation in clay soils with high total C and N content, with potential to increase sugarcane yield, but without reducing N-fertilizer requirement.

Made available in DSpace on 2017-07-10T17:37:23Z (GMT). No. of bitstreams: 1 Jesica_F_S_Genero.pdf: 1260489 bytes, checksum: da1efddef812f7eb843220dbd5d18a24 (MD5) Previous issue date: 2016-08-02 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The bacteria Azospirillum brasilense and Herbaspirillum seropedicae are nitrogen fixers and promoting plant growth, able to promote partnerships with the trigo. O objective of this study was to evaluate the crop wheat agronomic performance in response to inoculation with Azospirillum brasilense diazotrophs and Herbaspirillum seropedicae with or without nitrogen fertilization and quantify the number of viable cells of bacteria of the wheat plant roots. The experiment was conducted in the crop year 2014/2014 and 2015/2015. The experimental design was a randomized block in factorial 2 x 4, with four repetitions. The first factor relates to the nitrogen topdressing applications (with and without nitrogen). The second factor relates to inoculating seeds with diazotrophs [without inoculation treatment (control), strain A. brasilense, H. seropedicae strain and combination of the two strains. The combined inoculation of strains A. brasilense + H. seropedicae with nitrogen fertilization promoted an increase of 48% and 13% in grain yield, in the 2014 season and 2015 season, respectively, for the plants that were not inoculated. You can check the inoculation Herbaspirillum, showed better total amount of viable cells with the application of nitrogen. The reduction in the number of CFU of Azospirillum under nitrogen fertilization condition can point to a lower competitiveness of these individuals by exudates and nutrients / As bactérias Azospirillum brasilense e Herbaspirillum seropedicae são fixadoras de nitrogênio e promotoras de crescimento vegetal, capazes de promover associações com a cultura do trigo. O objetivo deste trabalho foi avaliar o desempenho agronômico do trigo em resposta a inoculação de bactérias diazotróficas com Azospirillum brasilense e Herbaspirillum seropedicae associada ou não à adubação nitrogenada e quantificar o número de células viáveis de bactérias das raízes de plantas de trigo. O experimento foi realizado nas safras agrícolas 2014/2014 e 2015/2015. O delineamento experimental foi de blocos casualizados em esquema fatorial 2 x 4, com 4 repetições. O primeiro fator refere-se às aplicações de nitrogênio em cobertura (com e sem nitrogênio). O segundo fator refere-se à inoculação de sementes com bactérias diazotróficas [tratamento sem inoculação (testemunha), estirpe de A. brasilense, estirpe de H. seropedicae e combinação das duas estirpes. Na ausência da inoculação, a adubação nitrogenada em cobertura promoveu um acréscimo de 18% no diâmetro do colmo. Na ausência da adubação nitrogenada em cobertura, a inoculação com H. seropedicae promoveu um acréscimo de 16% no diâmetro de colmo, e na presença da adubação nitrogenada esta bactéria promoveu um acréscimo de 24% no número de espiguetas por espiga em relação às plantas que não foram inoculadas. A inoculação combinada das estirpes A. brasilense + H. seropedicae com adubação nitrogenada em cobertura promoveu um acréscimo de 26% no número de espiguetas por espiga e o dobro do número de grãos obtidos, em relação à combinação sem adubação. A inoculação combinada das estirpes A. brasilense + H. seropedicae com adubação nitrogenada em cobertura promoveu um acréscimo de 48% e 13% na produtividade de grãos, na safra de 2014 e na safra de 2015, respectivamente, em relação as plantas que não foram inoculadas. A inoculação de H. seropedicae apresentou maior quantidade de células viáveis totais com a aplicação de nitrogênio. A redução do número de UFC de A. brasilense sob condição de adubação nitrogenada pode apontar para uma menor competitividade destes indivíduos por exsudatos e nutrientes

A adoção da rotação de culturas com leguminosas em áreas de implantação e reforma de canaviais é uma estratégia promissora para recuperação e manutenção da fertilidade do solo, e tem potencial no aproveitamento do N para as soqueiras. A análise mais detalhada do comportamento do N no solo durante o ano agrícola e dos parâmetros de produção em locais que possuem solo e clima contrastantes, permitem aprimorar o manejo da adubação nitrogenada da cana-de-açúcar. As hipóteses deste trabalho são (1) a rotação de culturas irá aumentar a disponibilidade de N no solo, a qual suprirá parte da demanda do N das soqueiras subsequentes e (2) a rotação de culturas promoverá redução da resposta das soqueiras à adubação nitrogenada. Objetivou-se avaliar o efeito da rotação de culturas nas transformações do N no solo (N mineral, N-ISNT e N e C da biomassa microbiana) e na produtividade da primeira soqueira. Os experimentos foram instalados em quatro regiões representativas de cultivo de cana-de-açúcar na região Centro Sul do Brasil: Quatá/SP (Local A); Iracemápolis/SP (Local B); Chapadão do Céu/GO (Local C) e Quirinópolis/GO (Local D). Em cada local, foi instalado um experimento em esquema de parcelas subdivididas no delineamento blocos ao acaso com quatro repetições, em áreas submetidas (com rotação) ou não (sem rotação) ao plantio de leguminosa do gênero crotalária. Após a colheita da cana planta, foram implantados os seguintes tratamentos: 60, 120 e 180 kg ha-1 de N e um controle (sem N), aplicados superficialmente sobre a palhada. Foram realizadas três amostragens para determinação do conteúdo de N mineral, N-ISNT e C e N da biomassa microbiana. Entre 150 a 180 dias após a colheita da cana-planta, foram realizadas avaliações de perfilhamento e índice SPAD. Na colheita, avaliou-se matéria seca da parte aérea, produtividade e atributos tecnológicos. Os resultados demonstraram que a mineralização dos resíduos da crotalária influenciaram a dinâmica do C e N e os reservatórios de N no perfil do solo ao longo do ciclo da primeira soqueira, com destaque para o solo de textura média (Local A). Apesar da rotação de culturas ter modificado o conteúdo de N mineral, N-ISNT, e aumentado o perfilhamento e índice SPAD no local A, somente nos solos argilosos (Locais C e D), a rotação aumentou a produtividade da soqueira. A hipótese de que a rotação de culturas reduz a demanda de fertilizantes nitrogenados não foi aceita; houve resposta à adubação nitrogenada nos quatro locais, independentemente da rotação de culturas. Porém, a rotação demonstrou potencial em aumentar a produção de biomassa com manutenção da dose de N. Esta pesquisa demonstrou a viabilidade de rotação de culturas em solos argilosos com elevado conteúdo de C e N total, com potencial de aumentar a produtividade do canavial, porém sem reduzir a demanda de adubação nitrogenada. / The adoption of crop rotation with sugarcane is a promising strategy for the recovery and maintenance of soil fertility, and it shows potential in the use of N to the sugarcane ratoon. A more detailed analysis of the transformations N in soil during the agricultural year and production parameters in sites that have contrasting soil texture and climate, allows to improve management of N fertilization for sugarcane. The hypothesis of this work are (1) crop rotation will increase soil N availability, which will satisfy part of N requirements of sugarcane ratoon and (2) crop rotation will reduce the sugarcane ratoon response to N fertilization. The objective was to evaluate the effects of sunn hemp rotation in the transformations of N in soil (mineral N content, ISNT-N and microbial biomass C and N) and sugarcane ratoon yield. The field trials were installed in four representative regions of cultivation of sugarcane in Brazil: Quatá/SP (Site A); Iracemápolis/SP (Site B); Chapadão do Céu/GO (Site C) e Quirinópolis/GO (Site D). At each site, a field trial was installed in split plot in a randomized block experimental design with four replications, in areas sowed (rotation) and not sowed (no rotation) with sunn hemp. After the harvest of cane-plant, the following treatments were applied: 60, 120 and 180 kg ha-1 of N and a control (without N) over the straw. Three sampling were performed to determine the soil inorganic N content, ISNT-N, C and N of microbial biomass. Between 150 to 180 days after the harvest of the cane-plant, tillering was counted and SPAD index was measured. At harvest of the sugarcane ratoon, biometric parameters, yield and quality parameters were evaluated. The results showed that mineralization of residues of sunn hemp influenced the dynamics of C and N and N content in the soil profile throughout the cycle of the sugarcane ratoon, with emphasis on medium-textured soil (Site A). Despite crop rotation has modified mineral N stocks, ISNT-N, and increased tillering and SPAD index in site A, crop rotation only increased the sugarcane ratoon yield on clay soils (Sites C and D). The hypothesis that crop rotation reduces N-fertilizer requirement was not accepted; there was response to N fertilization in four sites, regardless of crop rotation. However, the rotation showed potential in increase biomass production with maintenance of N rate. This research demonstrated the viability of crop rotation in clay soils with high total C and N content, with potential to increase sugarcane yield, but without reducing N-fertilizer requirement.

Approximately 25% of households in the U.S. treat their wastewater onsite using conventional onsite wastewater treatment systems (OWTS). These systems typically include a septic tank or a series of septic tanks followed by a soil absorption system. They effectively remove biochemical oxygen demand (BOD), total suspended solids (TSS), fats and grease but are not designed to remove significant amounts of nitrogen. High nitrogen loading to coastal and ground waters can be dangerous to aquatic life and public health. Hence, there is a need for advanced onsite wastewater treatment systems that can effectively remove nitrogen. Making enhanced nitrogen removal for OWTS as our primary goal, a laboratory scale Hybrid Adsorption and Biological Treatment Systems (HABiTS) was developed and upon observation of its effective nitrogen removal capacity, a pilot demonstration study with two side-by-side HABiTS, one with recirculation and one without recirculation (only forward flow) were constructed and tested at the Northwest Regional Water Reclamation Facility in Hillsborough County (Florida). HABiTS employ biological nitrogen removal and ion exchange for effective nitrogen removal. HABiTS is a two-stage process which uses nitrification for the oxidation of ammonium to nitrate and ion exchange for ammonium adsorption that helps buffer transient loading and also acts as a biofilm carrier in its stage 1 biofilter and it uses tire-sulfur hybrid adsorption denitrification (T-SHAD) in its stage 2 biofilter. These sulfur pellets help promote sulfur oxidation denitrification (SOD) and tire chips are used for nitrate adsorption during transient loading conditions, as biofilm carriers for denitrifying bacteria, and can also be used as organic carbon source to promote heterotrophic denitrification because they leach organic carbon. For this research, HABiTS without recirculation is considered as the control system and the performance of HABiTS with recirculation was tested for its ability to further enhance nitrogen removal from HABiTS. Nitrified effluent recirculation is a common strategy employed in wastewater treatment for enhanced nitrogen removal. It is the reintroduction of semi-treated wastewater to pass through an anoxic pre-treatment chamber to achieve better quality effluent. Recirculation is said to improve and consistently remove nitrogen at any hydraulic loading rate and/or nitrogen concentration. This is because of the dilution of high BOD septic tank effluent with nitrified effluent which lowers COD:TKN ratio and also improves mass transfer of substrates in the stage 1 biofilter. Recirculation also provides some pre-denitrification in the pre-treatment chamber, thereby reducing nitrogen load on the system. The HABiTS with recirculation (R) was run at 1:1 ratio of nitrified effluent recirculation rate to the influent flow rate for 50 days, and at 3:1 ratio for the remaining period of this research (200 days). The forward flow system (FF) was run under constant conditions throughout the research and comparisons between the two systems were made for different water quality parameters (pH, DO, conductivity, alkalinity, TSS, chemical oxygen demand (COD), total nitrogen (TN), total phosphorus (TP) and various nitrogen species). The final effluent ammonium results showed that the system with recirculation removed consistently > 80% NH4+-N during 1:1 and 3:1 recirculation ratios whereas the forward flow system achieved 57% removal. Further, an average of 81% total inorganic nitrogen (TIN) removal from the system influent was seen in the recirculation system’s final effluent when compared to an average of 55% in forward flow system’s final effluent. This research explains in detail, the impact of nitrified effluent recirculation on enhanced nitrogen removal in onsite systems and the results presented in this thesis proved that nitrified effluent recirculation provides promising enhanced nitrogen removal in an onsite wastewater treatment system.

Stable isotope probing (SIP) is an established technique that can be applied to identify the metabolically active micro-organisms within a microbial population. The SIP method utilises an isotopically-labelled substrate and PCR techniques to discern the members of a microbial community that incorporate the isotope into their DNA or RNA. The current literature gap around using 15N isotopes with RNA-SIP offers real potential and advantages for targeting and identifying active members from mixed communities involved in global biogeochemical nitrogen cycling. This study specifically investigated whether nitrogen based compounds can be used as substrates in RNA-SIP methodologies and whether they can in turn be used to probe mixed community environments known to be actively fixing nitrogen. The nitrogen-limited systems targeted represented an ideal opportunity to assess the suitability of 15N-RNA-SIP approaches due to their known high nitrogen fixation rates. Identifying these nitrogen-fixing bacteria could provide a better representation analysis of the community, leading to an improved prediction on how to manage and optimise the treatment performance of target waste systems and to exploit the unique bioconversion properties of these types of organisms. Initially, the project undertook methodological proof of concept by using a soluble nitrogen source, 15NH4Cl, to label the RNA of Novosphingobium nitrogenifigens and a mixed microbial community. Successful separation of the 14N- (control) and 15N-RNA was achieved for both pure and mixed communities using isopycnic caesium trifluoroacetate (CsTFA) gradients in an ultracentrifuge. The usefulness of this technique to identify active diazotrophs in real environmental samples was tested using a nitrogen-fixing community from a pulp and paper wastewater treatment system. After growing the mixed culture with 15N2 as the sole nitrogen source, the labelled RNA was extracted and fractionated using isopycnic centrifugation in CsTFA gradients. The community composition of the active nitrogen-fixing community in the 15N2 enriched fraction was analysed by establishing a 16S rRNA gene clone library containing over 200 members. These were analysed by comparison with published sequences and by phylogenetic analysis. It was found that the more isotopic label substrate incorporated, the further the buoyant density (BD) separation between 15N- and 14N-RNA. Novosphingobium nitrogenifigens gave an average BD shift of 0.03 + 0.004 g ml-1 (95.0 atom % 15N) with 15NH4Cl. For mixed communities the average BD shift was 0.02 + 0.004 g ml-1 (80.0 atom % 15N) with 15NH4Cl and 0.013 + 0.002 g ml-1 (32.6 atom % 15N) when using 15N2. Clone library analysis of 16S rRNA genes present in the enriched 15N-RNA fraction of the mixed community was shown to consist of a diverse population of bacteria as indicated by a Shannon Weaver index value of gt;2.8. Three dominant genera (Aeromonas, Pseudomonas and Bacillus) were identified by comparison with published sequences and phylogenetic analysis. Many other groups not known as archetypal nitrogen-fixing bacteria were also identified, demonstrating that 15N2-RNA-SIP provides a useful tool for the identification of important and previously unknown contributors to nitrogen fixation in a range of environments. Overall, this project has established that nitrogen based RNA-SIP is a powerful tool that can be used successfully and reproducibly with both pure and complex mixed microbial communities to study active diazotrophs in environmental samples.

On the Canadian prairies, organic production generally includes the use of annual green manure (GrM) crops, which are terminated using tillage to add nutrients and organic matter to the soil. However, in a GrM plough-down year, farmers face loss of income. As an alternative to growing traditional GrM crops, legumes can be grown alone or intercropped with cereals and harvested as green feed forage (GF) for use on-farm or for sale to other producers without depleting soil nitrogen (N) for the subsequent crop. We hypothesized that the GF system would have similar biomass, and N yield, and ultimately would return N into the soil. Furthermore, by intercropping a legume with a cereal, biological N2-fixation will be enhanced in the legume.<p> Field experiments, conducted over two years, were established at Vonda and Delisle, Saskatchewan, Canada. The experiment was conducted using a randomized complete block design (RCBD) with 16 treatments and four replicates in which field pea (<i>Pisum sativum</i> cv 40-10 silage pea), oat (<i>Avena sativa</i> L.cv AC Morgan), and triticale (X <i>Triticosecale</i> Wittmack cv Pika) were grown alone or in combination and managed as GrM or GF. Wheat and tillage fallow served as cropped and uncropped controls, respectively. The tillage fallow-control system was tilled twice in the growing season using a small tractor disc. The intercropped oat was seeded at three densities (50, 100, and 150 plants m-2) to determine whether increasing cereal density stimulated N2-fixation in the field pea.<p> The GrM system was sampled and incorporated (when the field pea was at full bloom) two weeks earlier than the GF system. Consequently, at both sites, all treatments in the GF system consistently yielded more dry matter and accumulated more N than treatments in the GrM system. At the Delisle site, where percent nitrogen derived from the atmosphere (%Ndfa) was compared, increasing cereal density did not increase N2-fixation in both management systems. However, pea in the GF system accumulated more than twice the amount of N (kg ha-1) from fixation as compared to pea in the GrM system, presumably because of the longer growth period.<p> Wheat grown following the GrM treatments produced more biomass and accumulated more N than wheat following the GF treatments. Wheat grown after the monoculture field pea as a GrM had greater yield than all treatments. As well, the GrM system returned more N to the soil than did the GF system. The extra two weeks of growth in the GF system resulted in the extraction of significant amounts of nutrients and probably moisture from the soil, which adversely affected yield and nutrient composition of the following wheat crop.<p> Although organic farmers may lose income in the plough-down year, on a longterm soil sustainability basis, the GrM system is a better option than the GF system as it returns nutrients to the soil, thus providing improved plant biomass, and N accumulation of subsequent crops. However, organic farmers growing GF for hay may benefit from the increased productivity of this system on a short-term basis. Thus, farmers pursuing GF options may need to adopt other means of sustaining soil productivity on a longer term. The tilled fallow-control system resulted in high amounts of biomass and N accumulation by the subsequent wheat crop, probably due to the fact that there were no nutrients taken up in the previous year and moisture was conserved in these treatments. However, this system may have less long-term benefits compared to the GrM regime, as no nutrients are returned through ploughing down a crop.

On the Canadian prairies, organic production generally includes the use of annual green manure (GrM) crops, which are terminated using tillage to add nutrients and organic matter to the soil. However, in a GrM plough-down year, farmers face loss of income. As an alternative to growing traditional GrM crops, legumes can be grown alone or intercropped with cereals and harvested as green feed forage (GF) for use on-farm or for sale to other producers without depleting soil nitrogen (N) for the subsequent crop. We hypothesized that the GF system would have similar biomass, and N yield, and ultimately would return N into the soil. Furthermore, by intercropping a legume with a cereal, biological N2-fixation will be enhanced in the legume.<p> Field experiments, conducted over two years, were established at Vonda and Delisle, Saskatchewan, Canada. The experiment was conducted using a randomized complete block design (RCBD) with 16 treatments and four replicates in which field pea (<i>Pisum sativum</i> cv 40-10 silage pea), oat (<i>Avena sativa</i> L.cv AC Morgan), and triticale (X <i>Triticosecale</i> Wittmack cv Pika) were grown alone or in combination and managed as GrM or GF. Wheat and tillage fallow served as cropped and uncropped controls, respectively. The tillage fallow-control system was tilled twice in the growing season using a small tractor disc. The intercropped oat was seeded at three densities (50, 100, and 150 plants m-2) to determine whether increasing cereal density stimulated N2-fixation in the field pea.<p> The GrM system was sampled and incorporated (when the field pea was at full bloom) two weeks earlier than the GF system. Consequently, at both sites, all treatments in the GF system consistently yielded more dry matter and accumulated more N than treatments in the GrM system. At the Delisle site, where percent nitrogen derived from the atmosphere (%Ndfa) was compared, increasing cereal density did not increase N2-fixation in both management systems. However, pea in the GF system accumulated more than twice the amount of N (kg ha-1) from fixation as compared to pea in the GrM system, presumably because of the longer growth period.<p> Wheat grown following the GrM treatments produced more biomass and accumulated more N than wheat following the GF treatments. Wheat grown after the monoculture field pea as a GrM had greater yield than all treatments. As well, the GrM system returned more N to the soil than did the GF system. The extra two weeks of growth in the GF system resulted in the extraction of significant amounts of nutrients and probably moisture from the soil, which adversely affected yield and nutrient composition of the following wheat crop.<p> Although organic farmers may lose income in the plough-down year, on a longterm soil sustainability basis, the GrM system is a better option than the GF system as it returns nutrients to the soil, thus providing improved plant biomass, and N accumulation of subsequent crops. However, organic farmers growing GF for hay may benefit from the increased productivity of this system on a short-term basis. Thus, farmers pursuing GF options may need to adopt other means of sustaining soil productivity on a longer term. The tilled fallow-control system resulted in high amounts of biomass and N accumulation by the subsequent wheat crop, probably due to the fact that there were no nutrients taken up in the previous year and moisture was conserved in these treatments. However, this system may have less long-term benefits compared to the GrM regime, as no nutrients are returned through ploughing down a crop.

High strength wastewater (HSWW) generated in food processing industries is characterized by high organic carbon and nitrogen content, and thus high oxygen demand. Biological nitrogen removal (BNR) is a technology widely used for the treatment of HSWW. Food processing facilities practice sanitation to keep food contact surfaces clean and pathogen-free. Benzalkonium chlorides (BACs) are cationic quaternary ammonium antimicrobial compounds (QACs) common in industrial antimicrobial formulations. BAC-bearing wastewater generated during sanitation applications in food processing facilities is combined with other wastewater streams and typically treated in BNR systems. The poor selectivity and target specificity of the antimicrobial BACs negatively impact the performance of BNR systems due to the susceptibility of BNR microbial populations to BAC. Objectives of the research were: a) assessment and quantification of the inhibitory effect of QACs on the microbial groups, which mediate BNR in HSWW treatment systems while treating QAC-bearing HSWW; b) evaluation of the degree and extent of the contribution of QAC adsorption, inhibition, and biotransformation on the fate and effect of QACs in BNR systems. A laboratory-scale, multi-stage BNR system was continuously fed with real poultry processing wastewater amended with a mixture of three benzalkonium chlorides. The nitrogen removal efficiency initially deteriorated at a BAC feed concentration of 5 mg/L due to complete inhibition of nitrification. However, the system recovered after 27 days of operation achieving high nitrogen removal efficiency, even after the feed BAC concentration was stepwise increased up to120 mg/L. Batch assays performed using the mixed liquors of the BNR system reactors, before, during, and post BAC exposure, showed that the development of BAC biotransformation capacity and the acquisition of resistance to BAC contributed to the recovery of nitrification and nitrogen removal. Kinetic analysis based on sub-models representing BNR processes showed that BAC inhibition of denitrification and nitrification is correlated with BAC liquid-phase and solid-phase concentrations, respectively. Simulations using a comprehensive mathematical BNR model developed for this research showed that BAC degradation and the level of nitrification inhibition by BAC were dynamic brought about by acclimation and enrichment of the heterotrophic and nitrifying microbial populations, respectively. The fate and effect of BACs in the BNR system were accurately described when the interactions between adsorption, inhibition, and resistance/biotransformation were considered within the conditions prevailing in each reactor. This work is the first study on the fate and effect of antimicrobial QACs in a continuous-flow, multi-stage BNR system, and the first study to quantify and report parameter values related to BAC inhibition of nitrification and denitrification. Results of this study enable the rational design and operation of BNR systems for the efficient treatment of QAC-bearing wastewater. The outcome of this research provides information presently lacking, supporting the continuous use of QACs as antimicrobial agents in food processing facilities, when and where needed, while avoiding any negative impacts on biological treatment systems and the environment.