Non-point sources (NPS) of pollution are non-discernable, diffuse sources of pollution that are often difficult to localize and in turn mitigate. NPS can include stormwater runoff, agricultural/aquaculture wastes and wastes from small decentralized wastewater treatment systems, such as conventional septic systems. The mitigation of these NPS is imperative to reduce their potential detrimental effects on the water environment. This dissertation addresses novel treatment technologies for the mitigation of nutrients, particularly nitrogen, in Recirculating Aquaculture Systems (RAS) and onsite wastewater treatment systems (OWTS). The removal of trace organics limiting RAS production and water reuse were also investigated. The first question this dissertation addressed is: Can the application of a UV-TiO2 reactor reduce the concentration of off-flavor compounds in RAS? In the UV-TiO2 reactor, spray-coated TiO2 plates were placed in an aluminum reactor and exposed to UV light. The process was applied in both a full-scale sturgeon RAS and a bench-scale RAS for the degradation of Geosmin (GSM) and 2-methylisoborneol (MIB). Improved performance on the removal of GSM and MIB was observed when the UV-TiO2 was applied as a batch reactor since it allowed for a longer treatment time without the effect of constant production of the compounds in the biological treatment processes. Treatment performance of UV-TiO2 was affected by GSM and MIB concentrations and dissolved oxygen. No harmful effects were observed on other water quality parameters when the UV-TiO2 reactor was operated as a batch or side stream process. The second question this dissertation addressed is: Does the application of Tire-Sulfur Hybrid Adsorption Denitrification (T-SHAD) in RAS improve nutrient and off-flavor compound removal when compared to conventional heterotrophic denitrification? T-SHAD combines tire mulch as an adsorbent and sulfur oxidizing denitrification for the removal of NO3--N from the aquaculture waters. Adsorption studies showed the tire has significant adsorption capacity for the off-flavor compounds GSM and MIB but can be limited by contact time and, possibly, the presence of competing organic matter in RAS. The application of T-SHAD as an effluent polishing step in RAS with a high empty bed contact time (EBCT) of 720 min removed 96.6% of NO3--N and 69.6% of GSM. The application of T-SHAD within RAS as denitrification side treatment for NO3--N removal resulted in lower EBCT (185 min) that limited NO3--N removal to 21% and showed no significant removal of off-flavor compounds. The comparison between T-SHAD and a molasses fed heterotrophic upflow packed bed reactor (UPBR), showed no significant differences in N species concentrations as well as off-flavor compound removal. However, high production of SO42- resulted from sulfur oxidizing denitrification (SOD) processes was noted. Hybrid Adsorption and Biological Treatment Systems (HABiTS), is composed of two biofilters in series employing ion exchange (IX) and nitrification for removal of NH4+ and tire scrap coupled with sulfur chips and oyster shells for both adsorption and SOD of NO3-. The third question addressed in this dissertation is: What IX/adsorption media best balances both ammonium removal and cost effectiveness for application in OWTS? Adsorption isotherms performed with different media materials showed that the zeolite material, clinoptilolite, was the best medium for the nitrification stage of HABiTS due to its high IX capacity for NH4+and cost. An adsorption capacity of 11.69 mg g-1 NH4+-N when in competition with other cations present in septic tank effluents was determined by the IX model fit to the data. The cost of clinoptilolite is significantly higher than the other media materials tested. However, the high adsorption capacity would allow for low dosages that can be combined with non-adsorptive material reducing overall costs. The fourth question this dissertation addressed is: How is the BNR process within HABiTS affected by IX? Results from side-by-side biofilter studies with HABiTS and a conventional nitrification/denitrification biofilter showed that the combined IX and nitrification in HABiTS can allow for faster startup, sustain variable loading, and achieve over 80% removal of NH4+ at a hydraulic loading rate of 0.34 m3 m-2-d-1 when compared to the conventional biofilter with 73% removal. Under lower loading rates the biological treatment was enhanced and dominated the NH4+ removal processes in both columns. The addition of a denitrification stage decreased Total Inorganic Nitrogen (TIN) by 53.54% and 40.97%, for the HABiTS treatment and the control treatment, respectively, under loading rates of 0.21 m3 m-2-d-1. Further decrease of NH4+-N loading rates results in high desorption of exchanged NH4+ in the clinoptilolite, resulting in lower TIN removal efficiencies (28.7%) when compared to the conventional control treatment (62%). The final question addressed in this dissertation is: Does the proposed hybrid system enhance the removal of TIN in OWTS under transient loading conditions? Further studies with HABiTS and the conventional biofilter were performed to determine N removal performance on an hourly basis. It was found that the performance of HABiTS varies with daily and hourly loads, particularly when recovering from periods of very low loading to high loadings and vice versa. If recovering from low loading periods, IX is observed for HABiTS and the biofilter outperforms the conventional treatment in overall TIN removal. However, recovery from a high loading period results in release of NH4+-N stored in the clinoptilolite and increased production of NO3--N that could affect the performance of the denitrification stage.

Enterobactérias são comumente encontradas em simbiose com plantas, promovendo o crescimento vegetal, através da redução do nitrogênio atmosférico a amônia. Entretanto, são os principais patógenos na medicina humana e veterinária. O objetivo deste estudo foi buscar marcadores genéticos e fisiológicos que caracterizem espécies de enterobactérias isoladas de cana-de-açúcar (n=24) e de origem clínica (n=15). Foram submetidas a testes moleculares e fisiológicos como: capacidade de reduzir N2; perfil de susceptibilidade a antibióticos; atividade hemolítica; produção de substâncias promotoras de crescimento e liberação de enzimas extracelulares. Atividades metabólicas e fisiológicas altamente conservadas, sustentadas pelo ponto vista genético, permitem a distribuição destas espécies em diferentes ecossistemas, e, em condições favoráveis, esta adaptação pode contribuir para o estabelecimento de processos patogênicos ou simbióticos em plantas. Então, enterobacterias ambientais podem se tornar patogênicas, enquanto isolados clínicos podem se adaptar a condições ambientais. / Enterobacteria are commonly found in symbiosis with plants, promoting their growth by reducing atmospheric nitrogen to ammonia. However, they are also major pathogens in human and veterinary medicine. The aim of this study was to characterize physiological and genetic markers that distinguish enterobacteria species isolated from sugarcane (n= 24) and clinical sources (n= 15). The samples were characterized regarding their molecular and physiological assays as: ability to reduce N2, antibiotic susceptibility profile; screen for hemolytic activity; production of plant-growth promoting compounds and release of extracellular enzymes. Finally, highly conserved physiological and metabolic activities are supported on genetic backgrounds that allow a wide distribution along different ecosystems and, in favorable conditions, this adaptation could contribute to the establishment of a pathogenic process or symbiosis in plants. So, environmental enterobacteria isolates can become pathogenic, whereas clinical strains can adapt to environmental conditions.

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.

Groundwater and surface water contamination has been linked in the past to inadequate or failing on-site wastewater treatment and disposal systems. The on-site wastewater systems installed in coastal areas have more potential for inflicting this kind of environmental damage. This work studied the regulatory compliance and environmental protection of the four types of on-site wastewater disposal systems found on the Mississippi Gulf Coast; i.e., vegetative rock filter, subsurface drip irrigation, sand mound, and sprinkler systems, by statistical techniques. Compliance was also evaluated for groundwater samples collected from monitoring wells installed at four corners of a disposal field. This work eventually culminated in formulation of strategy for modifying the aerobic treatment prior to disposal to help reduce nitrogen loading on the discharging environment. Process modeling and simulations were performed to optimize conditions for biological nitrogen reduction in the treatment unit by efficient management of aeration. Two separate proposals were developed, such as either running the aerator unit in a low operating dissolved oxygen concentration or intermittent aeration mode.

No description available.

Diazotrophs supply legumes such as soybean (Glycine max L. Merr) with nitrogen (N) needed for protein synthesis through biological nitrogen fixation (BNF). Through BNF, these bacteria such as Bradyrhizobium that reside in soybean root nodules, convert atmospheric nitrogen (N2) into ammonia (NH3/ NH4), a form that is biologically available for use by the plants, in return for photosynthate carbon from the plant. Abiotic stresses such as drought disrupt BNF and subsequently affects soybean yield. In addition, increasing demand for soybean is leading to supplementing its growth with synthetic N fertilizer. However, fertilizer application is known for its detrimental effects on the environment causing waterways eutrophication contributing to global warming. On the other hand, diazotrophs can supply soybean with up to 90% of N need. As such, improving the understanding and exploiting the relationship between soybean and diazotrophs is key to promoting the sustainable growing of soybean. This dissertation here investigates three main questions. First, how the soybean-diazotrophs respond to changes in water such as rainfall and irrigation. Second, how changes in these bacterial diazotrophs are related to levels of BNF, and N-related soybean molecular markers. Finally, as my colleagues and I found non-diazotrophs in the nodules of some soybean plants, I was curious about the role they are playing inside the nodules in concert with the diazotrophs. The main hypotheses tested in this dissertation are that root nodule bacterial community (bacteriome) would (1) vary by plant type, (2) respond to changes in water, and (3) be related to BNF. To answer the research questions, I devised the dissertation as follows. In Chapter 2, my colleagues and I used nine commercial cultivars of soybean that vary in drought tolerance and agronomic traits. We show that soybean sometimes, but not always, harbor a consortium of non-nitrogen fixing bacteria belonging to Pseudomonadaceae and Enterobacteriaceae families. However, as expected, nodules diazotrophs rather than non-diazotrophs responded most to changes in soil water status. In chapter 3, I used a collection of 24 genotypes of soybean that vary in their ability to fix nitrogen. The results revealed that the bacteriome diazotroph alpha diversity metrics, phylogenetic richness and evenness, was correlated with changes in BNF. Moreover, few N-related molecular markers were associated with some of the bacteria. However, we have also observed a strong effect of the environment on the diazotroph driven process of BNF (i.e. 39%-75%). For chapter 4, we sequenced three of the Pseudomonas spp. strains that were subsequently recovered again from a diversity of soybean nodules in field trials. I found that one of the strains has the ability to adapt to the nodule's unique hypoxic conditions, supporting Bradyrhizobium nodulation and possibly nodule iron. The results include the draft assembly of the proposed Pseudomonas nodulensis sp. nov. as a novel species of nodule adapted bacteria belonging to the P. fluorescens complex. The results of this dissertation contribute to the basic knowledge needed to advance sustainable breeding and management of soybean. Nodule diazotrophs are sensitive to water status e.g. drought, and other experiments have shown that the nodule bacteriome is the driver of BNF. Thus, improving the understanding and exploiting the nodule bacteriome will support developing more resilient cultivars of soybean that are efficient in BNF, and tolerant of stress. Identifying and testing diazotrophs and atypical nodule bacteria will provide a platform for developing new inoculants and biofertilizers. / Doctor of Philosophy / Soybean, the top harvested crop in the USA and 4th worldwide, is an important protein input of the livestock industry and an affordable alternative protein source for human consumption. Soybean depends on Nitrogen (N), provided by bacteria helpers, diazotrophs, that reside in nodules on soybean roots, to synthesize protein. While N makes up 80% of air, it is not suitable in its breathable form for use by most living organisms. Diazotrophs, converts this N to ammonium, a form more useful by soybean, through a process called biological nitrogen fixation (BNF). Root nodules provide a special habitat to support BNF, where soybean provides the diazotrophs with carbon as an energy source in return for the fixed ammonium. BNF is sensitive to environmental stress such as drought, which in turn affects soybean yield. While synthetic fertilizer supplementation may help reduce yield loss, it contributes to global warming and water systems pollution. Understanding the associations between soybean and diazotrophs has the potential to improve the sustainable growing of soybean. In this dissertation, we first determine the changes in the soybean root nodule bacteria in response to different water treatments. We then study how the bacterial community inside the nodules change based on different rates of BNF. After that, we look for the connections between soybean-based nitrogen molecular markers and these bacteria. Finally, we take a deeper look at how some different types of bacteria can help support N fixation. Our results have revealed that soybean hosts non-nitrogen fixing bacteria, and in high abundances. These bacteria seem to be supporting soybean growth. However, the soybean-diazotroph relationship is more sensitive to changes in water. We also found variation in nodule bacterial diversity that is related to N fixation. As well, we found that these, previously undescribed, non-nitrogen fixing bacteria are capable of living inside the nodules and they could help support the diazotrophs, under certain conditions. We provide some possible explanations to how these, previously undescribed and novel, bacteria may have adapted to the nodules. These results are very useful in the development of new inoculation products that would serve as biofertilizers for soybean, thus improving the sustainability of the agriculture industry.

Many plants, especially grasses, have Nitrogen (N) as their growth-limiting nutrient. Large amounts of N fertilizer (>100 kg N ha-1) are used in managed systems to maximize crop productivity. However, the plant captures less than 50% of the (~12 million tons per year, U.S.) applied N-fertilizer. The remaining mobile N lost through leaching and denitrification accumulates in waterways and the atmosphere, respectively. Losses of fertilizers create environmental and economic concerns globally and create conditions that support the invasion of exotic plants in the natural landscapes. There is thus a need to come up with biological solutions to better manage nitrogen for plant growth and ecosystem sustainability. Microbial communities in the rhizosphere are known to potentially have beneficial effects on plant growth. Diazotrophs, for example, are bacteria that can convert the atmospheric nitrogen to ammonia, a process called 'nitrogen fixation.' Utilizing the natural process of associative nitrogen fixation to support most of the plant's N needs would substantially reduce fertilizer use and thus reduce production and environmental costs. The goal of this dissertation was to determine the structure and function of root-zone microbial communities for increasing productivity of native plants. Towards this end, we study the root-zone bacterial and fungal communities of native and exotic invasive plants. This study identifies that shifts in rhizosphere microbial communities are associated with invasion and highlights the importance of rhizosphere associated structure and function of microbes. A study of root-zone associated microbes in switchgrass (Panicum virgatum L.) - a U.S. native, warm-season, perennial, bioenergy crop indicates that high biomass yield and taller growth are associated with increased plant N-demand and supportive of bacteria with greater rates of N2-fixation in the rhizosphere. Another crucial outcome of the thesis is a better description of the core and cultivar-specific taxa that comprise the switchgrass root-zone associated microbiome. The work in this dissertation has brought us closer to designing N supply strategies by utilizing the natural microbial communities to balance the N-cycle in agroecosystems and support a sustainable environment. / Ph. D.

Data from Hampton Roads Sanitation District was used to calculate nitrification and denitrification rates for the A²/O mode (1987) and the VIP mode (1988) of operation. Nitrification and denitrification rates compared to literature values for similar sludge ages. The mean VIP nitrification rate was eight percent less compared to the A²/O mode. Denitrification varied with the amount of nitrate loading to the anoxic zone and the rate of total nitrate recycle. The amount of denitrification that occurred in each zone during the different operations was determined. Process mode variations caused different percentages in each zone. Anaerobic and anoxic denitrification was a linear function of the mass of nitrate recycled to the anoxic zone. Fifty to seventy-five percent of the denitrification took place in the aerobic basin during both process modes, but more aerobic denitrification occurred for the A²/O operation. Secondary clarifier nitrate varied inversely with the nitrate recycle similarly for both process modes. The differences in sludge production between the VIP and A²/O process could be explained by the differences in mean cell residence time. / Master of Science

Orientador: Marcelo Carvalho Minhoto Teixeira Filho / Resumo: A utilização de bactérias promotoras de crescimento de plantas (BPCPs) pode promover aumento na eficiência da fixação biológica de nitrogênio (FBN), com benefícios no desenvolvimento e produtividade de grãos de soja. Diante o exposto, objetivou-se avaliar os efeitos da coinoculação entre rizóbios e seis BPCPs (Azospirillum brasilense, Bacillus amyloliquefacens, B. licheniformis, B. pumilus, B. subtilis e Pseudomonas fluorescens), inoculadas via semente ou em jato dirigido na base da planta no estádio V3, sobre a nodulação, nutrição, acúmulo de nutrientes e produtividade de grãos de soja no Cerrado. Os experimentos foram conduzidos em sistema plantio direto na Fazenda Experimenal da Faculdade Unesp, Campus de Ilha Solteira, localizada no município de Selvíria – MS, Brasil. num Latossolo Vermelho Distrófico de textura argilosa, em cultivo irrigado. O delineamento experimental foi em blocos ao acaso com quatro repetições, dispostos em esquema fatorial 6x2+3, sendo coinoculação de rizóbios com seis BPCPs (Azospirillum brasilense, Bacillus amyloliquefacens, B. licheniformis, B. pumilus, B. subtilis e Pseudomonas fluorescens), inoculadas na semente ou em jato dirigido na base da planta no estádio V3 da soja; além da testemunha sem inoculação e N, inoculação de sementes apenas com rizóbios (Bradyrhizobium japonicum), e tratamento com adubação nitrogenada (ureia). A coinoculação da soja com as bactérias promotoras de crescimentos via semente ou em estádio V3 proporciona maior número ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The use of plant growth promoting communities may increase the biological nitrogen efficiency, with benefits in the development and productivity of soybean grains. Given the above, the objective of this study was to evaluate the effects of co-inoculation between rhizobia and six species of growth promoting bacteria (Azospirillum brasilense, Bacillus amyloliquefacens, B. licheniformis, B. pumilus, B. subtilis e Pseudomonas fluorescens), inoculated via seeds or directed jet at the base of the plant in V3 stage, on nodulation, nutrition, nutrient accumulation and yield of soybean grains at Cerrado. The experiments were conducted under no-tillage system at the Experimenal Farm belonging to Unesp-Ilha Solteira, located in Selvíria-MS, Brazil, in irrigated cultivation. The experimental design adopted was randomized blocks, with four replicates, arranged in 6x2+4 factorial outline, being co-inoculation of rhizobia with six species of growth promoting bacteria (Azospirillum brasilense, Bacillus amyloliquefacens, B. licheniformis, B. pumilus, B. subtilis e Pseudomonas fluorescens) inoculated in the seed or directed jet at the base of the plant in V3 stage in soybean, beyond the control without inoculation and nitrogen, seed inoculation only with rihzobia (Bradyrhizobium japonicum), and treatment with nitrogen fertilization (urea). The co-inoculation of soybean with growth promoting bacteria via seed or in the V3 stage provides greater numbers of nodules per plant relative to conventio... (Complete abstract click electronic access below) / Mestre

O Brasil é o segundo maior produtor mundial de soja. Novas tecnologias que possibilitem um aumento na produção desse grão, sem causar impactos no ecossistema, são de grande interesse ambiental, social e econômico. Com objetivo de desenvolver um pacote biotecnológico foram realizados quatro grupos de estudos. Em ensaios in vitro e in vivo foram selecionados, um rizóbio (Ensifer fredii) e duas bactérias promotoras de crescimento vegetal (Pseudomonas fluorescens e Paenibacillus edaphicus) para serem co-inoculadas em sementes de soja. Por meio de estudos em casa-de-vegetação foram desenvolvidas duas tecnologias aplicadas ao inoculante contendo o rizóbio ( fatores Nod e cobamamida), que foram avaliados em conjunto (pacote biotecnológico) em campo de cultivo de soja, que mostrou-se capaz de aumentar a nodulação, o desenvolvimento da planta e rendimento dos grãos dessa leguminosa. / Brazil is the worlds second largest producer of soybean. New technologies that increase the production of this grain, without causing environmental impacts, they are of great enviromental, social and economic importance. In order to develop a biotechnological package it was conducted four study groups. Using in vitro and in vivo assays were selected, one rhizobium (Ensifer fredii) and two plant growth promoters bacterias (Pseudomonas fluorescens e Paenibacillus edaphicus) to b eco-inoculated in soybean seeds. Through studies in greenhouses, two technologies applied to agricultural inoculant have been developed (Nod factors and cobamamide), which were evaluated together (biotecnhological package) in soybean crop field, wich proved capable to increase nodulation, plant growth and yield grain of this legume.