by Yung-Ho Chow. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1997. / Includes bibliographical references (leaves 162-173). / ACKNOWLEDGEMENT --- p.i / ABSTRACT --- p.ii / LIST OF TABLES --- p.iii / LIST OF FIGURES --- p.iv / ABBREVIATION --- p.vi / Chapter CHAPTER 1 --- INTRODUCTION --- p.1 / Chapter 1.1 --- Azo dyes --- p.3 / Chapter 1.2 --- Chemistry of azo dyes --- p.3 / Chapter 1.2.1 --- Synthesis of azo dyes --- p.3 / Chapter 1.2.2 --- Oxidation and reduction --- p.4 / Chapter 1.2.3 --- Dyeing --- p.4 / Chapter 1.2.4 --- Staining to biological materials --- p.5 / Chapter 1.3 --- Toxicity of azo dyes --- p.5 / Chapter 1.3.1 --- Toxicity to mammals --- p.6 / Chapter 1.3.2 --- Toxicity to microorganisms --- p.6 / Chapter 1.4 --- Degradation of azo dyes --- p.9 / Chapter 1.4.1 --- Degradation of azo dyes by mammalian system --- p.9 / Chapter 1.4.2 --- Degradation of azo dyes by fungi system --- p.10 / Chapter 1.4.3 --- Degradation of azo dyes by bacteria --- p.11 / Chapter 1.4.3.1 --- Requirement of cofactors --- p.12 / Chapter 1.4.3.2 --- Effect of oxygen --- p.13 / Chapter 1.4.3.3 --- Effect of cell permeability --- p.14 / Chapter 1.4.3.4 --- Redox potential and rate of dye degradation --- p.15 / Chapter 1.4.3.5 --- Rate of dye degradation --- p.15 / Chapter 1.4.4 --- Azo-reductase --- p.18 / Chapter 1.4.4.1 --- Microsomal azo-reductase --- p.18 / Chapter 1.4.4.2 --- Bacterial azo-reductase --- p.19 / Chapter 1.5 --- Immobilization of microorganisms --- p.19 / Chapter 1.5.1 --- Gel matrix for entrapment --- p.20 / Chapter 1.5.2 --- Effect of gel entrapment to microbial cells --- p.21 / Chapter 1.5.2.1 --- Reduced diffusion of substrates in gel --- p.22 / Chapter 1.5.2.2 --- Effects in growth patterns --- p.22 / Chapter 1.5.2.3 --- Protection of entrapped microbial cells --- p.23 / Chapter 1.5.2.4 --- Increase metabolic activities --- p.26 / Chapter 1.5.2.5 --- Reduction of water activity --- p.27 / Chapter 1.5.2.6 --- Prolongation of products formation --- p.27 / Chapter 1.6 --- Application of immobilized microorganisms in bio-remediation of azo dyes --- p.28 / Chapter 1.7 --- Purpose of study --- p.28 / Chapter CHAPTER 2 --- MATERIALS AND METHODS --- p.29 / Chapter 2.1 --- Materials --- p.31 / Chapter 2.1.1 --- Chemicals --- p.31 / Chapter 2.1.2 --- Bacteria --- p.36 / Chapter 2.1.3 --- Instruments --- p.36 / Chapter 2.1.4 --- Media --- p.37 / Chapter 2.1.4.1 --- Luria Broth medium --- p.37 / Chapter 2.1.4.2 --- Minimal medium --- p.37 / Chapter 2.2 --- Methods --- p.38 / Chapter 2.2.1 --- Culture of Pseudomonas sp. ADD16-2 --- p.38 / Chapter 2.2.2 --- Purification and characterization of azo-reductase --- p.38 / Chapter 2.2.2.1 --- Preparation of crude extract --- p.38 / Chapter 2.2.2.2 --- Purification of azo-reductase --- p.39 / Chapter 2.2.2.2a --- Preparation of SDS-polyacrylamide gel --- p.40 / Chapter 2.2.2.2b --- Sample preparation and application --- p.41 / Chapter 2.2.2.2c --- Electrophoresis condition --- p.41 / Chapter 2.2.2.2d --- Staining of gel by Commasie blue --- p.41 / Chapter 2.2.2.3 --- Measurement of azo-reductase activity --- p.41 / Chapter 2.2.2.4 --- Determination of effect of pH to azo- reductase activity --- p.42 / Chapter 2.2.3 --- Measurement of azo dye decolourization rate by whole cells of Pseudomonas sp. ADD16-2 --- p.42 / Chapter 2.2.3.1 --- Preparation of cells --- p.42 / Chapter 2.2.3.2 --- Measurement of azo dye decolourization rate --- p.43 / Chapter 2.2.4 --- Measurement of azo dye decolourization rate by crude extract of Pseudomonas sp. ADD16-2 --- p.43 / Chapter 2.2.5 --- Determination of dye degradation products by High Performance Liquid Chromatography (HPLC) --- p.46 / Chapter 2.2.6 --- Measurement of redox potential of azo dyes --- p.47 / Chapter 2.2.7 --- Determination of the effect of cell permeation agents on dye degradation --- p.48 / Chapter 2.2.8 --- Determination of cell permeability --- p.48 / Chapter 2.2.9 --- To study the effect of the presence of dye degradation products or added aromatic amines to dye degradation --- p.49 / Chapter 2.2.9.1 --- Whole cell reactions --- p.50 / Chapter 2.2.9.2 --- Crude extract or purified azo-reductase reaction --- p.50 / Chapter 2.2.10 --- Immobilization of cells by different matrix --- p.50 / Chapter 2.2.10.1 --- Preparation of cells for immobilization --- p.50 / Chapter 2.2.10.2 --- Immobilization by calcium alginate --- p.51 / Chapter 2.2.10.3 --- Immobilization by K-carrageenan --- p.51 / Chapter 2.2.10.4 --- Immobilization by polyacrylamide gel --- p.52 / Chapter 2.2.10.5 --- Immobilization by agarose gel --- p.52 / Chapter 2.2.10.6 --- Measurement of viability of immobilized cells --- p.53 / Chapter 2.2.10.7 --- Measurement of azo dye degradation rate in immobilized cell system --- p.53 / Chapter 2.2.10.8 --- Measurement of intracellular K in calcium alginate immobilized cells --- p.53 / Chapter 2.2.10.9 --- Long term batch culture of immobilized cells --- p.53 / Chapter 2.2.11 --- Determination of toxicities of azo dyes and aromatic amines --- p.54 / Chapter CHAPTER 3 --- RESULTS --- p.55 / Chapter 3.1 --- Purification of azo-reductase 、 --- p.56 / Chapter 3.2 --- Properties of azo-reductase --- p.63 / Chapter 3.3 --- Degradation of azo dyes --- p.73 / Chapter 3.3.1 --- Degradation profiles --- p.73 / Chapter 3.3.2 --- Products of dye degradation --- p.80 / Chapter 3.3.3 --- Effect of cell permeability on dye degradation rate --- p.94 / Chapter 3.3.4 --- Induction of dye degradation rate by prior dye degradation exercise or by direct addition of aromatic amines --- p.97 / Chapter 3.4 --- Effect of immobilization --- p.114 / Chapter 3.4.1 --- Effect of different immobilization matrix --- p.114 / Chapter 3.4.2 --- Toxicities of different azo dyes and aromatic amines to free and immobilized cells --- p.124 / Chapter 3.4.3 --- Effect of azo dyes and aromatic amines at high concentrations on free and on immobilized cells --- p.124 / Chapter CHAPTER 4 --- DISCUSSION --- p.145 / Chapter 4.1 --- Degradation of azo dyes by Pseudomonas sp. ADD16-2 --- p.146 / Chapter 4.2 --- Permeability of azo dyes in Pseudomonas sp. ADD 16-2 --- p.150 / Chapter 4.3 --- Induction of dye degradation rate --- p.155 / Chapter 4.4 --- Effect of immobilization --- p.159 / CONCLUSION --- p.161 / REFERENCE --- p.162 / APPENDIX --- p.174 / appendix 1 Structures of azo dyes that have similar structures to Orange G --- p.175 / appendix 2 Absorption profiles of azo dye degradation products taken at different time intervals --- p.178 / appendix 3 Effect of pre-incubation time to dye degradation rate of Orange I by Pseudomonas sp. ADD16-2 --- p.183 / appendix 4 Effect of calcium ions (0-0.2 M) to (A) dye degradation and (B) viability of cells --- p.185 / appendix 5 Effect of ATP on induction effect of Orange I on whole cells of Pseudomonas sp. ADD16-2 --- p.187 / appendix 6 Summary of azo dyes that were degraded by Pseudomonas putida AD1 cells --- p.189

by Fung King-yuen Debera. / On t.p., "2+" is superscript. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1994. / Includes bibliographical references (leaves 102-112). / Acknowledgement --- p.i / Abstract --- p.ii / Table of Content --- p.iv / Chapter 1. --- Introduction --- p.1 / Chapter 1.1 --- Literature review --- p.1 / Chapter 1.1.1 --- Problems of heavy metals in the environment --- p.1 / Chapter 1.1.2 --- Methods of removal of heavy metal from industrial effluent --- p.5 / Chapter 1.1.3 --- The properties of magnetites --- p.10 / Chapter 1.1.4 --- Role of magnetites in water treatment --- p.12 / Chapter 1.1.5 --- The advantages of using magnetites and further application of magnetites --- p.16 / Chapter 1.2 --- Objectives of the study --- p.21 / Chapter 2. --- Materials and methods --- p.23 / Chapter 2.1 --- Selection of the organisms --- p.23 / Chapter 2.2 --- Culture media and chemicals --- p.23 / Chapter 2.3 --- Growth of the bacterial cells --- p.25 / Chapter 2.4 --- Immobilization of the bacterial cells on magnetites --- p.27 / Chapter 2.4.1 --- Effects of chemical and physical factors on the immobilization of the bacterial cells on magnetites --- p.27 / Chapter 2.4.2 --- Effect of pH on the desorption of cells from magnetites --- p.28 / Chapter 2.5 --- Nickel ion uptake experiments --- p.28 / Chapter 2.6 --- Effects of operational conditions on the nickel removal capacity of the magnetite-immobilized bacterial cells --- p.29 / Chapter 2 .6.1 --- Effect of physical factors --- p.29 / Chapter 2.6.2 --- Effect of chemical factors --- p.30 / Chapter 2.7 --- Optimization of the nickel removal efficiency --- p.30 / Chapter 2.8 --- Nickel adsorption isotherm of the magnetite- immobilized cells of Enterobacter sp4-2 --- p.30 / Chapter 2.9 --- Recovery of adsorbed Ni2+ from the magnetite- immobilized cells of Enterobacter sp4-2 --- p.31 / Chapter 2.9.1 --- Multiple adsorption-desorption cycles of Ni2+ by using citrate buffer --- p.32 / Chapter 2.9.2 --- Multiple adsorption-desorption cycles of Ni2+ by using ethylenediaminetetraacetic acid (EDTA) --- p.33 / Chapter 2.10 --- Effect of acidic treatment --- p.33 / Chapter 2.10.1 --- Effect of acidic treatment on the nickel removal capacity of the magnetites and the magnetite- immobilized cells of Enterobacter sp4-2 --- p.33 / Chapter 2.10.2 --- Effect of acidic treatment on the recovery of the adsorbed Ni2+ from magnetites and the magnetite- immobilized cells Enterobacter sp4-2 --- p.34 / Chapter 2.11 --- Removal and recovery of Ni2+ from the electroplating effluent --- p.34 / Chapter 3. --- Results --- p.36 / Chapter 3.1 --- Effects of chemical and physical factors on the immobilization of the bacterial cells on magnetites --- p.36 / Chapter 3.1.1 --- Effect of pH --- p.36 / Chapter 3.1.2 --- Effect of cells to magnetites ratio --- p.36 / Chapter 3.1.3 --- Effect of temperature --- p.39 / Chapter 3.2 --- Effect of pH on the desorption of cells from magnetites --- p.39 / Chapter 3.3 --- Nickel ion uptake experiments --- p.44 / Chapter 3.4 --- Effects of operational conditions on the nickel removal capacity of the magnetite-immobilized bacterial cells --- p.44 / Chapter 3.4.1 --- Effect of reaction temperature --- p.44 / Chapter 3.4.2 --- Effect of retention time --- p.44 / Chapter 3.4.3 --- Effect of pH --- p.47 / Chapter 3.4.4 --- Effect of the presence of cations --- p.50 / Chapter 3.4.5 --- Effect of the presence of anions --- p.50 / Chapter 3.5 --- Optimization of the nickel removal efficiency --- p.55 / Chapter 3.6 --- Nickel adsorption isotherm of the magnetite- immobilized cells of Enterobacter sp4-2 --- p.55 / Chapter 3.7 --- Recovery of adsorbed Ni2+ from the magnetite- immobilized cells of Enterobacter sp4-2 --- p.59 / Chapter 3.7.1 --- Multiple adsorption-desorption cycles of Ni2+ by using citrate buffer --- p.59 / Chapter 3.7.2 --- Multiple adsorption-desorption cycles of Ni2+ by using ethylenediaminetetraacetic acid (EDTA) --- p.63 / Chapter 3.8 --- Effect of acidic treatment --- p.63 / Chapter 3.8.1 --- Effect of acidic treatment on the nickel removal capacity of the magnetites and the magnetite-immobilized cells of Enterobacter sp4-2 --- p.63 / Chapter 3.8.2 --- Effect of acidic treatment on the recovery of the adsorbed Ni2+ from the magnetites and the magnetite-immobilized cells of Enterobacter sp4-2 --- p.66 / Chapter 3.9 --- Removal and recovery of Ni2+ from the electroplating effluent --- p.69 / Chapter 4. --- Discussion --- p.72 / Chapter 4.1 --- Selection of the organisms --- p.72 / Chapter 4.2 --- Effects of chemical and physical factors on the immobilization of the bacterial cells on magnetites --- p.72 / Chapter 4.2.1 --- Effect of pH --- p.72 / Chapter 4.2.2 --- Effect of cells to magnetites ratio --- p.74 / Chapter 4.2.3 --- Effect of temperature --- p.75 / Chapter 4.2.4 --- Effect of pH on the desorption of cells from magnetites --- p.76 / Chapter 4.3 --- Nickel ion uptake experiments --- p.78 / Chapter 4.4 --- Effects of operational conditions on the nickel removal capacity of the magnetite-immobilized bacterial cells --- p.80 / Chapter 4.4.1 --- Effect of reaction temperature --- p.80 / Chapter 4.4.2 --- Effect of retention time --- p.81 / Chapter 4.4.3 --- Effect of pH --- p.82 / Chapter 4.4.4 --- Effect of the presence of cations --- p.83 / Chapter 4.4.5 --- Effect of the presence of anions --- p.84 / Chapter 4.5 --- Optimization of the nickel removal efficiency --- p.85 / Chapter 4.6 --- Nickel adsorption isotherm of the magnetite- immobilized cells of Enterobacter sp4-2 --- p.86 / Chapter 4.7 --- Recovery of adsorbed Ni2+ from the magnetite- immobilized cells of Enterobacter sp4-2 --- p.87 / Chapter 4.7.1 --- Multiple adsorption-desorption of Ni2+ --- p.89 / Chapter 4.7.2 --- Effect of acidic treatment on the nickel removal capacity and recovery --- p.91 / Chapter 4.8 --- Removal and recovery of Ni2+ from the electroplating effluent --- p.93 / Chapter 5. --- Conclusion --- p.96 / Chapter 6. --- Summary --- p.99 / Chapter 7. --- References --- p.102

Orientador : Helia Harumi Sato / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-08T02:25:10Z (GMT). No. of bitstreams: 1 Kawaguti_HaroldoYukio_D.pdf: 24058276 bytes, checksum: a6cb1016d8d9d61e1418acf5a2867097 (MD5) Previous issue date: 2007 / Resumo: A isomaltulose é um dissacarídeo redutor, isômero da sacarose, que possui um sabor adocicado suave e propriedades físicas e sensoriais muito similares, que tem sido considerado um substituto promissor da sacarose na indústria de alimentos, devido a algumas características como baixo potencial cariogênico e baixo índice glicêmico, promoção do crescimento de bifidobactérias benéficas da microbiota intestinal, e por apresentar maior estabilidade em relação à sacarose em alimentos e bebidas acidificados, além de poder ser convertido para isomalte, um açúcar álcool dietético e não cariogênico aplicado na indústria de alimentos e farmacêutica. Os objetivos deste trabalho foram otimizar um meio de cultivo, de menor custo, para a produção da enzima glicosiltransferase pela linhagem Erwinia sp. D12 e estudar a produção de isomaltulose a partir de sacarose utilizando-se células livres e células imobilizadas em alginato de cálcio. Na otimização do meio de cultivo, em frascos sob agitação, a máxima atividade obtida foi de 12,4 UA de glicosiltransferase/mL de meio de cultivo após 8 horas de fermentação a 30ºC, em meio composto de 150 g/L de melaço de cana-de-açúcar, 20 g/L de água de maceração de milho- Milhocina®, 15 g/L de extrato de levedura Prodex Lac SDÒ, e pH ajustado a 7,5. No estudo da produção de glicosiltransferase, em fermentador de 6,6 litros, utilizando-se o meio de cultivo otimizado foi obtida máxima atividade de 22,5 UA de glicosiltransferase/mL de meio de cultivo, após 8 horas de fermentação a 27oC. No estudo da produção de isomaltulose por células íntegras imobilizadas de Erwinia sp. D12 em alginato de cálcio foi verificado que o tratamento dos grânulos de células imobilizadas com 0,06% de glutaraldeído, promoveu uma maior taxa de conversão, sendo obtido cerca de 72,3% de isomaltulose, após 12 horas de incubação em frascos sob agitação a 30ºC. As células íntegras imobilizadas e tratadas com 0,06% de glutaraldeído, em colunas de leito empacotado, apresentaram maior estabilidade do que àquelas imobilizadas sem tratamento com o aditivo, e mantiveram a conversão de sacarose em isomaltulose entre 50-60% por 10 dias, a partir de solução de sacarose 35% e fluxo de 0,56 mL/min a 30ºC. Foram estudados diferentes tratamentos para a preparação de células íntegras, células lisadas e extrato enzimático bruto imobilizados em alginato de cálcio. Os métodos que mostraram melhores resultados, em processo em batelada, foi o extrato enzimático bruto imobilizado em alginato de cálcio (EEI), em que foram obtidas taxas de conversão entre 59,7% e 63,3%; e células lisadas por sonicação e imobilizadas (CSI), com taxas de conversão entre 47,6% e 62,3%. A coluna de leito empacotado contendo grânulos de células lisadas imobilizadas (CSI) apresentou maior estabilidade do que a coluna contendo os grânulos de extrato enzimático bruto imobilizado (EEI). A coluna de leito empacotado de CSI converteu 53-59% de sacarose em isomaltulose durante sete dias, posteriormente houve queda lenta e gradual da conversão não havendo mais transformação em isomaltulose após 21 dias. No estudo da produção de isomaltulose utilizando-se células livres de Erwinia sp. D12, em processo em batelada, foi verificado o efeito do pH, da temperatura, da concentração do substrato sacarose e da concentração de massa celular em frascos agitados a 150 rpm e 30ºC. A conversão de sacarose em isomaltulose foi favorecida utilizando-se temperaturas superiores a 30ºC, pH entre 6,0-6,5, massa celular entre 7,5- 12,5% e solução de sacarose de 20-35%, obtendo-se rendimentos de isomaltulose acima de 50%. No estudo da vida útil das células livres em escala de bancada, utilizando-se frascos Erlenmeyers sob agitação, foi verificado que os parâmetros de conversão fixados a: temperatura de 35ºC, pH 6,5, concentração de substrato sacarose 35% e concentração de massa celular 10% foram os mais favoráveis, promovendo um alto rendimento em isomaltulose entre 70-75%, por 16 bateladas. Os ensaios realizados em escala piloto demonstraram a viabilidade da conversão de sacarose em isomaltulose por células livres, em que foram obtidos cerca de 114 litros de xarope com alto teor de isomaltulose (63,40%). Os cristais de isomaltulose, após clarificação e purificação do xarope convertido, apresentaram pureza de 96,5% / Abstract: Isomaltulose is a reducing disaccharide and a structural isomer of sucrose. It has a mild sweet flavour and very similar physical and sensorial properties and has been considered as a promising substitute for sucrose in the food industry, due to some of its characteristics such as a low cariogenic potential and low glycemic index and the promotion of beneficial bifid bacteria in the intestinal microbial flora. It also shows greater stability than sucrose in acidified foods and drinks, and can be converted into isomalt, a dietetic sugar alcohol with no cariogenic potential for use in the food and pharmaceutical industries. The objectives of this research were the optimisation of a culture medium with reduced costs for the production of the enzyme glucosyltransferase by the strain Erwinia sp. D12, and the study of isomaltulose production from sucrose by free and immobilized cells. In the optimisation of the culture medium in shaken flasks, the highest glucosyltransferase activity achieved was 12.4 UA/mL of culture medium after 8 hours of fermentation at 30ºC, in a medium composed of 150 g/L of sugar cane molasses, 20 g/L of corn steep liquor- Milhocina® and 15 g/L of yeast extract Prodex Lac SD®, with the pH adjusted to 7.5. In the study for glucosyltransferase production in a 6.6-liter reactor using the optimised culture medium, the highest glucosyltransferase production achieved was 22.5 UA/mL of culture medium, after 8 hours of fermentation at 27ºC. In the study for isomaltulose production using Erwinia sp. D12 cells immobilized in calcium alginate, it was shown that the addition of 0.06% glutaraldehyde during the immobilization process, promoted a higher conversion rate, reaching about 72.3% isomaltulose after 12 hours of incubation at 30°C in shaken flasks. The immobilized whole cells treated with 0.06% glutaraldehyde, used in packed-bed reactors, presented greater stability than those immobilized without the addition of the additive, and maintained the conversion of sucrose into isomaltulose between 50-60% for 10 days, using a 35% sucrose solution with a flow rate of 0.56 mL/min at 30ºC. Different treatments were studied for the preparation of whole cells, lysed cells and a crude enzyme extract immobilized in calcium alginate. The methods that showed the best results in batch processes were the crude enzyme extract immobilized in calcium alginate (EEI), where conversion rates between 59.7% and 63.3% were achieved; and immobilized lysed cells (CSI), with conversion rates between 47.6% and 62.3%. The packed bed column containing granules of immobilized lysed cells (CSI) presented greater stability than that containing granules of immobilized crude enzymatic extract (EEI). The packed bed column with CSI converted 53-59% of sucrose into isomaltulose during seven days, and then showed a gradual decline in conversion, ceasing completely after 21 days. In the study of isomaltulose production using free Erwinia sp. D12 cells in a batch process, the effects of pH, temperature, sucrose substrate concentration and cell mass concentration were determined in shaken flasks at 150 rpm and 30ºC. The following conditions favoured the conversion of sucrose into isomaltulose: temperatures above 30ºC, pH between 6.0-6.5, cell mass between 7.5-12.5% and a sucrose concentration between 20-35%; when isomaltulose yields above 50% were obtained. The half-life of the free cells was studied on a bench scale in shaken Erlenmeyers flasks and it was shown that the following fixed conversion parameters were the most favourable: temperature of 35ºC, pH 6.5, 35% sucrose substrate concentration and 10% cell mass concentration; promoting high isomaltulose yields between 70-75%, for 16 batches. The pilot scale assays demonstrated the viability of the conversion of sucrose into isomaltulose by free cells, obtaining about 114 liters of high isomaltulose syrup (63.40%). The isomaltulose crystals, after clarification and purification of the converted syrup, showed a purity of 96.5% / Doutorado / Mestre em Ciência de Alimentos

O presente trabalho teve por objetivo avaliar condições de imobilização da levedura Scheffersomyces stipitis NRRL Y-7124 pelo método do aprisionamento em gel de alginato de cálcio visando à produção de bioetanol em biorreator STR tipo cesta à partir de hidrolisado hemicelulósico de bagaço de cana-de-açúcar. Primeiramente, realizou-se as etapas de obtenção, destoxificação e caracterização do hidrolisado hemicelulósico de bagaço de cana-de-açúcar. Realizou-se em seguida um screening objetivando a seleção de um meio de cultivo adequado para a produção de etanol por esta levedura. O meio escolhido foi aquele onde se suplementou o hidrolisado com extrato de levedura (3,0 g/L), peptona (5,0 g/L), (NH4)2SO4 (2,0 g/L) e CaCl2 (0,1 g/L), onde verificou-se um fator de conversão de xilose à etanol (Yp/s) de 0,33 g/g. As condições de imobilização da levedura foram então avaliadas por planejamento fatorial 23 completo onde os fatores concentração de alginato de sódio, concentração do cloreto de cálcio e tempo de cura foram investigados. Após a análise estatística, as condições 2% de alginato de sódio, 0,1M de cloreto de cálcio e tempo de cura de 12 horas foram fixadas para as etapas seguintes. Nestas condições, avaliou-se então a influência da concentração de células à serem imobilizadas e agitação durante a fermentação a partir de um planejamento fatorial 22 completo, definindo-se assim 10 g/L de células e 100 rpm como condições ideais. Após a determinação das condições de imobilização do processo, verificou-se a estabilidade das células imobilizadas em repetidos ciclos fermentativos, para isso cinco bateladas repetidas em frascos Erlenmeyer foram realizadas. Observou-se que apesar da levedura assimilar xilose e produzir etanol em todos os ensaios, uma diminuição na eficiência da fermentação foi verificada, diminuindo em 24% da terceira para a quarta batelada, indicando assim que a levedura imobilizada era viável para o sistema de batelada repetida em até 3 ciclos nas condições estudadas. Iniciou-se então ensaios fermentativos em biorreator STR tipo cesta, realizando-se ensaios em meio sintético e em hidrolisado hemicelulósico. Observou-se reprodutibilidade nos ensaios utilizando os diferentes meios, com um valor de Yp/s de 0,21g/g e uma produtividade volumétrica de 0,15 g/L.h em ambos os ensaios. Fermentações em sistema de bateladas repetidas foram realizadas neste biorreator STR tipo cesta. Realizou-se cinco ciclos consecutivos, ao final dos quais observou-se comportamento semelhante às bateladas repetidas realizadas em frascos Erlenmeyer, na qual a partir de três ciclos a capacidade fermentativa da levedura S. stipitis diminuiu, apresentando uma produtividade volumétrica em torno de 0,16 g/L.h nas três primeiras bateladas. O gel de alginato de cálcio apresentou considerável estabilidade em sistema de bateladas repetidas indicando a possibilidade de sua utilização nesse processo. Embora os resultados obtidos neste trabalho sejam inferiores aos observados com células livres por outros autores, os mesmos demonstraram o potencial do emprego do gel de alginato de cálcio e da levedura Scheffersomyces stipitis imobilizada para a produção de bioetanol a partir de bagaço de cana-de-açúcar e contribuíram para os conhecimentos sobre a fermentação de hidrolisado hemicelulósico à etanol. / This study aimed to evaluate immobilization conditions for the yeast Scheffersomyces stipitis NRRL Y-7124 entrapped in calcium alginate gel in basket type of STR bioreactor for ethanol production from sugarcane bagasse hemicellulosic hydrolysate. For this purpose, first the steps to obtain the hydrolysate by dilute acid pretreatment, detoxification and characterization of hydrolysate was performed. Then, a screening aiming the selection of a suitable culture medium suitable for ethanol production by this yeast was carried out. The medium which showed maximum ethanol production (Yp/s, 0.33 g/g) was selected to continue the further studies. It was composed by the hydrolyzate supplemented with yeast extract (3.0 g/L), peptone (5.0 g/L), (NH4)2SO4 (2.0 g/L), CaCl2 (0.1g/L). The immobilization conditions of the yeast were then evaluated through a 23 factorial design where the three process variables i.e. concentration of sodium alginate, concentration of calcium chloride and reaction time were investigated. After statistical analysis, the optimum set of conditions (2% of sodium alginate, 0.1 M of calcium chloride and a reaction time of 12 hrs) were set to perform the following steps of this study. Subsequently, the influence of the cell concentration for immobilization and agitation during fermentation were studied considering a factorial design 22. This study revealed that 10 g/L of cells and 100 rpm were the optimum conditions for ethanol production via immobilized systems. After determination of the conditions for immobilization procedure, the stability of the immobilized cells were evaluated by repeated fermentation cycles, for that five repeated batches were performed in Erlenmeyer flasks. It was observed that despite the yeast assimilates xylose and produces ethanol in all assays, a decrease in the efficiency of the fermentation was verified from the third batch, revealing the 65% efficiency in the second batch and 39% in the fourth batch. This behavior indicates that the immobilized yeast is viable for repeated batch system only up to 3 cycles under the employed conditions. Fermentation tests in basket type STR bioreactor were carried out using synthetic medium and hemicellulosic hydrolysate as carbon source. Reproducibility was observed in assays using the different medium with ethanol yield (Yp/s) of 0.21 g/g and a volumetric productivity of 0.15 g/L.h in both assays. Fermentation assay in repeated batch system were carried out in STR basket type bioreactor. Five consecutive fermentation cycles were performed which eventually showed the similar behavior with the repeated batches conducted in Erlenmeyer flasks. The fermentative efficiency of the yeast S. stipitis was considerably good up to three cycles with a volumetric productivity of 0.16 g/L.h followed by a concomitant down fall. The calcium alginate gel showed a considerable stability in the experiments, indicating the viability of its application in repeated batch system. Although the results of this work are inferior to that observed by other authors using free cells, the calcium alginate gel potential is evident and the yeast Scheffersomyces stipitis showed to be capable to produce ethanol in immobilized form, contributing with knowledge for second generation ethanol production from sugarcane bagasse hemicellulosic hydrolysate adopting biochemical platform.

O presente trabalho teve por objetivo avaliar condições de imobilização da levedura Scheffersomyces stipitis NRRL Y-7124 pelo método do aprisionamento em gel de alginato de cálcio visando à produção de bioetanol em biorreator STR tipo cesta à partir de hidrolisado hemicelulósico de bagaço de cana-de-açúcar. Primeiramente, realizou-se as etapas de obtenção, destoxificação e caracterização do hidrolisado hemicelulósico de bagaço de cana-de-açúcar. Realizou-se em seguida um screening objetivando a seleção de um meio de cultivo adequado para a produção de etanol por esta levedura. O meio escolhido foi aquele onde se suplementou o hidrolisado com extrato de levedura (3,0 g/L), peptona (5,0 g/L), (NH4)2SO4 (2,0 g/L) e CaCl2 (0,1 g/L), onde verificou-se um fator de conversão de xilose à etanol (Yp/s) de 0,33 g/g. As condições de imobilização da levedura foram então avaliadas por planejamento fatorial 23 completo onde os fatores concentração de alginato de sódio, concentração do cloreto de cálcio e tempo de cura foram investigados. Após a análise estatística, as condições 2% de alginato de sódio, 0,1M de cloreto de cálcio e tempo de cura de 12 horas foram fixadas para as etapas seguintes. Nestas condições, avaliou-se então a influência da concentração de células à serem imobilizadas e agitação durante a fermentação a partir de um planejamento fatorial 22 completo, definindo-se assim 10 g/L de células e 100 rpm como condições ideais. Após a determinação das condições de imobilização do processo, verificou-se a estabilidade das células imobilizadas em repetidos ciclos fermentativos, para isso cinco bateladas repetidas em frascos Erlenmeyer foram realizadas. Observou-se que apesar da levedura assimilar xilose e produzir etanol em todos os ensaios, uma diminuição na eficiência da fermentação foi verificada, diminuindo em 24% da terceira para a quarta batelada, indicando assim que a levedura imobilizada era viável para o sistema de batelada repetida em até 3 ciclos nas condições estudadas. Iniciou-se então ensaios fermentativos em biorreator STR tipo cesta, realizando-se ensaios em meio sintético e em hidrolisado hemicelulósico. Observou-se reprodutibilidade nos ensaios utilizando os diferentes meios, com um valor de Yp/s de 0,21g/g e uma produtividade volumétrica de 0,15 g/L.h em ambos os ensaios. Fermentações em sistema de bateladas repetidas foram realizadas neste biorreator STR tipo cesta. Realizou-se cinco ciclos consecutivos, ao final dos quais observou-se comportamento semelhante às bateladas repetidas realizadas em frascos Erlenmeyer, na qual a partir de três ciclos a capacidade fermentativa da levedura S. stipitis diminuiu, apresentando uma produtividade volumétrica em torno de 0,16 g/L.h nas três primeiras bateladas. O gel de alginato de cálcio apresentou considerável estabilidade em sistema de bateladas repetidas indicando a possibilidade de sua utilização nesse processo. Embora os resultados obtidos neste trabalho sejam inferiores aos observados com células livres por outros autores, os mesmos demonstraram o potencial do emprego do gel de alginato de cálcio e da levedura Scheffersomyces stipitis imobilizada para a produção de bioetanol a partir de bagaço de cana-de-açúcar e contribuíram para os conhecimentos sobre a fermentação de hidrolisado hemicelulósico à etanol. / This study aimed to evaluate immobilization conditions for the yeast Scheffersomyces stipitis NRRL Y-7124 entrapped in calcium alginate gel in basket type of STR bioreactor for ethanol production from sugarcane bagasse hemicellulosic hydrolysate. For this purpose, first the steps to obtain the hydrolysate by dilute acid pretreatment, detoxification and characterization of hydrolysate was performed. Then, a screening aiming the selection of a suitable culture medium suitable for ethanol production by this yeast was carried out. The medium which showed maximum ethanol production (Yp/s, 0.33 g/g) was selected to continue the further studies. It was composed by the hydrolyzate supplemented with yeast extract (3.0 g/L), peptone (5.0 g/L), (NH4)2SO4 (2.0 g/L), CaCl2 (0.1g/L). The immobilization conditions of the yeast were then evaluated through a 23 factorial design where the three process variables i.e. concentration of sodium alginate, concentration of calcium chloride and reaction time were investigated. After statistical analysis, the optimum set of conditions (2% of sodium alginate, 0.1 M of calcium chloride and a reaction time of 12 hrs) were set to perform the following steps of this study. Subsequently, the influence of the cell concentration for immobilization and agitation during fermentation were studied considering a factorial design 22. This study revealed that 10 g/L of cells and 100 rpm were the optimum conditions for ethanol production via immobilized systems. After determination of the conditions for immobilization procedure, the stability of the immobilized cells were evaluated by repeated fermentation cycles, for that five repeated batches were performed in Erlenmeyer flasks. It was observed that despite the yeast assimilates xylose and produces ethanol in all assays, a decrease in the efficiency of the fermentation was verified from the third batch, revealing the 65% efficiency in the second batch and 39% in the fourth batch. This behavior indicates that the immobilized yeast is viable for repeated batch system only up to 3 cycles under the employed conditions. Fermentation tests in basket type STR bioreactor were carried out using synthetic medium and hemicellulosic hydrolysate as carbon source. Reproducibility was observed in assays using the different medium with ethanol yield (Yp/s) of 0.21 g/g and a volumetric productivity of 0.15 g/L.h in both assays. Fermentation assay in repeated batch system were carried out in STR basket type bioreactor. Five consecutive fermentation cycles were performed which eventually showed the similar behavior with the repeated batches conducted in Erlenmeyer flasks. The fermentative efficiency of the yeast S. stipitis was considerably good up to three cycles with a volumetric productivity of 0.16 g/L.h followed by a concomitant down fall. The calcium alginate gel showed a considerable stability in the experiments, indicating the viability of its application in repeated batch system. Although the results of this work are inferior to that observed by other authors using free cells, the calcium alginate gel potential is evident and the yeast Scheffersomyces stipitis showed to be capable to produce ethanol in immobilized form, contributing with knowledge for second generation ethanol production from sugarcane bagasse hemicellulosic hydrolysate adopting biochemical platform.

Se(VI) and Se(IV), as the two major species of selenium in water, are toxic to aquatic lives and may cause adverse health effects to humans at high levels. Biological reduction of Se(VI) is a two-stage process first from Se(VI) to Se(IV) and then from Se(IV) to Se(0) with potential accumulation of the more toxic Se(IV) due to the slower rate of the second stage. Selenium reduction was first evaluated with batch cultures of Shigella fergusonii strain TB42616 (TB) and Pantoea vagans strain EWB32213-2 (EWB) isolated in our laboratory from sludge and coal slurry sediment samples, respectively. In order to facilitate Se(VI) reduction and reduce Se(IV) accumulation, the Se(VI)-reducing strain TB was co-cultured with a Se(IV)-reducing strain EWB. Although Se(VI) reduction rate was not affected, Se(IV) reduction was significantly enhanced with low Se(IV) accumulation in the defined co-culture. Effects of culture composition as well as nitrate and arsenate on Se(VI) reduction were also investigated. A co-culture composition of 10:1 (EWB:TB) ratio was observed to achieve the best total selenium reduction. In addition, nitrate at 50 mg/L was observed to inhibit Se(IV) reduction but not Se(VI) reduction, while arsenate at 200 mg/L exhibited slight inhibition on both Se(VI) and Se(IV) reduction. Biokinetic parameters were optimized with a Monod-type kinetic model using batch pure culture data through the Robust Global Optimization Algorithm embedded in a computer package. Se(VI) reduction by the defined co-culture was then simulated and verified over a range of culture compositions and initial Se(VI) concentrations, respectively. An inter-species inhibition term was incorporated into the model to illustrate the competition for Se(IV) during Se(VI) reduction in the co-culture. The model showed a significant increase of Se(IV) accumulation with higher initial Se(VI) concentration. However, Se(IV) accumulation can be reduced with increasing population ratio of EWB to TB in the defined co-culture. The relatively high correlation coefficients suggested that the model was robust and applicable in simulating Se(VI) reduction by the defined co-culture. Since activated alumina was reported to be more effective for Se(IV) adsorption than Se(VI), the effect of biological activities on selenium removal was investigated using continuous-flow reactors packed with alum-impregnated activated alumina (AIAA) and cultured with a Se(VI)-reducing strain TB under various influent Se(VI) concentrations and hydraulic retention times (HRTs). A selenium removal efficiency of 92% was achieved in a bioreactor with initial biomass of 2.2×106 cells/g-AIAA after a 70-day operation period. Little improvement was observed by lowering the influent Se(VI) concentration from 50 to 10 mg/L while the removal efficiency was significantly enhanced by either extending the hydraulic retention time from 3.2 to 5.0 days or increasing the attached biomass during the startup. An increase in mass ratios of Se(VI) reduction by immobilized cells to adsorption by AIAA was also observed with increasing cell mass during the operation. Se(VI) reduction using continuous-flow reactors packed with strain TB immobilized Ca2+-alginate beads was investigated under various hydraulic retention times (HRT) and influent Se(VI) concentrations. A high removal efficiency up to 98.7% was achieved under an HRT of 5 days and an influent Se(VI) concentration of 400 mg/L. The results showed that the overall selenium removal was positively correlated to the bed height of the reactor and the HRT but not related to the influent Se(VI) concentration. The steady state was analyzed using a mathematical model based on Monod-type equations with four biokinetic parameters optimized including the half-velocity constants and maximum specific reduction rates. The relatively high correlation coefficients indicate that the model is robust and valid to simulate Se(VI) reduction in the gel-beads-packed continuous-flow system.

Many industrial activities discharge sulphate- and metal-containing wastewaters, including the manufacture of pulp and paper, mining and mineral processing, and petrochemical industries. Acid mine drainage (AMD) is an example of such sulphate- and metal-containing waste streams. Formation of AMD is generally the result of uncontrolled oxidation of the sulphide minerals present in the terrain in which the drainage flows with concomitant leaching of the metals. Acid mine drainage (AMD) and other sulphate- and metal-containing waste streams are amenable to active biological treatment. Anaerobic reduction of sulphate, reaction of produced sulphide with metal ions present in the waste stream, and biooxidation of excess sulphide are three main sub-processes involved in the active biotreatment of AMD. Anaerobic reduction of sulphate can be achieved in continuous stirred tank bioreactors with freely suspended cells or in immobilized cell bioreactors. The application of freely suspended cells in a continuous system dictates a high residence time to prevent cell wash-out, unless a biomass recycle stream is used. In an immobilized cell system biomass residence time becomes uncoupled from the hydraulic residence time, thus operation of bioreactor at shorter residence times becomes possible. In the present work, kinetics of anaerobic sulphate reduction was studied in continuous immobilized cell packed-bed bioreactors. Effects of carrier matrix, concentration of sulphate in the feed and sulphate volumetric loading rate on the performance of the bioreactor were investigated. The bioreactor performance, in terms of sulphate reduction rate, was dependent on the nature of the carrier matrix, specifically the total surface area which was provided by the matrix for the establishment of biofilm. Among the three tested carrier matrices, sand displayed the superior performance and the maximum volumetric reduction rate of 1.7 g/L-h was achieved at the shortest residence time of 0.5 h. This volumetric reduction rate was 40 and 8 folds faster than the volumetric reduction rates obtained with glass beads (0.04 g/L-h; residence time: 28.6 h) and foam BSP (0.2 g/L-h; residence time: 5.3 h), respectively. Further kinetic studies with sand as a carrier matrix indicated that the extent of volumetric reduction rate was dependent on the feed sulphate concentration and volumetric loading rate. At a constant feed sulphate concentration, increases in volumetric loading rate caused the volumetric reduction rate to pass through a maximum, while increases in feed sulphate concentrations from 1.0 g/L to 5.0 g/L led to lower volumetric reduction rates. The maximum volumetric reduction rates achieved in the bioreactors fed with initial sulphate concentration of 1.0, 2.5 and 5.0 g/L were 1.71, 0.82 and 0.68 g/L-h, respectively. The coupling of lactate utilization to sulphate reduction was observed in all experimental runs and the rates calculated based on the experimental data were in close agreement with calculated theoretical rates, using the stoichiometry of the reactions involved. The maximum volumetric reduction rates achieved in the immobilized cell bioreactors were significantly faster than those reported for freely suspended cells employed in the stirred tank bioreactors.

Many industrial activities discharge sulphate- and metal-containing wastewaters, including the manufacture of pulp and paper, mining and mineral processing, and petrochemical industries. Acid mine drainage (AMD) is an example of such sulphate- and metal-containing waste streams. Formation of AMD is generally the result of uncontrolled oxidation of the sulphide minerals present in the terrain in which the drainage flows with concomitant leaching of the metals. Acid mine drainage (AMD) and other sulphate- and metal-containing waste streams are amenable to active biological treatment. Anaerobic reduction of sulphate, reaction of produced sulphide with metal ions present in the waste stream, and biooxidation of excess sulphide are three main sub-processes involved in the active biotreatment of AMD. Anaerobic reduction of sulphate can be achieved in continuous stirred tank bioreactors with freely suspended cells or in immobilized cell bioreactors. The application of freely suspended cells in a continuous system dictates a high residence time to prevent cell wash-out, unless a biomass recycle stream is used. In an immobilized cell system biomass residence time becomes uncoupled from the hydraulic residence time, thus operation of bioreactor at shorter residence times becomes possible. In the present work, kinetics of anaerobic sulphate reduction was studied in continuous immobilized cell packed-bed bioreactors. Effects of carrier matrix, concentration of sulphate in the feed and sulphate volumetric loading rate on the performance of the bioreactor were investigated. The bioreactor performance, in terms of sulphate reduction rate, was dependent on the nature of the carrier matrix, specifically the total surface area which was provided by the matrix for the establishment of biofilm. Among the three tested carrier matrices, sand displayed the superior performance and the maximum volumetric reduction rate of 1.7 g/L-h was achieved at the shortest residence time of 0.5 h. This volumetric reduction rate was 40 and 8 folds faster than the volumetric reduction rates obtained with glass beads (0.04 g/L-h; residence time: 28.6 h) and foam BSP (0.2 g/L-h; residence time: 5.3 h), respectively. Further kinetic studies with sand as a carrier matrix indicated that the extent of volumetric reduction rate was dependent on the feed sulphate concentration and volumetric loading rate. At a constant feed sulphate concentration, increases in volumetric loading rate caused the volumetric reduction rate to pass through a maximum, while increases in feed sulphate concentrations from 1.0 g/L to 5.0 g/L led to lower volumetric reduction rates. The maximum volumetric reduction rates achieved in the bioreactors fed with initial sulphate concentration of 1.0, 2.5 and 5.0 g/L were 1.71, 0.82 and 0.68 g/L-h, respectively. The coupling of lactate utilization to sulphate reduction was observed in all experimental runs and the rates calculated based on the experimental data were in close agreement with calculated theoretical rates, using the stoichiometry of the reactions involved. The maximum volumetric reduction rates achieved in the immobilized cell bioreactors were significantly faster than those reported for freely suspended cells employed in the stirred tank bioreactors.

Cyanide is a known toxic chemical, the production of plastics, electroplating, tanning, chemical syntheses, etc. At short-term exposure, cyanide causes rapid breathing, tremors, and long-term exposure to cyanide cause weight loss, thyroid effects, nerve damage and death. Although chemical and physical processes can be employed to degrade cyanide and its related compounds, they are often expensive and complex to operate. A proven alternative to these processes is biological treatment, which typically relies upon the acclimation and enhancement of indigenous microorganisms. Biological degradation of cyanide has often been offered as a potentially inexpensive and environmentally friendly alternative to conventional processes. The aims of first part of study were to evaluate the biodegradability of tetracyanonickelate (TCN) by Klebsiella oxytoca under anaerobic conditions. Results reveal that TCN can be biotransformed to methane by resting cells of K. oxytoca. Results also show that TCN biodegradation was inhibited by the addition of nitrate, nitrite, or ammonia at higher concentrations (5 and 10 mM). Moreover, it was found that the optimum pH for TCN conversion by K. oxytoca was about 7.1. Results from the fermenter experiment show that TCN can be completely degraded within 14 days. K. oxytoca is capable of using TCN as the nitrogen source under anaerobic conditions. TCN could be biotransformed to non-toxic end product (methane) by resting cells of K. oxytoca. Those studies provide us insight into the characteristics of TCN conversion by K. oxytoca under anaerobic conditions. In second part of this study, the technology of immobilized cells can be applied in biological treatment to enhance the efficiency and effectiveness of biodegradation. In this study, potassium cyanide (KCN) was used as the target compound and both alginate (AL) and cellulose triacetate (CT) gels were applied for the preparation of immobilized cells. The free suspension systems reveal that the cell viability was highly affected by initial KCN concentration and pH. Results show that immobilized cell systems could tolerate a higher level of KCN concentration and wider ranges of pH. In the batch experiments, the maximum KCN removal rates using alginate and cellulose triacetate immobilized beads were 0.108 and 0.101 mM h-1 at pH 7, respectively. Results also indicate that immobilized system can support a higher biomass concentration. Complete KCN degradation was observed after the operation of four consecutive degradation experiments with the same batch of immobilized cells. This suggests that the activity of immobilized cells can be maintained and KCN can be used as the nitrogen source throughout KCN degradation experiments. The maximum KCN removal rates using AL and CT immobilized beads in continuous-column system were 0.224 and 0.192 mM h-1 with initial KCN concentration of 3 mM, respectively. In third part of this study, a microbial process for the degradation of propionitrile by K. oxytoca was studied. The free and immobilized cells of K. oxytoca were then examined for their capabilities on degrading propionitrile under various conditions. The efficiency and produced metabolic intermediates and end-products of propionitrile degradation were monitored in bath and continuous bioreactor experiments. Results reveal that up to 100 mM and 150 mM of propionitrile could be removed completely by the free and immobilized cell systems, respectively. Furthermore, AL and CT immobilized cell systems show higher removal efficiencies in wider ranges of temperature (20-40¢XC) and pH (6-8) compared with the free cell system. Results also indicate that immobilized cell system could support a higher cell density to enhance the removal efficiency of propionitrile. Immobilized cells were reused in five consecutive degradation experiments, and up to 99% of propionitrile degradation was observed in each batch test. This suggests that the activity of immobilized cells can be maintained and reused throughout different propionitrile degradation processes. A two-step pathway was observed for the biodegradation of propionitrile. Propionamide was first produced followed by propionic acid and ammonia. Results suggest that nitrile hydratase and amidase were involved in the degradation pathways of K. oxytoca. In the continuous bioreactor, both immobilized cells were capable of removing 150 mM of propionitriles completely within 16 h, and the maximum propionitriles removal rates using AL and CT immobilized beads were 5.04 and 4.98 mM h-1, respectively. Comparing the removal rates obtained from batch experiments with immobilized cells (AL and CT were 1.57 and 2.18 mM h-1 at 150 mM of propionitrile, respectively), the continuous-flow bioreactor show higher potential for practical application. These findings would be helpful in designing a practical system inoculated with K. oxytoca for the treatment of cyanide-containing wastewater.

Resumo: O presente trabalho apresenta um estudo de produção de ácido lático a partir de Lactobacillus rhamnosus imobilizado por aprisionamento em alginato de cálcio, com a utilização de soro de queijo como fonte de carbono alternativa à glicose encontrada tradicionalmente no meio MRS para bactérias láticas. A imobilização foi efetiva com 2 % de alginato, tendo eficiência de 99,99 %, e taxa de saída de células de 0,25 %, utilizando MRS como meio de cultivo. Estudou-se também o uso de fontes alternativas de nitrogênio como água de maceração de milho, Pro-Floo®, autolisado de levedura e sulfato de amônio. Os melhores resultados de produção e rendimento foram obtidos a partir da utilização de soro de queijo com as fontes de nitrogênio do MRS (extrato de levedura, peptona e extrato de carne), chegando a um rendimento (Yp/s) de 0,83, com produtividade de 0,90 g.L-1.h-1, seguido do cultivo com água de maceração de milho (AMM) e Pro-Floo®, com Yp/s de 0,72 e 0,57 respectivamente. No cultivo com água de maceração de milho a produção de ácido lático atingiu 119,04 g/L em 48 h. Com células livres, o melhor resultado de rendimento foi 0,73 quando de utilizou água de maceração de milho, com produtividade de 2,25 g.L-1.h-1 e produção de ácido lático de 107,89 g/L. Foram realizados dois ensaios utilizando uma modificação no alginato com ácido palmítico, para melhoria na viabilidade das células imobilizadas. Houve melhora no Yp/s quando se utilizou a alginato modificado com ácido palmítico passando de 0,72 para 0,79 no cultivo com AMM e de 0,57 para 0,67 quando se utilizou Pro-Floo®. Outro cultivo foi conduzido em reator de leito empacotado com imobilização em alginato recoberto de polietilenoimina, utilizando meio MRS. No reator pode-se observar a produção contínua de ácido lático até 72 horas com rendimento de 0,88 em 4 horas de cultivo atingindo uma concentração de 11,79 g/L de ácido lático. / Abstract: This work presents a study of lactic acid production by Lactobacillus rhamnosus immobilized by entrapment technique in calcium alginate, using whey as alternative carbon source, avoiding glucose use in the traditional MRS medium for lactic acid bacteria. Cell immobilization was effective using 2% of alginate, with efficiency of 99.99% and rate of cell release of 0.25 %. Alternative nitrogen sources like corn steep liquor (CSL), Pro-Floo®, autolyzed yeast and ammonium sulfate was also studied. The higher values of production and yield (Yp/s) were obtained in the cultivation with whey and the MRS nitrogen sources (yeast extract, peptone and meat extract), reaching an Yp/s of 0.83, and productivity of 0.90 g.L-1.h-1, followed by the cultivation with corn steep liquor and Pro-Floo®, with Yp/s of 0.72 and 0.57 respectively. With corn steep liquor, the lactic acid production reached 119.04 g/L in 48 h. In the culture with free cells the yield was 0.73 with corn steep liquor in 48 h, the productivity was 2.25 g.L-1.h-1 and production 107.89 g/L. Two experiments were done with a palmitolation of alginate to improve of immobilized cell viability. Increase in yield was obtained when palmitolation was employed; the yield increased from 0.72 to 0.79 in the cultivation using corn steep liquor, and from 0.57 to 0.67 when Pro-Floo® was used an alternative nitrogen source. Another experiment was realized in a packed-bed continuous reactor, with polyethyleneimine coated alginate beads, using MRS as culture medium. It was observed continuous lactic acid production until 72 h, with a yield of 0.88 in 4 hours reaching a lactic acid concentration of 11.79 g/L. / Orientador: Jonas Contiero / Coorientador: Cristina Jacques Bolner de Lima / Banca: Rubens Monti / Banca: Eliana Setsuko Kamimura / Mestre