Tetracyanonickelate (K2(Ni(CN)4), TCN) is one of the most toxic organics to living organisms. In this study, Klebsiella oxytoca (K. oxytoca) SYSU-011 (a cyanide- degrading bacterium), which was isolated from the wastewater of a metal-plating plant, was shown to be able to biodegrade TCN under anaerobic conditions. Two different media (Burk and NFG) were used to grow K. oxytoca. Results indicate that higher TCN biodegradation rate was observed when Burk medium was used as the growth media for K. oxytoca. In the nitrogen source addition experiment, TCN degradation was inhibited by the addition of nitrite. In the carbon source addition experiment, TCN degradation was enhanced by the addition of glucose and fructose. These findings would be helpful in designing a practical in situ or on-site treatment system inoculated with K. oxytoca for the treatment of TCN-containing wastewater.

Univ., Diss.--München, 1999.

The cyanide-degrading bacterium Klebsiella oxytoca SYSU-011 was isolated from the waste water of a metal-plating plant. In this study, we found out that K. oxytoca was capable of utilizing tetracyanonickelate {K2[Ni(CN)4]}(TCN) as its sole nitrogen source. This organism could degrade TCN both aerobically (D.O.¡×100¢H) and anaerobically (D.O.¡×0¢H).The addition of ammonia (5 mM) in the growth medium would inhibit TCN-degrading. The TCN-degrading by-product, a greenish precipitate, was found in the spent medium and was identified as nickel cyanide [Ni(CN)2] by FT-IR spectroscopic studies. Ammonia was demonstrated as a product of the TCN-degrading process by K. oxytoca resting cells. The addition of glucose could greatly enhance the TCN-degradation. Nitrogenase was found to be the cyanide degrading enzyme in this organism. The activity of nitrogenase was inhibited by ammonia but could be induced by the addition of TCN or KCN.

L'optimisation des procédés utilisant un substrat bon marché et abondant est considéré comme un facteur affectant le prix de production du 2,3-BD. Les valeurs optimales du pH, du taux d'aération, de la vitesse d'agitation, et de la concentration en substrat (maltodextrine) pour la production de 2,3-BD à partir de maltodextrines par la souche génétiquement modifiée Klebsiella oxytoca KMS005 ont été déterminées par une méthode conventionnellefacteur par facteur ainsi que par l’utilisation de la méthode des surfaces de réponse avec un plan d’expériences de Box-Behnken. Les résultats ont montré que les valeurs optimales de pH, taux d'aération, vitesse d'agitation, et concentration en substrat (maltodextrine) ont été respectivement de 6,0, 0,8 wm, 400tr/min et 150 g/L. Les surfaces de réponses ont permis de montrer que la vitesse d'agitation était le paramètre le plus influent pour la production de 2,3-BD. La mise en oeuvre d’un procédé fed-batch a permis d’obtenir en 78 h une concentration en 2,3-BD de 88,1±0,2 g/L avec un rendement de 0,412±0,001 g/g et une productivité de 1,13±0,01 g/L/h. L’influence des conditions de micro-aération sur la croissance des microorganismes et sur la production de 2,3-BD a été étudiée. En bioréacteurs batch, le transfert d’oxygène a été caractérisé via la mesure kLa en faisant varier le débit d'air et la vitesse d'agitation. La quantité optimale d'oxygène fournie aété évaluée à 9,5 g correspondant à un kLa de 25,2 h-1. Ensuite, un Ensuite, un procédé fed-batch a été étudié avec différentes stratégies de débit d'alimentation en glucose. Les meilleurs résultats ont été obtenus pour un débit d'alimentation constant en glucose de 2 g/h après une phase batch de croissance de 48 heures, et suivie d'une phase batch finale de 40 heures. Il en est résulté une concentration finale de 2,3-BD de 74,7 g/L avec une productivité de 0,64 g/L/h et peu de sous-produits formés (environ 3 g/L d’acide succinique, acétate et éthanol). D’autre part, les résultats issus des expérimentations en bioréacteur de 2 L ont été mis en oeuvre à l’échelle pilote. La production de 2,3-BD de maltodextrine a été réalisée dans des bioréacteurs de 10, 90 et 300 L pour différentes vitessesd’agitation et un taux d'aération fixé à 0,8 vvm.). Une concentration de 2,3- BD de 53,8 g/L et un rendement de 0,40 g/g de sucre consommé en 48 hont été obtenus avec une vitesse d’agitation constante de 295 tr/min en réacteur de 10 L. Pour le réacteur de 90 L, une concentration en 2,3-BD de52,53 g/L et un rendement de 0,43 g/g de sucre consommé ont été atteints en 72 h pour une vitesse d’agitation constante à 130 tr/min. Les meilleuresconditions d’inoculation ont été obtenues pour les précultures en phase exponentielle de croissance (12 h d’incubation) et une DO550 égale à 4 avant transfert dans le bioréacteur de 90 L. Pour le réacteur de 300 L, la concentration en 2,3-BD était de 45,02 g/L pour un rendement de 0,43 g/g de sucre consommé, obtenusaprès 72 h. / An optimization process with a cheap and abundant substrate is considered one of the factors affecting the price of commercial 2,3-Butanediol (2,3-BD) production. The optimized levels of pH, aeration rate, agitation speed, and substrate concentration (maltodextrin)were optimized by a conventional method and Response Surface Methodology (RSM) with Box-Behnken design in which metabolically engineered Klebsiell oxytoca KMS005 utilized maltodextrin to produce 2,3-BD. Results revealed that pH, aeration rate, agitation speed,and maltodextrin concentration at levels of 6.0, 0.8 vvm, 400 rpm, and 150 g/L, respectively, were the optimal conditions. RSM indicated that the agitation speed was the most influential parameter when either agitation and aeration interaction or agitation and substrate concentrationinteraction played important roles for 2,3-BD production. Under interim fedbatch fermentation, 2,3-BD concentration, yield, and productivitywere obtained at 88.1±0.2 g/L, 0.412±0.001 g/g sugar supplied, and 1.13±0.01 g/L/h, respectively, within 78 h. The influence of micro-aerobicconditions on microbial growth and 2,3-BD production was also studied. In batch bioreactors, air flow rate and agitation rate characterized through kLa measurement were tested. The optimal amount of oxygen supply was evaluated at 9.5 g corresponding to a kLa of 25.2 h-1 for cell growth and 2,3-BD production. Then, a fed-batch process was investigated by different glucose feeding rate strategies. Fedbatch with a glucose feeding rate of 2 g/h starting at the end of the growth phase during 48 h, followed by a final batch phase of 40 h was found satisfactory. It resulted in a final 2,3- BD concentration of 74.7 g/L with a productivity of 0.64 g/L/h but few byproducts formed (about 3 g/L including succinate, acetate and ethanol). Validated information in the 2 L bioreactor was further applied in a larger scale production of 2,3-BD with series of bioreactors from 10, 90 and 300 L vessels. Batch experiments were conducted based on various agitation speeds with the fixedaeration rate at 0.8 vvm. As a result, 2,3-BD concentration, and yield were achieved at 53.8 g/L, and 0.40 g/g sugar supplied within 48 h, respectively, under the constant tip speed at 295 rpm using a 10 L vessel. Its concentration of 52.53 g/L and yield of 0.43 g/g sugar consumedwithin 72 h were attained under the condition of the constant tip speed at 130 rpm using a 90 L fermenter. An appropriate seed inoculum condition was found with an optical cell density (OD550) around 4 at the log phase (12 h incubation) prior to transferring of the inoculum into the 90 L fermenter. Under the constant tip speed at 70 rpm, 2,3-BD concentration and yield were obtained at 45.02 g/L and 0.43 g/g sugar consumed in the pilot scale of 300 L bioreactor after 72 h incubation.

Potassium cyanide (KCN) is an inhibitor that reduces the activity of terminal oxidases in electron transport system of Klebsiella oxytoca. Previous research verified that K. oxytoca could induce cyanide-resistant respiration pathway when cells were grown in KCN condition. To address the role of cyanide-resistant pathway in regulating the respiration of bacterium in KCN incubation, 8-hydroxyquinoline (8-HQ), an inhibitor of cyanide-resistant pathway, was added to the bacterial suspension pretreated with KCN. This experiment was devised into 4 groups as below: (1). TSB (without KCN or 8-HQ), (2). TSB + 1 mM KCN, (3). TSB + 100 £gM 8-HQ, and (4). TSB + 1 mM KCN+ 100 £gM 8-HQ. Our results show 100 £gM 8-HQ exerted it slight toxicity to bacterial growth. However, the bacterial growth was severely impaired when the cells treated with KCN and 8-HQ concurrently as evidenced by the lower oxygen uptake rate of cells in comparison with the control group (without addition of 8-HQ). Furthermore, K. oxytoca grown in growth medium containing 100 £gM 8-HQ produced more significant H2O2. Thus we suggested that cyanide-resistant respiration of K. oxytoca could protect the cells from H2O2 damage. Since cytochrome d has been implicated in having an important role in KCN degradation in the K. oxytoca, we constructed cyd- mutant to explore the possible role in KCN degradation. In this study the sequence of the genes encoding this terminal oxidase (cydAB) of K. oxytoca mutant were deduced. Results showed that cytochrome d oxidase of K. oxytoca is not a cyanide-insensitive oxidase, but playing an important role in KCN degradation.

The cyanide-degradation bacteria Klebsiella oxytoca SYSU-110 was isolated from the waste water of a metal-plating plant in southern Taiwan. K. oxytoca can utilize many nitrile compounds [including acetonitrile (100 mM), benzonitrile (1 mM), butyronitrile (100 mM), glutaronitrile (50 mM), methacrylnitrile (100 mM), phenylacetonitrile (1 mM), propionitrile (25 mM), succinonitrile (25 mM) and valeronitrile (50 mM)] as its sole nitrogen source. In this study, we found out that K. oxytoca was capable of degrading acetonitrile and propionitrile. Frome GC analysis, we recognized amide was an intermediate compound, while the carboxylic acid and ammonia were the final end-products. Therefore, we presume that K. oxytoca biodegraded nitrile compounds by two enzymes, the nitrile hydratase and amidase. We also analyzed the total cell proteins by 2-D polyacrylamide gel electrophoresis after the cells were cultured in medium containing 25mM succinonitrile. There were 23 proteins could be induced or overexpressed by nitrile and we had identified 11 by Mascot Peptide mass Fingerprint and Blast. Six proteins that can protect the cells from oxidative damage are: superoxide dismutase, glutathione s-transferase, dyp-type peroxidase, metal binding protein PsaA (that can transport metal ions into the cells), LraI, and FepA (used to transport inorganic ions into the cells). Three enzymes glutamine synthetase, methylenetetrahydrofolate reductase,¡@and dihydroxyacid dehydratase were used to synthesize amino acids. One protein was identified as ribosomal protein L9. The last identified protein is nucleoside triphosphates kinase which can convert nucleoside diphosphates to nucleoside triphosphates non-specifically. From the activity analysis, superoxide dismutase and glutathione S-transferase activities were escalated when the cells were cultured in 25mM succinonitrile, and the concentration of ROS has rise. These results suggested that succinonitrile could cause oxidative damage to the cells and induce some anti-oxidative damage proteins to protect them.

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.

Submitted by Marco Antônio de Ramos Chagas (mchagas@ufv.br) on 2017-04-12T11:44:31Z No. of bitstreams: 1 texto completo.pdf: 10511825 bytes, checksum: ff27fb15316ce502d8d27e94ab3c9b16 (MD5) / Made available in DSpace on 2017-04-12T11:44:31Z (GMT). No. of bitstreams: 1 texto completo.pdf: 10511825 bytes, checksum: ff27fb15316ce502d8d27e94ab3c9b16 (MD5) Previous issue date: 1997-03-25 / A produção de etanol a partir de soro de queijo suplementado com LB ou 0,5% de extrato de Ievedura por Klebsiella oxytoca M5A1, recombinante P2 (Kb.P2), em valores de pH controlado em 6,0; 6,5; 6,9; 7,0; e 7,3, apresentou baixo rendimento, o que indica ineficiência de estirpe Kb.P2 em utilizar a lactose do soro como substrato. A temperatura ótima para produção de enzima β-D-galactosidade foi 30°C. O pH e a temperatura de atividade ótima da enzima β-D-galactosidade foram 7,0 e 45°C, respectivamente. A enzima não apresentou estabilidade em temperatura superior a 35°C. Houve repressão catabólica de enzima, com a adição de glicose 10 minutos após a adição do indutor lPTG. A galactose adicionada em uma cultura paralela, no mesmo tempo, inibiu ligeiramente a produção de enzima. A β-D-galactosidase de Kb.P2 apresentou 37% de atividade em relação à E. coli K12, quando induzida com IPTG, indicando que Kb.P2 possui uma baixa expressão de enzima. As células rompidas com a prensa francesa ou as células permeabilizadas com tolueno apresentaram 1,3 vez mais atividade de β-D-galactosidase 30 minutos após a adição do indutor, comparado a células induzidas e não tratadas, o que indica baixa eficiência na entrada do substrato nas células. Os resultados permitem evidenciar que a fermentação de soro de queijo por Kb.P2 depende, ainda, do conhecimento e da manipulação do sistema da bactéria, visando utilizar lactose. / The fermentation of cheese whey, supplemented with LB or 0.5% yeast extract, by ethanologenic Klebsiella oxyfoca M5A1, recombinant P2 (Kb.P2), in media with the pH controlled at 6.0; 6.5; 6.9; 7.0. and 7.3, resulted in low ethanol yield. These results indicated that Kb.P2 was not expressing the Lac- operon efficiently. The optimum temperature for β-D-galactosidase production was 30°C. The best pH and temperature for β-D-galactosidase activity were 7.0 and 45°C, respectively. However, enzyme stability was reduced at temperatures higher than 35°C. The addition of glucose repressed β-D-galactosidase more than the addition of galactose. When compared to Escherichia coli K12. B-D-galactosidase induction with IPTG in Kb.P2 corresponded to only 37% of the activity observed for K12. This result showed that the expression of Lac-operon genes was weak. Cultures induced for 30 min with IPTG, and, then, treated in a French press or with toluene had 1.3 times more β-D-galactosidase activity than induced cells that were not ruptured, showing inefficient transport of the substrate into the cells. The possibility of Kb.P2 use for cheese whey fermentation depends on a better understanding of Lac-operon.

Submitted by Marco Antônio de Ramos Chagas (mchagas@ufv.br) on 2016-06-01T14:13:34Z No. of bitstreams: 1 texto completo.pdf: 11919399 bytes, checksum: 7b65e307cd59fdd0169cabc3a06ca449 (MD5) / Made available in DSpace on 2016-06-01T14:13:34Z (GMT). No. of bitstreams: 1 texto completo.pdf: 11919399 bytes, checksum: 7b65e307cd59fdd0169cabc3a06ca449 (MD5) Previous issue date: 1999-08-13 / Fundação de Amparo à Pesquisa do Estado de Minas Gerais / A presença e a distribuição de bactérias e leveduras em frutos de café, desinfestados superficialmente, foram observadas, ulilizando-se técnicas de microscopia com colorações biológicas e fluorocromo, inclusão em parafina e técnicas imunológicas. As preparações a fresco e de inclusão em parafina possibilitaram observar a existência de um gradiente de distribuição de bactérias e leveduras na polpa do café, com predomínio de leveduras em sua porção mais externa. Foram encontradas, também, bactérias nas sementes de café. A aplicação de fluoresceína diacetato, em cortes a fresco, não se mostrou eficiente, dada a dificuldade de visualização dos microrganismos in situ e a inespecificidade da reação. Os anticorpos específicos, para a bactéria Klebsiella oxytoca, foram obtidos; porém, a aplicação de imunofluorescência usando anticorpos como sonda, para detecção das bactérias,em tecidos de frutos de café, necessita de adaptações. / The presence and distribution of bacteria and yeast in coffee beans disinfected superficially were observed microscopically with biological and fluorochromic dyes, pemianent slides and immunological techniques. Fresh preparations and permanent slides permitted observation of the existence of a distribution gradient of bacteria and yeast within the coffee pulp, with a predominance of yeast in the outer portion. Bacteria were also found in the coffee seeds. Application of fluorescein diacetate in fresh cuts was not found efficient due to difficulty in visualizing the microorganisms in situ and the non specificity of the reaction. Specific antibodies for the bacteria Klebsiella oxytoca were obtained but the application of immunofluorescence using antibodies as probes for the detection of bacteria in coffee bean tissues requires further adaptations. / Dissertação antiga escaneada.