The goal of this work was to contribute to the fed-batch process optimisation task by deriving parameters that have considerable impact on productivity as well as product quality The chosen parameters were I) the design of the glucose feed profile, II) the choice of induction strategy, with respect to the method of addition, and III) the time of the induction, with respect to the specific glucose consumption rate. The present fed-batch experiments using the lacUV5-promoter, for production of b-galactosidase, have shown that a high glucose feed rate gives a specific production rate, qp, that is twice as high, after induction, compared to a feed rate that is 2.5 times lower. The constant accumulation of lacZ-mRNA indicates that the translational capacity is initially limiting the synthesis machinery, but after four hours of maximum specific production and a corresponding drop in lacZ-mRNA production, the cultivation is likely to be transcription limited. The high feed-rate system resulted in high accumulation of β-galactosidase, corresponding to 40% of total cellular proteins. By design of feed profiles in a fed-batch process the detrimental effects of overflow metabolism, giving acetic acid formation, can be avoided. However, the results show that a one-dose addition of isopropyl-β-D-galactopyranoside (IPTG), provokes a non-growth associated production of acetic acid. This response can be alleviated by; lowering the inducer concentration (in this case to below 165 μM), by further reducing the feed rate of glucose or by using alternative induction methods. The use of a stepwise addition or a feed of IPTG thus delayed and reduced the level of acetic acid accumulation. It was also shown that a small change in the time-point of induction lead to large variability, regarding both productivity and acetic acid accumulation, in a fed-batch cultivation, In order to further investigate the protein quality two additional proteins were studied in fed-batch cultivations using high and low glucose feed. The aim was to prove the hypothesis that the feed related change in the rate of synthesis of the nascent polypeptide controls the product quality. For the two proteins: Zb-MalE (wt) and Zb-MalE31 (mutant), the transcription rate, in terms of amount of IPTG, and translation rate, in terms of changes in feed rate, influences the percentage of inclusion body formation and degradation of nascent polypeptide. The data show a higher rate of inclusion body formation for the model protein Zb-MalE31 during high feed rate cultivations, as well as at high levels of inducer. Furthermore, the rate of proteolysis was significantly higher for a high feed rate. The high feed rate thus results in a higher rate of synthesis but a lower corresponding quality, for the model proteins studied. In the present investigation of fed-batch cultivations using several different expression vectors, it was found that the central alarmone guanosine tetraphosphate (ppGpp) was formed at both high and low feed rates upon induction. It could be shown, however, that by secretion of Zb-MalE to the periplasm, the stringent response could be avoided. This might be due to the decreased burden on the host where the secretion of product further seems to make the cell able to redirect the carbon flux from overflow metabolism, since no acetic acid was produced. The secretion also demonstrates that the growth arrest could be aborted, which is otherwise gained in the PmalK production system. A novel fed-batch process based on the promoters for the universal stress proteins A and B (PuspA, PuspB) was designed to make use of these powerful promoters in an industrial production context. It was concluded that the process had to start from a high specific growth rate and induction was performed once a limiting feed started. This was done to purposely induce the stringent response and/or acetic acid accumulation since this was required for induction. In the suggested system, induction has to be performed and maintained at continuous substrate feeding, whilst avoiding exceeding the cellular capacity, since the stationary phase starvation alone did not lead to production. In conclusion, a new stress induction based production system was achieved resulting in high accumulations of product protein without any detected metabolic side effects. / <p>QC 20101008</p>

The objective of this study was to investigate the physiology and productivity in Escherichia colicultures with emphasis on the temperature-limited fed-batch (TLFB) culture. The TLFB techniquecontrols the oxygen consumption rate of the growing culture by a gradually declining temperaturefrom 37-35 °C down to 20-18 °C. The temperature regulated the DOT around a set-point (30 % airsat.), and all nutrients were in excess. Glucose was fed into the culture continuously, however, highacetate formation was avoided by keeping the glucose at a low, yet excessive, concentration. Thebiomass productivity was approximately the same in TLFB as in glucose-limited fed-batch (GLFB)cultures, since the specific growth rate and the oxygen consumption rate are limited by the oxygentransfer capacity of the reactor in both techniques.High concentrations of endotoxins were found in the medium of E. coli fed-batch cultures at lowspecific growth rates (below 0.1 h-1) and severe glucose limitation. In this thesis the TLFB techniquewas found to suppress the endotoxin release even at low specific growth rates. The repressed release of endotoxins in TLFB cultures was due to the high availability of glucose and not to the low growthrate or the lower temperature. The conclusion was drawn from comparing with the GLFB technique performed at 20 °C, which resulted in high endotoxin release.Extensive release of endotoxin, accompanied with high concentrations of soluble proteins was foundin a TLFB culture exposed to a higher energy dissipation rate, 16 kW m-3, instead of 2 kW m-3, due toa higher stirrer speed (1000 instead of 500 rpm). The hypothesis that this is a result of mechanicalstress is discussed in context with the common view that cells like E. coli, which are smaller than the Kolmogoroff’s microscale of turbulence, should not be influenced by the turbulence.TLFB cultured cells exhibited more stable cytoplasmic membranes when treated with osmotic shockas compared to the GLFB cultured cells. The concentrations of DNA and soluble proteins in the periplasmic extracts from the TLFB cultured cells were lower than from GLFB cultured cells. Inaddition, the specific productivity of periplasmic β-lactamase was higher in the TLFB cultures,suggesting that this technique could be an alternative for protein production. The solubility of apartially aggregated recombinant protein increased in the TLFB compared to the GLFB cultures.However some time after induction, in spite of the gradually declining temperature, the solublefraction decreased.For obtaining better understanding of the performance of the process and for identifying criticalparameters, a mathematical model was developed based on the growth, energy and overflowmetabolism at non-limiting nutrient conditions. The temperature-dependent maximum specific glucoseand oxygen uptake rates were determined in pH-auxostat cultures for temperatures ranging from 18 to37 °C. A dynamic simulation model of the TLFB technique was developed and the results were compared to experimental data. The simulation program was also used for sensitivity analysis of some physiological and process parameters to study the impact on biomass concentration and temperatureprofiles. An effect on the biomass concentration profile but not on the temperature profile wasobserved when changing the oxygen transfer coefficient. If the maximum specific glucose uptake ratewas altered, or if the glucose concentration was permitted to assume other values, the temperatureprofile but not the biomass concentration profile was influenced. Cell death affected both the biomassconcentration profile and the temperature profile. / QC 20100922

The present Master thesis describes the development and optimization of a fed-batch process for production of recombinant proteins in Escherichia coli BL21(DE3) in a multi-fermentor system. The system consists of six 1-liter fermentors, capable of producing 500-1500 μg/mL with present protocol. Response surface methodology (RSM) was used for multivariable optimization regarding cultivation time, pH, temperature and feed rate. Optimal protein expression conditions were found out to be 17.8 h cultivation time, 36.7 ºC, pH 6.8 and a feed rate corresponding to specific growth of 0.23 h-1, on glucose substrate. The aggregation of expressed proteins to inclusion bodies, could not be affected by the various growth conditions employed during cultivations. A study was conducted regarding growth conditions effect on phosphogluconoylation of expressed proteins. In ten fed-batch cultivations on glucose, LC/MS analysis showed a gluconoylated fraction with additional 178 Da mass, but no correlation between growth conditions and gluconoylation could be found. In two fed-batch cultivations on glycerol-feed, a lower feed rate resulted in no gluconoylation, while a higher did. An explanation would be that the lower amount of available intra-cellular carbon limits formation of gluconoylation precursors.

This study focuses on the molasses based complex medium design for benzaldehyde lyase production by recombinant E. coli BL21 and development of a feeding strategy based on the designed complex medium. For this purpose, firstly, the effects of molasses were investigated in laboratory scale bioreactors. As E. coli BL21 was not able to utilize sucrose, molasses was pretreated and hydrolyzed to fructose and glucose. Thereafter, effect of pretreated molasses concentration was investigated in the range of 16 to 56 kg m-3 by batch-bioreactor experiments / and the highest cell concentration and benzaldehyde lyase activity were obtained as CX=5.3 kg m-3 and A=1617 U cm-3, respectively, in the medium containing 7.5 kg m-3 glucose and 7.5 kg m-3 fructose. Then, different feeding strategies were developed to produce efficient cells with high concentration and BAL activity. In the first strategy, after 10 hours of batch-cultivation with molasses based medium having 7.5 kg m-3 glucose and 7.5 kg m-3 fructose concentration, based on the airflow rate, pretreated molasses was fed to the system. When air flow rate decreased considerably, fed was given to the system that results in increase in glucose and fructose concentration in the medium to 2.5 kg m-3. At the end of the process, the highest cell concentration obtained was CX=7.4 kg m-3. The maximum activity was reached at 20th hour as A=2360 U cm-3. On the other hand, as air flow variation only demonstrated the absence of glucose not fructose, a second strategy, based on the detection of the fructose and glucose concentrations during the process, was applied. In this strategy when glucose and fructose were depleted, fed was given to the system that results in increase in glucose and fructose concentration in the medium to 2.5 kg m-3 / and the highest BAL activity was obtained as 2370 U cm-3 at t= 26 h where the cell concentration was 7.5 kg m-3. At the last strategy, when glucose and fructose were depleted, fed was given to the system that results in increase in CGlucose=1.5 kg m-3 and CFructose=1.5 kg m-3 in the production medium to decrease the accumulation of acetic acid. By this strategy highest cell concentration was obtained as 8.04 kg m-3 at t=24 h and the highest BAL activity was 2315 U cm-3. These strategies could be accepted having the same BAL activity with little distinctions. However, cell concentration of the last one was higher than others and also the lowest amount of carbon source was used. Thus, last one could be chosen as the most favorable strategy.

In this study, the aim was to investigate the effects of exponential feeding strategy on benzaldehyde lyase (BAL) production by recombinant Escherichia coli BL21. For this purpose, the effects of medium components were investigated to optimize the initial medium composition of the fed-batch fermentations. For the batch bioreactor operations, the highest cell concentration and BAL activity were achieved in a media containing 30 g L-1 pretreated molasses, and 5 g L-1 (NH4)2HPO4 as 5.07 g L-1, and 1611 U ml-1 at t=8 h, respectively. Thereafter, in order to increase the cell growth and BAL production while avoiding acetate accumulation, fed-batch bioreactor operations were conducted with exponential feeding at different specific growth rates namely, 0.1 h-1 (mu-0.1), 0.15 h-1 (mu-0.15), and 0.2 h-1 (mu-0.2), and a combined exponential and constant feeding (mu-0.2+) strategy. In the experiments, 9 hours of batch-wise operation with the optimized production medium was followed by a fed-batch operation phase using the pre-determined exponential feeding profiles and for mu-0.2+ operation after 10 hours of exponential feeding as mu-0.2, where the feed rate was kept constant at 21.6 g h-1. Additionally, the plasmid stability was investigated using the feeding method of mu-0.2+ operation with antibiotics in the feed solution, and it was observed that the plasmid was stable. Among the three exponential feeding conditions, the highest cell concentration and BAL activity were determined in

No description available.

Hannover, Universiẗat, Diss., 2007.

Zugl.: Bonn, Univ., Diss., 2009

Techn. Universiẗat, Diss., 2004--Berlin.

Made available in DSpace on 2014-06-11T19:27:24Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-10-01Bitstream added on 2014-06-13T19:26:15Z : No. of bitstreams: 1 oliveira_ksm_me_rcla.pdf: 637533 bytes, checksum: f315d101c191461c95fee11e9fd4d042 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A goma xantana é um biopolímero produzido por Xanthomonas campestris muito utilizado como agente espessante. A síntese do biopolímero pode ocorrer em variadas condições, no entanto a qualidade a goma produzida também muda. Alguns trabalhos sugerem que a produção de goma xantana por batelada alimentada pode resultar num desempenho melhor em concentração de goma, rendimento e produtividade quando comparado à batelada simples. Nesse trabalho foram avaliados a influência de diferentes metodologias de oferta de sacarose na produção de goma xantana e sua qualidade, e também a produtividade e rendimento do processo. As melhores metodologias foram testadas em bioreator de 7,5L. Também foram avaliadas diferentes metodologias de separação do biopolímero (com o acréscimo de sais ao caldo ou ao etanol na etapa de extração da goma), bem como a influência de surfactantes e sais na viscosidade da goma em solução. Em incubadora com agitação orbital a quantidade e qualidade de goma xantana produzida a 25°C foram mais interessantes. Em fermentador, a produção de xantana a 30°C em meio contendo 2% de sacarose pode ter seu tempo reduzido sem que isso afete a concentração e a viscosidade da goma obtida. A viscosidade do biopolímero produzido a 25°C em meio contendo 4% de sacarose foi superior (365,9 cP) quando comparado ao biopolímero produzido a 30°C, em meio contendo 2% de sacarose. A produtividade e concentração de goma obtidos estão entre os encontrados na literatura (0,34 g xantana L-1 h-1 e 24 g xantana L-1). O uso de sais na extração da goma permite a redução do solvente utilizado e uma goma de melhor qualidade. Destacaram-se a utilização dos sais NaCl na concentração de 0,01% e CaCl2 a 0,05%. O tratamento térmico aumenta a quantidade e a viscosidade do biopolímero além de eliminar as células. A adição de 0,01% de SDS e de 0,001% de Tween 80 na solução de goma xantana aumenta sua viscosidade. / Xanthan gum is a biopolymer produced by Xanthomonas campestris, used as a thickener. Its synthesis can happen in different conditions, but quality of xanthan produced also changes. Studies suggest that fed batch xanthan gum production can result in better performance in gum concentration, yield and productivity, when compared to batch. This work evaluates the influence of different methods of sucrose supply on xanthan gum quantity and quality, as well its productivity and process efficiency. The best methods were tested in a bioreactor of 7.5 L. Were also evaluated different separation methods (with the addition of salt in broth or ethanol during the separation phase) and the influence of surfactants on viscosity on xanthan solution. In shaker incubator, the gum produced at 25°C was more interesting. In bioreactor, the xanthan production at 30°C using 2% sucrose can be reduced to 40 hours, without affecting concentration and viscosity of the gum obtained. The viscosity of the biopolymer produced at 25°C using 4% sucrose were higher (365.9 cP) when compared to biopolymer produced at 30°C with 2% sucrose. Concentration and yield are similar than those found in the literature (0.34 g xanthan L-1 h-1 and 24 g xanthan L-1). The use of salts in xanthan extraction reduces the input and a better quality gum. The salts NaCl (concentration of 0.01%) and CaCl2 (0.05%) showed best results. Heat treatment increases the xanthan quantity and viscosity and eliminates cells from broth. The addition of 0.01% SDS and 0.001% Tween 80 in the solution of xanthan gum increases its viscosity.