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1.
A novel feeding strategy in fedbatch recombinant yeast fermentation was developed to achieve high plasmid stability and protein productivity for fermentation using low-cost rich (non-selective) media. In batch fermentations with a recombinant yeast, Saccharomyces cerevisiae, which carried the plasmid pSXR125 for the production of beta-galactosidase, it was found that the fraction of plasmid-carrying cells decreased during the exponential growth phase but increased during the stationary phase. This fraction increase in the stationary phase was attributed to the death rate difference between the plasmid-free and plasmid-carrying cells caused by glucose starvation in the stationary phase. Plasmid-free cells grew faster than plasmid-carrying cells when there were plenty of growth substrate, but they also lysed or died faster upon the depletion of the growth substrate. Thus, pulse additions of the growth substrate (glucose) at appropriate time intervals allowing for significant starvation period between two consecutive feedings during fedbatch fermentation should have positive effects on stabilizing plasmid and enhancing protein production. A selective medium was used to grow cells in the initial batch fermentation, which was then followed with pulse feeding of concentrated non-selective media in fedbatch fermentation. Both experimental data and model simulation show that the periodic glucose starvation feeding strategy can maintain a stable plasmid-carrying cell fraction and a stable specific productivity of the recombinant protein, even with a non-selective medium feed for a long operation period. On the contrary, without glucose starvation, the fraction of plasmid-carrying cells and the specific productivity continue to drop during the fedbatch fermentation, which would greatly reduce the product yield and limit the duration that the fermentation can be effectively operated. The new feeding strategy would allow the economic use of a rich, non-selective medium in high cell density recombinant fedbatch fermentation. This new feeding strategy can be easily implemented with a simple IBM-PC based control system, which monitors either glucose or cell concentration in the fermentation broth. 相似文献
2.
Summary Fructokinase negativeZymomonas mobilis UQM 2864, was co-immobilised with invertase in alginate and incubated on sucrose-based media in batch and fedbatch culture. The highest fructose concentration achieved was 138 g/l using fed-batch cultivation with sugar-cane syrup-simultaneously producing 79.9 g/l or 10.1% (v/v) ethanol in less than 24 hours. The ethanol and fructose yields were 95 and 84% respectively. Co-immobilisation resulted in faster fermentation times, particularly for the batch fermentations, and complete utilisation of substrate. 相似文献
3.
Summary The production of acetate from the fermentation of lactate by Gluconobacter oxydans was studied. Batch experiments showed that glucose was the preferred substrate compared to lactate. A fed-batch culture was fed with a mixture of glucose and lactate followed by periodic addition of lactate. The maximum productivity of acetate was 0.16 g/l h but this value decreased during the fedbatch culture due to growth inhibition by acetate. 相似文献
4.
Summary A method is proposed to estimate the concentration of metabolically active cells of a microaerobic culture on-line from the
measurement of oxygen uptake rate (OUR) and respiratory quotient (RQ). With the cultivation ofEnterobacter aerogenes in a fedbatch mode the estimated active cell concentration agrees well with the viable cell concentration determined by microscopic
count and agar plate incubation. 相似文献
5.
Summary A recombinantEscherichia coli strain harboring the PHB biosynthesis genes fromAlcaligenes eutrophus was used to produce poly--hydroxybutyrate (PHB) by pH-stat fedbatch culture. Initial glucose concentration for optimal growth was found to be 20g/L from a series of flask cultures. A final PHB concentration of 88.8 g/L could be obtained after 42 hrs of cultivation. 相似文献
6.
Best practices in heterotrophic high-cell-density microalgal processes: achievements,potential and possible limitations 总被引:1,自引:0,他引:1
Bumbak F Cook S Zachleder V Hauser S Kovar K 《Applied microbiology and biotechnology》2011,91(1):31-46
Microalgae of numerous heterotrophic genera (obligate or facultative) exhibit considerable metabolic versatility and flexibility
but are currently underexploited in the biotechnological manufacturing of known plant-derived compounds, novel high-value
biomolecules or enriched biomass. Highly efficient production of microalgal biomass without the need for light is now feasible
in inexpensive, well-defined mineral medium, typically supplemented with glucose. Cell densities of more than 100 g l−1 cell dry weight have been achieved with Chlorella, Crypthecodinium and Galdieria species while controlling the addition of organic sources of carbon and energy in fedbatch mode. The ability of microalgae
to adapt their metabolism to varying culture conditions provides opportunities to modify, control and thereby maximise the
formation of targeted compounds with non-recombinant microalgae. This review outlines the critical aspects of cultivation
technology and current best practices in the heterotrophic high-cell-density cultivation of microalgae. The primary topics
include (1) the characteristics of microalgae that make them suitable for heterotrophic cultivation, (2) the appropriate chemical
composition of mineral growth media, (3) the different strategies for fedbatch cultivations and (4) the principles behind
the customisation of biomass composition. The review confirms that, although fundamental knowledge is now available, the development
of efficient, economically feasible large-scale bioprocesses remains an obstacle to the commercialisation of this promising
technology. 相似文献
7.
BACKGROUND: Important parameters during recombinant protein production in Escherichia coli, such as productivity and protein activity, are affected by the growth rate. This includes the translocation of protein over the membrane to gain better folding capacity or reduced proteolysis. To vary the growth rate two techniques are available: fedbatch and continuous cultivation, both controlled by the ingoing feed rate. RESULTS: During fedbatch cultivation, E. coli contains phosphatidylethanolamine, phosphatidylglycerol, cardiolipin and saturated fatty acids in amounts which are stable with growth rate. However, the levels of cardiolipin are very high compared to continuous cultivation. The reason for fedbatch triggering of this metabolism is not known but hypothesised to result from an additional need for carbon and energy. The reason could be the dynamic and sometimes rapid changes in growth rate to which the fedbatch cell has at all times to adjust. The membrane flexibility, essential for translocation of various components, is however to some degree sustained by production of increased amounts of unsaturated fatty acids in phosphatidylglycerol. The result is a functionally stiff membrane which generally promotes low cell lysis and is constant with respect to protein leakage to the medium. At comparatively high growth rates, when the further stabilising effect of cyclic fatty acids is gone, the high level of unsaturated fatty acids results in a pronounced effect upon sonication. This is very much in contrast to the membrane function in continuous cultivation which shows very specific characteristics as a function of growth rate. CONCLUSIONS: The stiff and unchanging fedbatch membrane should promote a stable behaviour during downstream processing and is less dependent on the time of harvest. However, optimisation of protein leakage can only be achieved in the continuously cultivated cell where leakage is twice as high compared to the constant leakage level in fedbatch. If leakage is undesired, continuous cultivation is also preferred since it can be designed to lead to the lowest values detected. Induction at low growth rate (<0.2 h-1) should be avoided with respect to productivity, in any system, since the specific and total protein production shows their lowest values at this point. 相似文献
8.
Boström M Markland K Sandén AM Hedhammar M Hober S Larsson G 《Applied microbiology and biotechnology》2005,68(1):82-90
The effect of changes in substrate feed rate during fedbatch cultivation was investigated with respect to soluble protein formation and transport of product to the periplasm in Escherichia coli. Production was transcribed from the PmalK promoter; and the cytoplasmic part of the production was compared with production from the PlacUV5 promoter. The fusion protein product, Zb-MalE, was at all times accumulated in the soluble protein fraction except during high-feed-rate production in the cytoplasm. This was due to a substantial degree of proteolysis in all production systems, as shown by the degradation pattern of the product. The product was also further subjected to inclusion body formation. Production in the periplasm resulted in accumulation of the full-length protein; and this production system led to a cellular physiology where the stringent response could be avoided. Furthermore, the secretion could be used to abort the diauxic growth phase resulting from use of the PmalK promoter. At high feed rate, the accumulation of acetic acid, due to overflow metabolism, could furthermore be completely avoided. 相似文献
9.
Zhi-Yong Zheng Shan-Jing Yao Xiaobei Zhan Chi Chung Lin 《Biotechnology and Bioprocess Engineering》2009,14(1):52-59
Tryptone has multiple and complex effects on cell physiology and process performance in pulse fed-batch cultivation of recombinant
Escherichia coli. By applying feedback control of dissolved oxygen signal responding to pulse in the feed rate, the production of acetate
was avoided and the optimization of production of recombinant human epidermal growth factor (hEGF) was successfully achieved.
With the addition of an optimum amount of tryptone along with glucose in the pulse fedbatch cultivation of E. coli, the ability of the cell to divide and the stability of the plasmid within the bacteria were improved. Consequently, segregation
of the cells into a viable but non-culturable physiological state was alleviated. Addition of tryptone also enhanced cell
respiration before and after hEGF expression and thus further benefited the production of recombinant hEGF. Excessive addition
of tryptone resulted in low sensitivity of the oscillation of dissolved oxygen signal and poor operability of pulse fed-batch
cultivation as this led to an accumulation of acetate, which weakened the dissolved oxygen signal responses. Consequently,
the production of recombinant protein was considerably reduced. By combining the process performance and the positive effect
of complex media pulse addition on bacterial metabolism, the optimal production conditions of hEGF were successfully determined.
A high cell density of 91 g/L dry cell weight was obtained under these optimal production conditions. Furthermore, a high
level of 0.24 g/L hEGF was attained leading to a 32.6% increase in product yield as compared to the controls. 相似文献
10.
Microbial degradation of phenol was studied using batch and fedbatch cultures of acclimatized activated sludge under a wide range of phenol (0-793 mg l−1) and biomass (0.74-6.7 g l−1) initial concentrations. As cell growth continued after total phenol removal, the production and later consumption of a main metabolic intermediate was considered the step governing the biodegradation kinetics. A model that takes explicitly into account the kinetics of the intermediate was developed by introducing a specific growth rate model associated with its consumption and the incorporation of a dual-substrate inhibitory effect on phenol degradation. Biomass growth and phenol removal were adequately predicted in all the cultures. Moreover, the model-based design of the fedbatch feeding strategies allowed driving separately the phenol degradation under substrate-limitation and substrate-inhibition modes. A sensitivity analysis was also performed in order to establish the importance of the parameters in the accuracy of model predictions. 相似文献
11.
12.
Flux response of glycolysis and storage metabolism during rapid feast/famine conditions in Penicillium chrysogenum using dynamic 13C labeling
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Lodewijk de Jonge Nicolaas A. A. Buijs Joseph J. Heijnen Walter M. van Gulik Alessandro Abate S. Aljoscha Wahl 《Biotechnology journal》2014,9(3):372-385
The scale‐up of fermentation processes frequently leads to a reduced productivity compared to small‐scale screening experiments. Large‐scale mixing limitations that lead to gradients in substrate and oxygen availability could influence the microorganism performance. Here, the impact of substrate gradients on a penicillin G producing Penicillium chrysogenum cultivation was analyzed using an intermittent glucose feeding regime. The intermittent feeding led to fluctuations in the extracellular glucose concentration between 400 μM down to 6.5 μM at the end of the cycle. The intracellular metabolite concentrations responded strongly and showed up to 100‐fold changes. The intracellular flux changes were estimated on the basis of dynamic 13C mass isotopomer measurements during three cycles of feast and famine using a novel hybrid modeling approach. The flux estimations indicated a high turnover of internal and external storage metabolites in P. chrysogenum under feast/famine conditions. The synthesis and degradation of storage requires cellular energy (ATP and UTP) in competition with other cellular functions including product formation. Especially, 38% of the incoming glucose was recycled once in storage metabolism. This result indicated that storage turnover is increased under dynamic cultivation conditions and contributes to the observed decrease in productivity compared to reference steady‐state conditions. 相似文献
13.
Jeong-Woo Choi Young-Kee Kim Won Hong Lee Henrik Pedersen Chee-Kok Chin 《Biotechnology and Bioprocess Engineering》1999,4(2):138-146
Optimal substrate feeding strategy in bioreactor operation was investigated to increase the production of secondary metabolite
in a high density culture of plant cell. It was accomplished by the previously proposed structured kinetic model that describes
the cell growth and synthesis of the secondary metabolite, berberine, in a batch suspension culture ofThalictrum rugosum. Four types of operation strategies for sugar feeding intoT. rugosum culture were proposed based on the model, which were the periodic fedbatch operations to maintain the cell activity, the
cell viability, and the specific production rate, and the perfusion operation to maintain the specific production rate. From
the simulation results of these strategies, it could be found that the periodic fed-batch operation and the perfusion operation
could achieve the higher volumetric production of berberine (mg berberine/L) and specific production yield (mg berberine/g
dry cell weight) than those of batch cultures. Although the highest productivity (mg berberine/day) of berberine could be
achieved by the periodic fed-batch operation to maintain the cell activity compared with the other strategies in the periodic
fed-batch operations, the specific production yield was low due to the higher maximum dry cell weight than other cases. The
periodic fed-batch operation to maintain cell viability resulted in the highest volumetric production of berberine and specific
production yield compared with the other strategies. In the cases of maintaining the specific production rate, the per-formance
of the periodic fed-batch operation was better than that of the perfusion operation in the respect of the volumetric production
and productivity of berberine. In order to increase the volumetric production of berberine and to get the highest specific
production yield, the periodic fed-batch operation to maintain cell viability could be chosen as the optimal operating strategy
in high density, culture ofT. rugosum plant cell. 相似文献
14.
Ruth C Zuellig T Mellitzer A Weis R Looser V Kovar K Glieder A 《Systems and synthetic biology》2010,4(3):181-191
Natural tools for recombinant protein production show technological limitations. Available natural promoters for gene expression
in Pichia pastoris are either constitutive, weak or require the use of undesirable substances or procedures for induction. Here we show the
application of deletion variants based on the well known methanol inducible AOX1 promoter and small synthetic promoters, where cis-acting elements were fused to core promoter fragments. They enable differently
regulated target protein expression and at the same time to replace methanol induction by a glucose or glycerol feeding strategy.
Trypsinogen, the precursor of the serine protease trypsin, was expressed using these different promoters. Depending on the
applied promoter the production window (i.e. the time of increasing product concentration) changed significantly. In fedbatch
processes trypsinogen yields before induction with methanol were up to 10 times higher if variants of the AOX1 promoter were applied. In addition, the starting point of autoproteolytic product degradation can be predetermined by the
promoter choice. 相似文献
15.
Jing Wang Long Liu Haiyan Zhou Jianghua Li Guocheng Du Jian Chen 《Biotechnology and Bioprocess Engineering》2011,16(5):916-922
High cell density cultivation was investigated for L-phenylalanine (L-Phe) production by an L-tyrosine (L-Tyr) auxotrophic
Escherichia coli WSH-BR165 (pAPB03). Dual exponential feeding of L-Tyr and glucose was adopted to achieve high cell density cultivation. The
control was completed without dual feeding. The processes where dual feeding and induction were commenced together and those
where induction began after dual feeding were studied and compared. The results indicated that the former dual feeding mode
was most favorable for enhanced L-Phe production. With an optimal specific growth rate of 0.09/h during the dual exponential
feeding period, the maximum dry cell weight reached 43.16 g/L (3.04 times that of the control) with a final L-Phe titer of
44.53 g/L (1.06 times that of the control) and an L-Phe productivity of 1.484 g/L/h (1.69 times that of the control). High
cell density cultivation via the feeding of L-Tyr and glucose exponentially after the induction point proved to be an efficient approach to enhance L-Phe
production. 相似文献
16.
R. González Matute D. Figlas N. Curvetto 《World journal of microbiology & biotechnology》2011,27(6):1331-1339
Agaricus blazei Murrill is usually cultivated using the same biphasic composting method employed for A. bisporus. Because cultivation of A. blazei on traditional A. bisporus composts poses some disadvantages, non-composted substrates were studied for A. blazei cultivation. Mycelial growth rate and productive performance of A. blazei were evaluated on substrates containing sunflower seed hulls, Pleurotus spp. spent mushroom substrate, or their combination, in the absence or in the presence of different supplements (vermicompost,
peat or brewery residues). Substrates were prepared by initially soaking them and then they were sterilized (1 atm for 120 min).
In addition, each substrate’s degradation was measured after cultivation by obtaining the lignin, cellulose, hemicellulose,
organic matter, total fiber, ash, carbon and nitrogen contents before spawn-run and at the end of two flushes of A. blazei. The cultivation of A. blazei on non-composted substrates is possible and with a low rate of contamination when using the spent mushroom substrate as the
main component or combined 50:50 with sunflower seed hulls. In addition, the best yields were obtained on those substrates
containing spent Pleurotus mushroom substrate with supplements and those mixtures with sunflower seed hulls and vermicompost. These yields were similar
to those reported on composted substrates. Substrate changes in composition measured at the end of two flushes indicate that
the lignin-hemicellulose fraction was preferentially used and that the substrates exhibiting the best yield showed greater
biodegradation of lignin-hemicellulose fraction than the others did. 相似文献
17.
Hiroshi Sata Hajime Taniguchi Yoshiharu Maruyama 《Bioscience, biotechnology, and biochemistry》2013,77(6):1521-1527
In the usual batch cultivation, Bacillus circulans F-2 produced amylase only when granular carbon sources such as raw starch or crosslinked starches (CLP) were added. In the dialysis cultivation, where CLP and partially purified amylase were incubated inside the dialysis tubing, the bacterium inoculated outside of the tubing grew and produced the amylase. Amylase production of this bacterium was further investigated in feeding cultivation, in which maltose was fed to the cultivation medium at various rates. The bacterial growth increased with the increase of the feeding rate of maltose, but maximum amylase production was observed at a feeding rate of 45 mg/hr/1. No amylase was produced on the media containing monosaccharides, sucrose, lactose, or isomaltose in the feeding cultivation although bacterial growth was observed. The amylase of this bacterium was found to be inducible. Replacement of 20% of the maltose with glucose resulted in a great decrease (70%) in the amylase production. This shows that the amylase synthesis of B. circulans F-2 is severely repressed by glucose. 相似文献
18.
Pedro Moñino Fernández Albert Vidal García Tanisha Jansen Wendy Evers Maria Barbosa Marcel Janssen 《Biotechnology and bioengineering》2023,120(6):1569-1583
Oxygen-balanced mixotrophy (OBM) is a novel type of microalgal cultivation that improves autotrophic productivity while reducing aeration costs and achieving high biomass yields on substrate. The scale-up of this process is not straightforward, as nonideal mixing in large photobioreactors might have unwanted effects in cell physiology. We simulated at lab scale dissolved oxygen and glucose fluctuations in a tubular photobioreactor operated under OBM where glucose is injected at the beginning of the tubular section. We ran repeated batch experiments with the strain Galdieria sulphuraria ACUF 064 under glucose pulse feeding of different lengths, representing different retention times: 112, 71, and 21 min. During the long and medium tube retention time simulations, dissolved oxygen was depleted 15–25 min after every glucose pulse. These periods of oxygen limitation resulted in the accumulation of coproporphyrin III in the supernatant, an indication of disruption in the chlorophyll synthesis pathway. Accordingly, the absorption cross-section of the cultures decreased steeply, going from values of 150–180 m2 kg−1 at the end of the first batch down to 50–70 m2 kg−1 in the last batches of both conditions. In the short tube retention time simulation, dissolved oxygen always stayed above 10% air saturation and no pigment reduction nor coproporphyrin III accumulation were observed. Concerning glucose utilization efficiency, glucose pulse feeding caused a reduction of biomass yield on substrate in the range of 4%–22% compared to the maximum levels previously obtained with continuous glucose feeding (0.9 C-g C-g−1). The missing carbon was excreted to the supernatant as extracellular polymeric substances constituted by carbohydrates and proteins. Overall, the results point out the importance of studying large-scale conditions in a controlled environment and the need for a highly controlled glucose feeding strategy in the scale-up of mixotrophic cultivation. 相似文献
19.
High cell density fed-batch cultivation of<Emphasis Type="Italic"> Escherichia coli</Emphasis> using exponential feeding combined with pH-stat 总被引:5,自引:0,他引:5
A new feeding strategy in fed-batch culture, exponential feeding combined with pH-stat is suggested to avoid the accumulation of substrate in culture broth. Exponential feeding was stopped whenever a predetermined amount of limiting substrate was supplied and then pH change was observed. When pH rose above an upper limit due to the depletion of substrate, feeding was restarted. With this feeding strategy, recombinant Escherichia coli could be grown to 101 g/l by controlling the specific growth rate at 0.1 h–1.An erratum to this article can be found at 相似文献
20.
Summary A recombinant Saccharomyces cerevisiae producing hepatitis B surface antigen (HBsAg) exhibited growth-assciated product formation. By controlling the medium feed rate, based on the calculated amount of medium required for 1 h, a constant specific growth rate was obtained in the range of 1.12-0.18 h–1. In order to prolong the exponential growth phase, the medium feed rate was increased exponentially. A fedbatch cultivation method based on the production kinetics of batch culture enhanced HBsAg production ten times more than in batch culture. The reason for the increase can be explained by the fact that the production of HBsAg is expressed as an exponential function of time when the specific growth rate is controlled to a constant value in growth-associated product fromation kinetics. In the scale-up of this culture to 91, the specific growth rate could also be maintained constant and the HBsAg production trend was similar to that in a 1-l culture. However, ethanol accumulation occurred at a late stage in fed-bach culture. Ethanol produced was not reutilized and inhibited further cell growth.
Offprint requests to: M. B. Gu 相似文献