Sven-Olof Enfors

Physiological responses to mixing in large scale bioreactors

Escherichia coli fed-batch cultivations at 22 m3 scale were compared to corresponding laboratory scale processes and cultivations using a scale-down reactor furnished with a high-glucose concentration zone to mimic the conditions in a feed zone of the large bioreactor. Formate accumulated in the large reactor, indicating the existence of oxygen limitation zones. It is suggested that the reduced biomass yield at large scale partly is due to repeated production/re-assimilation of acetate from overflow metabolism and mixed acid fermentation products due to local moving zones with oxygen limitation. The conditions that generated mixed-acid fermentation in the scale-down reactor also induced a number of stress responses, monitored by analysis of mRNA of selected stress induced genes. The stress responses were relaxed when the cells returned to the substrate limited and oxygen sufficient compartment of the reactor. Corresponding analysis in the large reactor showed that the concentration of mRNA of four stress induced genes was lowest at the sampling port most distant from the feed zone. It is assumed that repeated induction/relaxation of stress responses in a large bioreactor may contribute to altered physiological properties of the cells grown in large-scale bioreactor. Flow cytometric analysis revealed reduced damage with respect to cytoplasmic membrane potential and integrity in cells grown in the dynamic environments of the large scale reactor and the scale-down reactor.

Temperature limited fed-batch technique for control of proteolysis in Pichia pastoris bioreactor cultures

BackgroundA temperature limited fed-batch (TLFB) technique is described and used for Pichia pastoris Mut+ strain cultures and compared with the traditional methanol limited fed-batch (MLFB) technique. A recombinant fusion protein composed of a cellulose-binding module (CBM) from Neocallimastix patriciarum cellulase 6A and lipase B from Candida antarctica (CALB), was produced and secreted by this strain.ResultsA protein concentration of about 1 g L-1 was produced in the MLFB process. However, this product was considerably degraded by protease(s). By applying the TLFB process, the yield was increased to 2 g L-1 full-length product and no proteolytic degradation was observed. Flow cytometry analysis showed that the percentage of dead cells increased rapidly during the initial methanol feed phase in the MLFB process and reached a maximum of about 12% after about 40–70 hours of methanol feeding. In the TLFB process, cell death rate was low and constant and reached 4% dead cells at the end of cultivation (about 150 hours methanol feeding time). The lower cell death rate in the TLFB correlated with a lower protease activity in the culture supernatant. The specific alcohol oxidase (AOX) activity in the TLFB process was 3.5 times higher than in the MLFB process.ConclusionThree mechanisms that may contribute to the much higher accumulation of product in the TLFB process are: 1) reduced proteolysis due to lower temperature, 2) reduced proteolysis due to lower cell death and protease release to the medium, 3) increased synthesis rate due to higher AOX activity.

Modeling of Overflow Metabolism in Batch and Fed-Batch Cultures of Escherichia coli

A dynamic model of glucose overflow metabolism in batch and fed‐batch cultivations of Escherichia coli W3110 under fully aerobic conditions is presented. Simulation based on the model describes cell growth, respiration, and acetate formation as well as acetate reconsumption during batch cultures, the transition of batch to fed‐batch culture, and fed‐batch cultures. E. coli excreted acetate only when specific glucose uptake exceeded a critical rate corresponding to a maximum respiration rate. In batch cultures where the glucose uptake was unlimited, the overflow acetate made up to 9.0 ± 1.0% carbon/carbon of the glucose consumed. The applicability of the model to dynamic situations was tested by challenging the model with glucose and acetate pulses added during the fed‐batch part of the cultures. In the presence of a glucose feed, E. coli utilized acetate 3 times faster than in the absence of glucose. The cells showed no significant difference in maximum specific uptake rate of endogenous acetate produced by glucose overflow and exogenous acetate added to the culture, the value being 0.12−0.18 g g−1 h−1 during the entire fed‐batch culture period. Acetate inhibited the specific growth rate according to a noncompetitive model, with the inhibition constant (ki) being 9 g of acetate/L. This was due to the reduced rate of glucose uptake rather than the reduced yield of biomass.

Glucose overflow metabolism and mixed-acid fermentation in aerobic large-scale fed-batch processes with Escherichia coli.

Abstract Industrial 20-m3-scale and laboratory-scale aerobic fed-batch processes with Escherichia coli were compared. In the large-scale process the observed overall biomass yield was reduced by 12% at a cell density of 33 g/l and formate accumulated to 50 mg/l during the later constant-feeding stage of the process. Though the dissolved oxygen signal did not show any oxygen limitation, it is proposed that the lowered yield and the formate accumulation are caused by mixed-acid fermentation in local zones where a high glucose concentration induced oxygen limitation. The hypothesis was further investigated in a scale-down reactor with a controlled oxygen-limitation compartment. In this scale-down reactor similar results were obtained: i.e. an observed yield lowered by 12% and formate accumulation to 238 mg/l. The dynamics of glucose uptake and mixed-acid product formation (acetate, formate, d-lactate, succinate and ethanol) were investigated within the 54 s of passage time through the oxygen-limited compartment. Of these, all except succinate and ethanol were formed; however, the products were re-assimilated in the oxygen-sufficient reactor compartment. Formate was less readily assimilated, which accounts for its accumulation. The total volume of the induced-oxygen-limited zones was estimated to be 10% of the whole liquid volume in the large bioreactor. It is also suggested that repeated excretion and re-assimilation of mixed-acid products contribute to the reduced yield during scale-up and that formate analysis is useful for detecting local oxygen deficiency in large-scale E. coli processes.

The molecular basis of partitioning in aqueous two-phase systems

Protein purification based on partition in aqueous two-phase systems has attracted interest for many years. This approach has been advocated as a primary-stage unit operation in downstream processing. In reality, application has been strictly limited through inadequate understanding of the complex molecular forces involved in partitioning processes.

Substrate gradients in bioreactors: origin and consequences

Gradients of glucose in time and space are shown in a 30 m3 cultivation of Saccharomyces cerevisiae grown in minimal medium to a cell density of 20 gl−1. The fed-batch concept was used with glucose as the limiting component which was fed continuously to the process. As the mean glucose concentration declined throughout the process, the level of glucose was at all times different in three sampling ports (bottom/middle/top) of the reactor. These gradients were furthermore shown to depend on the feed position. This means that if the feed was supplied in the relatively stagnant mixing zone above the top impeller, the gradients were more pronounced than by feed in the well mixed bottom impeller zone. A rapid sampling system was constructed, and continuous glucose samples of every 0.15 s were analysed from a point of the reactor. Fifty samples were collected with this system, but the amount and frequency is possible to change. The results of these series show a variance of the glucose concentration where at one stage, a peak appeared of a relative difference in concentration of 40 mgl−1. The pattern of these rapid glucose fluctuations was shown to depend on the turbulence level at the location of the feed. It was shown, that the fluctuations were more pronounced when the feed was localised in a relatively stagnant area than in the well-mixed impeller area, where the deviation from the mean was negligible. The fluid flow, in the impeller (gassed and ungassed) and bulk area (ungassed) of the reactor, was characterised by turbulence measurements using thermal anemometry. These types of areas resembles well the different areas of sampling as mentioned above. The turbulent frequencies in these areas were in the range of 10−1 to 104 Hz with the highest amplitudes at low frequencies. The spectra depicts a uniform time scale for all zones, especially at the low frequencies. The dominance of low frequency, high amplitude flow variations and the observed short-time oscillations in substrate concentration support the hypothesis of substrate transport over fairly long distances without substantial mixing both in the impeller, but especially, in the bulk zone of the reactor.Simulations with an integrated CFD and biokinetic model were performed. The predictions of the glucose gradients of this model were compared to measurements.

Monitoring of genes that respond to process-related stress in large-scale bioprocesses.

In large-scale aerobic fed-batch processes, cells are exposed to local zones of high glucose concentrations that can also cause local oxygen limitations at high cell densities. The mRNA levels of four stress genes (clpB, dnaK, uspA, and proU) and three genes responding to oxygen limitation or glucose excess (pfl, frd, and ackA) were investigated in an industrial 20-m(3) Escherichia coli process and in a scale-down reactor with defined high-glucose and low-oxygen zones. The mRNA levels of ackA and proU were high during the batch growth phase, but declined drastically when glucose became limited, whereas the mRNA levels of the other stress genes were relatively constant throughout the process. In the industrial-scale reactor, the stress gene mRNA levels were, in most cases, highest in the middle part and at the top of the reactor, where the substrate was fed. Cells passing through the high glucose zone of the scale-down reactor had elevated mRNA levels for the oxygen limitation genes and had also elevated heat-shock gene mRNA levels. Both responses to stress occurred within seconds. The approach presented in this study offers a tool for monitoring process-related changes in the transcriptional regulation of genes.

Analysis and control of proteolysis of a fusion protein in Pichia pastoris fed-batch processes

A fusion protein composed of a cellulose-binding module (CBM) from Neocallimastix patriciarum cellulase 6A and lipase B from Candida antarctica (CALB), was produced by Pichia pastoris Mut(+) in high-cell density bioreactor cultures. The production was induced by switching from growth on glycerol to growth on methanol. The lipase activity in the culture supernatant increased at an almost constant rate up to a value corresponding to 1.3 g x l(-1) of CBM-CALB. However, only about 40% of the product was of full-length according to Western blot analysis. This loss was due to a cleavage of the protein in the linker between the CBM and the CALB moieties. The cleavage was catalyzed by serine proteases in the culture supernatant. The CALB-moiety was subjected to further slow degradation by cell-associated proteolysis. Different strategies were used to reduce the proteolysis. Previous efforts to shorten the linker region resulted in a stable protein but with ten times reduced product concentration in bioreactor cultures (Gustavsson et al. 2001, Protein Eng. 14, 711-715). Addition of rich medium for protease substrate competition had no effect on the proteolysis of CBM-CALB. The kinetics for the proteolytic reactions, with and without presence of cells were shown to be influenced by pH. The fastest reaction, cleavage in the linker, was substantially reduced at pH values below 5.0. Decreasing the pH from 5.0 to 4.0 in bioreactor cultures resulted in an increase of the fraction of full-length product from 40 to 90%. Further improvement was achieved by decreasing the temperature from 30 to 22 degrees C during the methanol feed phase. By combining the optimal pH and the low temperature almost all product (1.5 g x l(-1)) was obtained as full-length protein with a considerably higher purity in the culture supernatant compared with the original cultivation.

Electric chips for rapid detection and quantification of nucleic acids

A silicon chip-based electric detector coupled to bead-based sandwich hybridization (BBSH) is presented as an approach to perform rapid analysis of specific nucleic acids. A microfluidic platform incorporating paramagnetic beads with immobilized capture probes is used for the bio-recognition steps. The protocol involves simultaneous sandwich hybridization of a single-stranded nucleic acid target with the capture probe on the beads and with a detection probe in the reaction solution, followed by enzyme labeling of the detection probe, enzymatic reaction, and finally, potentiometric measurement of the enzyme product at the chip surface. Anti-DIG-alkaline phosphatase conjugate was used for the enzyme labeling of the DIG-labeled detection probe. p-Aminophenol phosphate (pAPP) was used as a substrate. The enzyme reaction product, p-aminophenol (pAP), is oxidized at the anode of the chip to quinoneimine that is reduced back to pAP at the cathode. The cycling oxidation and reduction of these compounds result in a current producing a characteristic signal that can be related to the concentration of the analyte. The performance of the different steps in the assay was characterized using in vitro synthesized RNA oligonucleotides and then the instrument was used for analysis of 16S rRNA in Escherichia coli extract. The assay time depends on the sensitivity required. Artificial RNA target and 16S rRNA, in amounts ranging from 10(11) to 10(10) molecules, were assayed within 25 min and 4 h, respectively.

Impact of plasmid presence and induction on cellular responses in fed batch cultures of Escherichia coli

Fed batch cultivations of plasmid-free and recombinant Escherichia coli were employed in order to determine cellular responses and effects of plasmid presence and induction on the host cell physiology. While plasmid presence was shown to have minor influence on overall biomass yield, induction with 0.1 mM IPTG led to a marked reduction. The number of dividing cells, measured as colony forming ability, was influenced by plasmid presence and to a larger extent by induction. The latter caused a decline in the number of dividing cells to less than 10% of the population within 10 h. However, this cell segregation did not affect the specific rate of product formation, which was approximately constant throughout the cultivations. Analysis of the in vivo degradation rate of the product indicated that it was proteolytically stable. The cellular content of the stringent response signal substance, ppGpp, peaked immediately after transition from batch to fed batch mode to stabilise at a higher value than in the batch phase. When the specific growth rate declined below 0.06 h-1 an additional rise in ppGpp concentration was observed.