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tl_files/kuhner/product/safety/Bilder/pdf.jpg Fed-Batch Mode in Shake Flasks by Slow-Release Technique (Biotechnology and Bioengineering, 10/2006)

FeedBeads/Tubes

Product

Slow Release Technology

Short description

FeedBead is a polymer-based slow release system for glucose.

Performance characteristics

 
 
+ Improves the screening security
+ Suitable for high throughput screening (HTS)
+ Easy handling
+ Fed-batch without tubing and pumps
+ No additional enzymes

Fed-batch operation

FeedBeads are polymer particles enabling the user to run fed-batch processes in shake flasks or microtiter plates. The user can now apply an additional screening routine (batch and fed-batch) under substrate limited condition.

In consequence FeedBeads can increase the probability to identify an optimal production strain during the screening phase.

Scale-up

Through the use of FeedBeads the main disadvantages of the batch-approaches (eg. Crabtree-Effect, Overflow Methabolism, ...) can be avoided, optimal production strains can be identified quickly, and the scale-up runs very smoothly.

 

Technical data

Slow Release Technology

Product information

   
Sizes

For more information
Substrate

glucose
Options

on request are also other cristalline nutrients as limited substrate available

Application fields

Slow Release Technology

FeedBeads: Controlled glucose delivery by slow release technology

FeedBeads provide substrate limited fed-batch conditions in the shaken bioreactor without the need for enzymes or additional equipment such as tubing or pumps.

tl_files/kuhner/product/shaker/add-ons/fb_fig1 (2).png

Fig. 1:  Released amount of glucose [mg] from the number of added FeedBeads (FB, 12 mm diameter, Art.-Nr. SMFB63319) per 250 mL shake flask; each with 25 mL 0,1 M phosphate buffer, pH 7, temperature 37°C, shaking frequency 280 rpm, shaking diameter 50 mm.


Crystalline glucose is embedded in a silicone matrix and released with a defined kinetic. The amount of glucose in the experiment can be increased simply by adding multiple beads. The beads do not interact.

Hansenula polymorpha: Screening under substrate limited conditions

The yeast Hansenula polymorpha is a well known protein producing microorganism. Green fluorescent protein (GFP) under control of appropriate promoters is a common reporter protein in Hansenula because it is easy to detect. Expression of GFP in Hansenula polymorpha is subject to catabolite repression in the presence of high glucose concentrations.

The majority of screening experiments are performed in batch mode, so GFP expression is limited to the short period before the glucose is almost completely consumed. This means it is often impossible to identify the best protein producing clones under substrate limited conditions during a batch screening setup.

FeedBeads mimic fed-batch conditions even under screening conditions. Catabolite repression can be circumvented and suitable production clones are rapidly identified.

tl_files/kuhner/product/shaker/add-ons/fb_fig2 (2).png

Fig. 2:  Hansenula polymorpha-production of green fluorescent protein (GFP) with slow-release substrate (FeedBeads) and in batch-mode (control);  medium SYN6 red with 5 g/L glucose, without glycerol, with 100 mM citrate, temperature 30 °C, shaking speed 800 rpm, shaking diameter 3 mm, 96 well microtiter plate, 6 mm FeedBeads (Art.-Nr. SMFB63318).


Without FeedBeads the short period between 8 and 12 hours cultivation time, in which the yeast is producing GFP, is clearly visible. Afterwards glucose is consumed completely and the cells metabolism succumbs to the lack of carbon source.

In contrast, the protein production of Hansenula can be significantly enhanced with FeedBeads. After the cells have grown, actual glucose concentration in the medium is negligible because all the released glucose is expended immediately by the cells. FeedBeads supply the yeast cells with a continuous carbon and energy source but the amount of free glucose is too low to induce catabolite repression.

With the application of FeedBeads catabolite repression in Hansenula polymorpha can be avoided and production of GFP is four times higher when compared with the control. All cultures are synchronized and results are reproducible, even from microplates. 

Escherichia coli: FeedBeads prevent aerobic acetate formation

If the bacterium Escherichia coli takes up more glucose than the central metabolism can process, the excess glucose is converted to acetate even in the presence of oxygen. This overflow metabolism occurs at high glucose concentrations. After complete consumption of the sugar, E. coli is able to utilize the acetate formed earlier as a carbon and energy source in a type of diauxic behavior, gaining an advantage over competing microorganisms.

However for various reasons, acetate is an unwanted by-product in fermentation processes: the weak organic acid uncouples the transmembrane pH-gradient. This interferes with energy metabolism and ∆pH-dependent carriers of the cell. Furthermore, the efficient conversion of glucose to the desired product is impaired.

Aerobic acetate production is prevented when FeedBeads generate substrate limiting conditions. After sufficient biomass has built up, glucose concentration in the medium is too low to elicit overflow metabolism. The toxic effects of acetate do not occur and substrate is used efficiently.

Below, the development of the pH and dissolved oxygen concentration of E. coli cultivated with
and without FeedBeads is demonstrated using the BPM-system:

tl_files/kuhner/product/shaker/add-ons/fb_fig3 (2).png

Fig. 3:  Dissolved oxygen concentration DO [%] and ph [- ] during cultivation of von Escherichia coli in 250 mL shake flasks with and without FeedBeads; Data collected by BPM-system; mineral medium with 20 g/L glucose (Batch) or 4 FeedBeads (Art.-Nr. SMFB63319); liquid volume 10 mL, shaking frequency 250 rpm, shaking diameter 50 mm.

 

 

In batch mode without FeedBeads, the bacteria oxidize glucose and form acetate as a by-product. This is can be seen as the decline in pH. When glucose is completely exhausted after 11 hours of cultivation, DO rises. The cells undergo a metabolic shift towards acetate exploitation, with pH rising accordingly. All energy sources are consumed after 25 hours, breathability of the cells declines with accumulating DO.

With FeedBeads all glucose in the medium is delivered solely by the slow-release substrate. During the growth phase the medium contains comparatively high levels of glucose for few cells. This induces the mechanisms of overflow metabolism and the formation of low concentrations of acetate, indicated by the slight pH decline in the first 7 hours of cultivation.

At this time glucose becomes the limiting factor because of the high number of cells. Breathability declines, oxygen saturation rises. Acetate which formed earlier and glucose are oxidized simultaneously, shown by the brief deviation of DO after 8 hours. Until the experiment is interrupted after 30 hours, E. coli metabolizes glucose released by the FeedBeads without any acetate formation and both pH and DO remain constant during the remaining cultivation time.

In contrast to the culture without slow-release substrate, with FeedBeads the oxygen concentration remains above 45 % saturation at all times. Hence, oxygen limitation does not occur.

Aerobic acetate formation of E. coli and the unwanted side effects can be eliminated when using FeedBeads and substrates are metabolized more effectively devoid of additional charge.