March 15, 2015 (Vol. 35, No. 6)

Tibor Anderlei Ph.D.
David Laidlaw CEO Kuhner Shaker
Kristina Bruellhoff CEO and Managing Director PS Biotech
Sebastian Selzer

Slow Release Technology for Small-Scale Bioreactor Operations

Fed-batch cultivation is a common production process in the biotech industry. Upstream research and screening efforts are, however, often carried out as batch processes due to a lack of feeding technology available at small scales.

A consequence of the batch/fed-batch inconsistency is that primary screening for production strains is performed under different physiological conditions than will later be used in production-scale cultivation. These process differences could result in a suboptimal choice of a production strain during the selection phase.

By matching cultivation parameters at small scale as close as possible to parameters used in production, the advancement of false-positives identified during screening into the next stage of production can be minimized.

To overcome this issue, different fed-batch techniques have been developed for small-scale cultivations. One such technique is to use complex compounds (e.g., starch), which are converted by enzymatic reactions into an easily available carbon source (C-source). This approach has been named simultaneous saccharification and fermentation (SSF) and by tuning the enzyme activity and reagent concentration a fed-batch process is mimicked.

Another approach to supply a C-source is to employ slow-release particles where substrate release into the media is driven by diffusion from an inert matrix. This release technology is named FeedBeads (for flasks) and Feed Plate® (for microtiter plates) and was developed at the University of Technology in Aachen, Germany.

FeedBeads and Feed Plate enable users to run fed-batch processes in shake flasks or microtiter plates without tubing, pumps or adding foreign enzymes and reagents to the media. FeedBeads and Feed Plate are polymer-based feeding systems that release substrates to the fermentation broth in a defined way. Both systems support screening for optimal production strains under relevant fed-batch process conditions and simplify scaleup from shaken bioreactors to stirred-tank bioreactors.

Through the use of these feeding systems many disadvantages of batch approaches such as crabtree-effect, overflow metabolism, or catabolite repression can be avoided. The matrix of both systems is silicon containing substrates such as crystalline glucose, although any crystalline substrate is possible. The substrate matrix in Feed Plate is fixed at the bottom of each well (Figure 1), FeedBeads are small pellets which swirl freely in the fermentation broth (Figure 2).

Figure 1. Feed Plate is a polymer-based release system for fed-batch feeding on high-throughput scale.

E. coli Case Study

If Escherichia coli uptakes more glucose than its central metabolism can process, excess glucose is shunted to acetate even in the presence of oxygen. This overflow metabolism occurs at high glucose concentrations and, after depletion of the soluble glucose, E. coli then utilizes 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, interfering  with energy metabolism and ΔpH-dependent transporters in the cell. Furthermore, the efficient conversion of glucose to the desired product is impaired.

By releasing glucose at substrate-limiting rates, FeedBeads and Feed Plate prevent aerobic acetate production. After sufficient biomass has built up, glucose concentrations in the medium remain too low to elicit overflow metabolism, and the toxic effects of acetate production do not occur. In this case the glucose substrate is used as efficiently as would be expected in later large-scale production processes. 

Figure 2. FeedBeads are polymer particles enabling the user to run fed-batch processes in shake flasks or microtiter plates.

Figure 3 illustrates the development of the pH and dissolved oxygen concentration of E. coli cultivated with and without FeedBeads using an online bioprocess monitoring system.

Polymer-based release systems allow a defined slow release of nutrient into the cultivation media and offer numerous advantages over enzymatic and physical reagent delivery approaches. Theoretically, the polymers allow incorporation of every crystalline substance into a matrix so that nitrogen, phosphate, and other nutrient sources may also be supplied.

Since the polymer-release technique is less sensitive to influencing parameters such as temperature or pH, they offer reliable release rates and enable high reproducibility. They can be used with standard lab equipment, eliminating the need for costly investments in fed-batch setups such as pumps, valves, or supply containers.

FeedBeads and Feed Plate show excellent storage properties and are easy to handle. Microbial side products such as amylases or proteases do not interfere with the matrices as is the case with SSF.
Microtiter plates are widely used tools for high-throughput applications such as screenings of libraries, enabling easy handling of large numbers of parallel samples at once. Due to the fact that FeedBeads and Feed Plate are based on the same technology both systems are comparable.

Thus, it seems appropriate to start screenings with Feed Plate and scale up using the FeedBeads. Existing processes and production conditions can be optimized easily. 

Figure 3. Dissolved oxygen concentration DO [%] and pH [-] during cultivation of Escherichia coli in 250 mL shake flasks with and without FeedBeads. Data collection system: BPM (Kuhner Shaker); mineral medium: 20 g/L glucose (Batch) or four FeedBeads (Art.-Nr. SMFB63319); liquid volume: 10 mL; shaking frequency: 250 rpm; shaking diameter: 50 mm (ISF1-X, Kuhner Shaker).

Tibor Anderlei, Ph.D. ([email protected]), is director of sales and business development at Adolf Kühner. David Laidlaw is CEO of U.S.-based Kuhner Shaker. Kristina Bruellhoff serves as CEO and  a managing director and Sebastian Selzer is chief technology officer and a managing director at PS Biotech.

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