E.Coli Scale-Up Fermentation: From 500 mL MiniBio to 3 L Laboratory Bioreactor

Summary:

Bioprocess scale-up refers to the transfer of a new or developing process from laboratory scale to production scale. Using a small-scale bioreactor to mimic large-scale conditions in order to reduce costs is the first step in improving a bioprocess. In this study, typical E. coli fermentation was carried out in Applikon bioreactors with nominal volumes of 500 mL and 3 L, and scale-up studies were performed from small to large scale. The same cultivation conditions were used for both bioreactors. Results were evaluated based on optical density (OD600) measurements and process control; parameter control (pH, T, dO2) and cell growth profile exhibited the same behavior at both scales.

Introduction

The starting point for commercial production of many biotechnological products such as antibiotics, alcohols, amino acids, enzymes and organic acids is the optimization of small-volume laboratory bioreactors to bioreactor scales of hundreds of thousands of liters. It is important to maintain the same physical and chemical conditions during the scaling process. Chemical conditions include pH, dissolved oxygen level, nutrient concentration and concentration of toxic metabolites. Physical conditions relate to bioreactor configuration and applied power. Small-scale fermenters are preferred in order to obtain large amounts of data in a short time for the purpose of bioprocess optimization. One of the strategies used for scaling among STR-type bioreactors is based on creating similar geometric configuration according to the following equation (1): DT2 / DT1 = (VL2 / VL1) 1/3 DT: inner diameter of culture vessel, VL: working volume; 1 and 2: bioreactors In this study, scalability between two small-scale fermenters was tested. Spargers, impellers and fixed culture vessel ratios were used to enable the application of the same gas supply strategies. For this purpose, a typical E. coli fermentation was carried out using a laboratory bioreactor with a working volume of 2 L and total volume of 3 L and a bioreactor with a working volume of 400 mL and total volume of 500 mL. Conventionally, a 3 L laboratory bioreactor is used in scale-up studies. The reason for this is that the bioreactor at this volume more closely resembles large-scale ones in terms of mixing and aeration, mixing types and parameters. On the other hand, the 500 mL bioreactor requires less bench space and less culture medium. At the same time, due to its smaller volume, it can be autoclaved faster with a smaller autoclave. The aim of this study is to investigate scalability between 3 L and 500 mL bioreactors, and to evaluate process parameters and growth profiles obtained during cultivation.

Materials and Methods

E. coli fermentations were carried out in 3 L and 500 mL bioreactors. The same configuration, pre-culture and culture medium were used for both bioreactors. Additionally, parameters such as pH, dissolved oxygen (dO2) and temperature were controlled equivalently for both systems. Biomass growth during the process was monitored by measuring optical density (OD600) at 600 nm at different time points.

A. Geometric Similarity

To confirm similar geometry between the two culture vessels, equation (1) described above was used: 1- 3 L culture vessel DT1 – 130 mm VL1- 2000 mL D T 2 / DT1 = (VL2 / VL1)1/3 2- 500 mL culture vessel DT2 – 71 mm VL2- 400 mL

B. Microorganism and Media

• Pre-culture of plasmid-free E. coli K12 was grown from glycerol stock in LB medium. The pre-culture was grown in a resonance acoustic incubator (RamBio, Applikon Biotechnology) at 30°C for 16 hours. • LB 2x medium (20 g / L tryptone; 10 g / L yeast extract; 20 g / L NaCl) was used as the culture medium for E. coli fermentation. After dissolution of all medium components, the pH was adjusted to 7 using 0.1 M NaOH solution. • Bioreactors were inoculated using the same pre-culture, with the starting point taken when the OD600 value was 0.2. C. 500 mL MiniBio • A glass autoclavable bioreactor (Applikon Biotechnology) with a working volume of 400 mL and total volume of 500 mL was used for batch fermentation of E. coli. In process control of the bioreactor, my-control software (Applikon Biotechnology), which was specially designed for this purpose, was used. The bioreactor configuration is shown in Figure 1A. The culture vessel configuration contains 2 Rushton impellers, an L-type sparger, DO2, pH and temperature sensors, a sample port and a liquid addition port for pH control. • Samples were taken by drawing 0.2 mL of culture through the sampling septum with a sterile syringe. Optical density (OD) was measured at 600 nm with a spectrophotometer.

D. 3 L Bioreactor

• A glass autoclavable bioreactor (Applikon Biotechnology) with a working volume of 2 L and total volume of 3 L was used for batch fermentation of E. coli. In process control of the bioreactor, ez-control software (Applikon Biotechnology), which was specially designed for this purpose, was used. The bioreactor configuration is shown in Figure 1B. The inserts used were the same as those in the 500 mL bioreactor. The bioreactor was equipped with dO2, temperature and pH sensors and these parameters were controlled with ez-control (Applikon Biotechnology) software. Aeration was performed with an open-tube L-type sparger. The culture vessel configuration contains 2 Rushton impellers, a sample port and an additional port for pH control. • Samples were taken by drawing 1.0 mL of culture through the sampling septum with a sterile syringe. Optical density (OD) was measured at 600 nm with a spectrophotometer.

E. Actuators and Set Points

• dO2 was controlled upward starting from 30% air saturation using an air valve for both bioreactors. • pH was adjusted to 7.0 using ammonia liquid solution (25% V / V). • Temperature was set at 37°C. Bioreactor temperature control was provided by heating / cooling Peltier for the 500 mL and by a heating blanket and cold finger for the 3 L. • Stirrer speed was set to 2000 rpm for the 500 mL and 1000 rpm for the 3 L. For the specified conditions, the KLa measured in both bioreactors was approximately 217 h-1. • Process values of parameters recorded from both controllers (Ez-control for the 3 L bioreactor, my-control for the 500 mL bioreactor) were collected using Applikon Biotechnology's SCADA software. Figure 1A. Configuration of mini-Bio with gas inlets, heating / cooling Peltier and sensors to control temperature, pH and dissolved oxygen. Figure 1B. STR-type (stirred) bioreactor 3 L with dissolved oxygen, temperature and pH measurement sensors.

Results and Discussion

To investigate scalability between 3 L and 500 mL bioreactors, parallel E. coli fermentations were carried out for 7 hours. Optical density (OD) results measured at different time points are shown in Figure 2. Figure 2. Semi-logarithmic representation of E. coli growth. The optical density value shown was obtained by dividing the natural log of the optical density at the time point by the optical density at the beginning. Growth rates obtained in the exponential phase were 0.854 for the 3L bioreactor and 0.859 for the 500 mL bioreactor. After 7 hours of batch fermentation, the growth curves obtained were similar for both scales. Final biomass obtained in the 3 L and 500 mL fermenters were 7.12 ± 0.07 and 6.81 ± 0.09, respectively. The results of this study showed that growth conditions in 3 L and 500 mL bioreactors are close to each other and are repeatable within themselves. The fact that the same KLa value could be obtained for both scales and that the two culture vessels had similar geometry showed that the said configurations were suitable for scale-up and scale-down.

Controlled Parameters

Figures 3A, 3B and 3C show the comparison between parameters controlled with ez-control (3 L bioreactor) and my-control (500 mL bioreactor). Figure 3A. Dissolved oxygen (% air saturation) in the 3 L bioreactor with ez-control and air valve as actuator - blue lines; dissolved oxygen (% air saturation) in the 500 mL bioreactor with my-control and air valve as actuator - green lines. dO2 During the 7-hour batch fermentation, air was used to set at 30%. The results in Figure 3A showed that, as expected, good control was achieved at the set point. The comparison between two different controllers in bioreactors of two different scales showed that both Ez-control and my-control successfully controlled parameters throughout the process. Additionally, the collected data showed that the culture had the same growth behavior at two different scales. These results strengthen the success of scalability between 3 L and 500 mL bioreactors. Figure 3B. pH in the 3 L fermenter with ez-control and an alkaline peristaltic pump as actuator - blue lines; pH in the 500 mL bioreactor with my-control and air valve as actuator - green lines. Figure 3B shows the pH profile during fermentation. While the set point was pH 7, only one actuator was used for upward control during the culture period, meaning that when the pH dropped due to acid produced during bacterial growth, base solution was added. The absence of an actuator for downward control in the pH loop explains why the pH value was above the set point. The proximity of pH values observed during batch fermentation (above the set point) confirmed that both bioreactors provided the same growth conditions, especially when there was no specific control. Figure 3C. Temperature in the 3 L fermenter with ez-control and cold finger and heating blanket used - blue lines; temperature in the 500 mL bioreactor with my-control and heating/cooling Peltier used - green lines. Figure 3C shows that temperature in both systems could be controlled at a set point of 37°C. Although different actuators were used for temperature control in both systems - 500 mL a heating/cooling Peltier element; 3 L bioreactor heating blanket (below set point) and cold finger (above set point) - optimum temperature control at the determined point was achieved in both systems.

Conclusions:

• Parallel E. coli fermentations were carried out in 3 L and 500 mL bioreactors using the same conditions. Biomass results showed that both bioreactors allowed similar growth rates. OD600 of 7.12 and 6.81 were obtained for 3 L and 500 mL bioreactors, respectively, and it was observed that the two bioreactors showed very close biomass results with a difference of 4.4%. • Two different Applikon controllers were used in the two bioreactors, ez-control for 3L and my-control for 500 mL. The only difference between the two configurations was temperature control. The results showed that dO2, pH and temperature parameters were controlled perfectly in two bioreactors of different scales and E. coli grew at the same level. • The study demonstrated scalability between Applikon's 3 L and 500 mL bioreactors and showed that both bioreactors are reliable options for small-scale processes.

Reference Carolina Santos Fernandes, MSc, Cristina Bernal Martinez, PhD, Paul ter Huurne, BSc, Timo Walvoort, MSc, "E.coli scale-up fermentation from mini to laboratory bioreactor" poster.

Hakan Dibek / Senior Chemist / Applikon Biotechnology / Product Manager / Ant Teknik Cihazlar