Introduction
While use of shake flasks, T-flasks, or roller bottles have become the lab standard in most fermentation and cell culture research labs, lack of controls for pH, dissolved oxygen and other parameters cause an inherent limitation in cell yields and protein expression. When higher yields are needed, rather than inoculate, feed and maintain cultures using dozens or even hundreds of bottles or flasks, a benchtop fermentor or cell-culture bioreactor can be used to increase yields by 10-fold or more.
This Application Report describes a step-by-step procedure for growing E. coli in New Brunswick Scientific's BioFlo® 310 benchtop fermentor. The BioFlo 310 is a versatile, powerful system, designed for high-yield growth of a wide variety of organisms, ranging from yeast and bacteria to mammalian, plant, and insect cells. The system is available with interchangeable vessels, sized 2.2–14.0 liters, and comes as a pre-packaged kit for ordering simplicity, or can be tailor-configured by selecting from dozens of standard options. Materials and Methods The Fermentor
A pre-configured BioFlo 310 Fermentor Kit with 7.5 L vessel with Rushton blades (NBS Catalog Number M1287-1172) was used for an E. coli fed-batch fermentation. This system includes a 15" industrial color touchscreen controller to regulate agitation, temperature, pH, DO, Foam/Level, three pumps, gases and much more. A Thermal Mass Flow Controller (TMFC) for regulating gas flow in a range of 0.4–20 Standard Liters per Minute (SLPM) is also standard, and options for multiple mass flow controllers and/or other gas flow settings are available as standard options. In this run, an optional BioCommand® Plus supervisory software package (NBS Catalog Number M1291-0000) was used to control the feed schedule and log data. To optimize your results, you can also connect and control numerous additional options directly to the BioFlo 310, including scales, analyzers and sensors for optical density (OD), CO2, Redox and more. This paper presents a fermentation run that was not fully optimized to obtain the highest yields possible–rather it is meant to serve as a guide for basic procedures and materials. Inoculum
The inoculum was prepared using LB broth at a 25-g/L concentration as the shake flask medium. The inoculum was cultivated overnight on an NBS orbital shaker at 240 rpm. OD600 was between 2 and 4 at the time of inoculation. Inoculum volume was 2-5% of the 5 L working volume, or 250 ml. Medium
The initial fermentor medium composition included:
Potassium phosphate monobasic, KH2PO4 3.5 g/L
Potassium phosphate dibasic, K2HPO4 5.0 g/L
Ammonium Sulfate, (NH4)2SO4 5.0 g/L
Yeast Extract
(note that we use Tastone 900AG from Sensient) 5.0 g/L
Antifoam, (Breox Foam Control Agent FMT 30) 1.00 ml/L
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