to BioTechniques free email alert service to receive content updates.

protocol
Basics of Benchtop Fermentor Operation for Growth of E. coli
Sponsored,vendor-submitted protocol   Sponsored by New Brunswick Scientific    Published in BioTechniques Protocol Guide 2008 (p.29)

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





More Information
 
Do you have question about this protocol? Would you like more information about this protocol?
Submit your questions here and Biotechniques will forward your question and contact information from our database to an expert at New Brunswick Scientific who will contact you.
Yes, please forward my question, along with my name, title, company, and e-mail address to New Brunswick Scientific
BioTechniques will forward your question to the company, who will contact you directly by email.
Request more information
 
Name Question
Job Title
Company
Email
Contact Information
Protocol Categories
rightcontent.jsp called otherwise condition rightcontent.jsp called 222222222222222222222
Search Protocols
Search our protocols for your specific technique.
Submit Your Protocols
BioTechniques is always looking for author-submitted protocols related to peer-reviewed papers, so submit your protocols today to our editorial team. Suppliers or advertisers interested in publishing their protocols should also submit.
Click here for more information