Technical Support

Animal-Product-Free LB Broths Technical Brief

Sheldon E. Broedel, Jr., Ph. D.
Athena Environmental Sciences, Inc., Baltimore , MD

Introduction

LB both has been the venerable culture medium formulation used by researchers for over 50 years. First introduced in 1955 by two groups,1,2,3 LB broth has three different variations, designated as LB Miller,4 LB Lennox, and LB Luria, named after the researchers who developed the formulations. LB is a simple medium composed of three ingredients: yeast extract, sodium chloride and casein hydrolysate. Table 1 gives the formulation for each of the three different variations.

Table 1.

Product Specifications
Ingredient Miller Lennox Luria
Yeast Extract 5 5 5
NaCl 10 5 0.5
Casein Hydrolysate 10 10 10

In recent years, the use of animal-derived raw materials has become a concern to manufacturers of biopharmaceutical products, because they can contain harmful contaminants.5 Initially, this concern was confined to the manufacturing end of the industry. As the regulatory environment has evolved with regard to the use of animal-derived materials, greater restrictions are being placed at earlier stages of product development. The consequence is a pressing need for media formulations which are not made from animal sources.

To address this issue, we have developed a non-animal protein hydrolysate to replace the casein hydrolysate. An obvious choice for the casein replacement would be plant-derived hydrolysates, however, no one plant protein source has the same amino acid composition as casein. Therefore, the direct replacement of casein hydrolysate with a plant protein hydrolysate would not yield a medium with the same nutritional composition. To overcome this limitation, we have designed a blended plant-based protein hydrolysate that matches casein hydrolysate in composition and in performance.

Results and Discussion

Using an in silico model, several candidate blends were devised. The algorithm applied first searched among 30 different plant protein hydrolysates for those with amino acid compositions that most closely matched casein’s composition for each individual amino acid. The model then used an iterative process of forming hypothetical blends that resulted in eight possible variations with compositions closely matching casein. These base formulations were then used to derive a mathematical model for optimizing the blending.

Growth curves for strain HMS174 cultured in various APF LB formulations

Figure 1. Growth curves for strain HMS174 cultured in various APF LB formulations. To 1mL of each medium in a 24-well culture dish, a 0.1mL aliquot of an overnight culture grown in LB (Miller) was added. The cultures were incubated in a Tecan Genios microplate reader with shaking at 37°C. The absorbance at 600nm was measured at 1 hour intervals. Duplicate wells of non-inoculated medium served as blanks. The net absorbance values were plotted to generate the growth curves.

To determine the best formulation, the candidate hydrolysates were blended according to a simplex lattice mixture design.6 Eleven prospective formulations were tested empirically by culturing E. coli strain HMS174 in medium composed of 5g/L yeast extract, 10 g/L NaCl and 15 g/L blended hydrolysate. Growth was monitored spectrophotometrically over a 24 hour period and the growth rate and yield calculated. Figure 1 shows the results of this initial screen. The growth parameter values were used to construct a mathematical model which to predicted blends that would produce a growth rate and yield that matched the LB reference medium. This model predicted two possible formulations, designated 12 and 13. Two additional formulations, designated 14 and 16, were predicted by examining the compositions that gave the same growth characteristics as the reference medium and selecting compositions intuitively based on this comparison. Figure 2 (top panel) shows the growth curves of strain HMS174 cultured in these formulations. The growth rates of strain HMS174 in all four formulations were statistically identical (p = 0.41) to the reference LB medium. Formulations numbered 14 and 16 gave the closest values and were selected for a second analysis (Fig. 2, bottom panel). In this test, the growth rates were again identical between the three media. The biomass yields were the same up to about 12 hours of growth after which time the absorbance values for the reference LB cultures appeared to decrease as compared to the new formulations. The basis for this is not known.

Growth curves of strain HMS174 cultured in APF LB formulas Growth curves of strain HMS174 cultured in APF LB formulas

Figure 2. Growth curves of strain HMS174 cultured in APF LB formulas predicted by the initial screen. The cultures were set up and incubated as described in Figure 1. The top panel shows growth in Formulas 12, 13 and 14 along with medium containing only yeast extract and NaCl (“No hydrolysate”). The bottom panel shows growth in Formulas 14 and 16 with seven replicate cultures.

The growth of other commonly used E. coli strains were tested using Formula 16. Figure 3 shows that a wild-type strain, W3110, along with four commonly used strains exhibited nearly identical growth characteristics in the animal-product-free Formula 16 as in the standard LB broth. There was a slightly higher yield in some cases for the APF Formula 16. Strain JM109 grew less well in the standard LB formula than in the APF formula, however, its growth rate and yield in this experiment was typical for strain JM109 in our laboratory (data not shown).

Growth curves for wild-type and four commonly used E. coli strain

Figure 3. Growth curves for wild-type and four commonly used E. coli strain. Duplicate cultures of each strain were grown as described in Figure 1 in LB (Miller) or APF LB Formula 16.

From the above experiments, APF Formula 16 was deemed to be the one which most closely matched the standard LB formulations. While there were formulations that showed higher growth rates and yields, the intent was to match the growth of various strains of E. coli to that observed in standard LB broth. In cases where higher growth rates and yields may be important, such as in the production of a recombinant protein, formulations giving higher biomass could be advantageous. Table 2 gives the composition of APF LB in the three different versions using the newly designed casein hydrolysate replacement and Table 3 shows the final amount of each amino acid per liter of culture.

Table 2.

Compositions of APF LB Broth (g/L)
Ingredient Miller Lennox Luria
Yeast Extract 5 5 5
NaCl 10 5 0.5
Atholate™ 15 15 15

Table 3.

Amino acid composition in LB media (mg/L)
ala arg asp cys glu gly met phe pro
Casein Hydrolysate 301 313 674 30 1858 189 272 398 884
Athena's APF Blend 16 367 660 868 125 2438 369 164 469 698
ser thr trp his lle leu lys tyr val
Casein Hydrolysate 514 418 98 222 436 746 677 282 590
Athena's APF Blend 16 479 341 54 206 386 672 414 302 428
Totals: Casein Hydrolysate 8,902
Athena's APF Blend 16 9,437

References

1. Luria, S. E. and J. W. Burrous. 1955. Hybridization between Escherichia coli and Shigella. J. Bacteriol. 74:461-476.
2. Lennox, E. S. 1955. Transduction of linked genetic characters of the host by bacteriophage P1. Virology. 1:190-206.
3. Luria, S. E., J. N. Adams, and R. C. ting. 1960. Transduction of lactoseutilizing ability among strain of E. coli and S. dysenteriae and the properties of the transducing phage particles. Virology. 12:348-390.
4. Miller, J. H. 1972. Experiments In Molecular Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York.
5. CDRH BSE Working Group. Guidance for FDA Reviewers and Industry: Medical Devices Containing Materials Derived from Animal Sources (Except for In Vitro Diagnostic Devices). Nov. 6, 1998. U.S. FDA. http://origin.www.fda.gov/cdrh/ode/88.html
6. Montgomery, D. C. 2001. Design and Analysis of Experiments. 5th Ed. John Wiley & Sons, Inc. Hoboken, NJ. ISBN 0-471-31649-0. pp 472-484.