Carbon as a Parameter in
Bacterial Systems Growth Limitation and
Substrate Utilization Studies
M. D. RICKARD, Research Engineer
Chemicals Laboratory
W. H. RILEY, Biochemist
Biochemical Research Laboratory
1701 Building
The Dow Chemical Company
Midland, Michigan
INTRODUCTION
The need for more definitive parameters for following bacterial growth and
substrate utilization has led the authors to the application of infrared carbon analysis as a parameter for the study of the behavior of bacterial systems. The
method is based on the rapid combustion of a micro sample in oxygen and measurement of the CO2 produced with a nondispersive infrared analyzer (1). Although
designed primarily for the analysis of the carbon content of waste waters (2), the
technique has application to the study of bacterial systems since it makes possible
a rapid, accurate determination of tne organic carbon content of biological samples in solution or suspension.
The object of this paper is to survey the utility of carbon analysis in following the metabolism of organic compounds during the growth of a bacterial culture
in a defined medium. The relationships obtained among cellular carbon, exogenous soluble carbon, and viable count during bacterial growth are shown along
with the rates of synthesis of cellular material.
MATERIALS AND METHODS
Culture Methods
Aerobacter aerogenes ATCC No. 8308 was used throughout these studies. The
organism was cultured in a glucose-mineral salts medium in an incubated shaker
at 30 C. The composition of the medium used is shown in Table I. The stock
culture was maintained on nutrient agar slants and was passed through three serial
transfers in the medium before the cells were used.
Experimental Treatment
Cells from a 24-hr culture were harvested by centrifugation and washed with
sterile phosphate buffer (M/15 - pH 7.0). These cells were then suspended in
fresh sterile medium for use as an inoculant.
The experiments were performed in a 16-liter fermenter which was maintained in a 30 C water bath. Aeration and agitation of the contents were accomplished through the use of an air diffuser placed in the bottom of the vessel. Filtered, compressed air was passed through the diffuser at a sufficient rate to insure
adequate aeration and mixing of the culture during respiration. The fermenter's
contents were sampled through the use of a positive siphon.
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