Use of heterotrophic CO 2 assimilation as a measure of metabolic activity in planktonic and sessile bacteria

Abstract

We have examined whether assimilation of CO 2 can be used as a measure of metabolic activity in planktonic and sessile heterotrophic bacteria. CO 2 assimilation by environmental samples and pure cultures of heterotrophic bacteria was studied using 14CO 2 and 13CO 2 as tracers. Heterotrophic growth on complex organic substrates resulted in assimilation of CO 2 into cell biomass by activated sludge, drinking water biofilm, and pure cultures of Escherichia coli ATCC 25922, Es. coli ATCC 13706, Rhodococcus ruber, Burkholderia sp., Bacillus circulans, Pseudomonas putida, Pseudomonas stutzeri, and Pseudomonas aeruginosa. Analysis of 13C-labelled phospholipid fatty acids (PLFAs) confirmed that heterotrophic bacteria may assimilate 13CO 2 into cell macromolecules such as membrane lipids. All major PLFAs extracted from activated sludge and drinking water biofilm samples were enriched in 13C after incubation with CO 2. Between 1.4% and 6.5% of the biomass produced by cultures of P. putida and a drinking water biofilm during growth in complex media was apparently derived from assimilation of CO 2. Resting cells assimilated less CO 2 compared to actively growing cells, and CO 2 assimilation activity correlated with the amount of biomass produced during heterotrophic growth. The 14CO 2 assimilation assay was evaluated as a tool to examine inhibitory effects of biocides on planktonic and sessile heterotrophs (biofilms). On the basis of 14CO 2 assimilation activity, the minimum inhibitory concentration (MIC) of benzalkonium chloride was estimated to 21.1 and 127.2 mg l −1 for planktonic and biofilm samples, respectively. The results indicate that assimilation of isotopically labelled CO 2 can be used as a relatively simple measure of metabolic activity in heterotrophic bacteria. CO 2 assimilation assays may be used to study the effects of antimicrobial agents on growth and survival of planktonic and sessile heterotrophic organisms.