Assessing Microbial Monitoring Methods for Challenging Environmental Strains and Cultures

Abstract

This paper focuses on the comparison of microbial biomass increase (cell culture growth) using field-relevant testing methods and moving away from colony counts. Challenges exist in exploring the antimicrobial growth of fastidious strains, poorly culturable bacteria and bacterial communities of environmental interest. Thus, various approaches have been explored to follow bacterial growth that can be efficient surrogates for classical optical density or colony-forming unit measurements. Here, six species grown in pure culture were monitored using optical density, ATP assays, DNA concentrations and 16S rRNA qPCR. Each of these methods have different advantages and disadvantages concerning the measurement of growth and activity in complex field samples. The species used as model systems for monitoring were: Acetobacterium woodii, Bacillus subtilis, Desulfovibrio vulgaris, Geoalkalibacter subterraneus, Pseudomonas putida and Thauera aromatica. All four techniques were found to successfully measure and detect cell biomass/activity differences, though the shape and accuracy of each technique varied between species. DNA concentrations were found to correlate the best with the other three assays (ATP, DNA concentrations and 16S rRNA-targeted qPCR) and provide the advantages of rapid extraction, consistency between replicates and the potential for downstream analysis. DNA concentrations were determined to be the best universal monitoring method for complex environmental samples.