Development of a high-throughput method for investigating carbon allocation in microbial pure cultures

Since the discovery of microbes, microbial carbon metabolism has been a central research focus, with significant progress in understanding their metabolic pathways under different carbon sources. However, most studies have primarily examined metabolic mechanisms and gene regulation, while how microbes allocate absorbed carbon to growth, respiration, and extracellular metabolism has rarely been quantified in its entirety. There is strongly rising interest in understanding and modeling soil microbial carbon use efficiency (CUE), but results are currently only based on data of growth and respiration, not accounting for extracellular product formation and total substrate uptake. This therefore potentially ignores a large fraction of stress- and resource-limited (extra)cellular metabolism. This gap limits a comprehensive understanding of microbial carbon allocation and its environmental adaptability, highlighting the need for innovative approaches to address this critical aspect.

To this end, we are currently developing high throughput methodology to measure growth, respiration, and the excretion of extracellular enzymes (EE), extracellular polysaccharides (EPs) and extracellular metabolites (EM) in Bacillus subtilis, Escherichia coli and Saccharomyces cerevisiae cultures. In response to carbon concentration, carbon: nutrient stoichiometry, temperature, and oxygen stress, we will assess microbial carbon allocation of three representative microbial species. For this regard, substrate uptake, growth, respiration, extracellular protein and extracellular polysaccharide production will be quantified in a microtiter plate format assay. The growth use efficiency of Bacillus subtilis cultured at 28°C with 0.4% glucose was determined to range between 20% and 30% with this assay. This study addresses a major gap in microbial carbon allocation research, revealing how environmental factors influence anabolic and catabolic transitions, and affect intracellular and extracellular metabolism, and providing important insights into microbial adaptation and ecological roles.