Abstract
In various (industrial) conditions, cells are in a non-growing but metabolically active state in which de novo protein synthesis capacity is limited. The production of a metabolite by such non-growing cells is dependent on the cellular condition and enzyme activities, such as the amount, stability, and degradation of the enzyme(s). For industrial fermentations in which the metabolites of interest are mainly formed after cells enter the stationary phase, the investigation of prolonged metabolite production is of great importance. However, current batch model systems do not allow prolonged measurements due to metabolite accumulation driving product-inhibition. Here we developed a protocol that allows high-throughput metabolic measurements to be followed in real-time over extended periods (weeks). As a validation model, sugar utilization and arginine consumption by a low density of translationally blocked Lactococcus lactis was designed in a defined medium. In this system L. lactis MG1363 was compared with its derivative HB60, a strain described to achieve higher metabolic yield through a shift toward heterofermentative metabolism. The results showed that in a non-growing state HB60 is able to utilize more arginine than MG1363, and for both strains the decay of the measured activities were dependent on pre-culture conditions. During the first 5 days of monitoring a ∼25-fold decrease in acidification rate was found for strain HB60 as compared to a ∼20-fold decrease for strain MG1363. Such measurements are relevant for the understanding of microbial metabolism and for optimizing applications in which cells are frequently exposed to long-term suboptimal conditions, such as microbial cell factories, fermentation ripening, and storage survival.
Highlights
The widespread use of bacteria in many biotechnological applications is attributed to their growth ability and to their metabolic persistence under non-growing/dormant condition
The glycolytic flux of Lactococcus lactis has been reported to run at maximal rate during balanced growth in batch culture (Koebmann et al, 2002b)
The novelty in the presented approach lies in the fact that real-time monitoring of lactic acid formation or ammonium accumulation due to arginine deamination can be performed for up to several weeks by a relatively straightforward and simple microplate-based assay
Summary
The widespread use of bacteria in many biotechnological applications is attributed to their growth ability and to their metabolic persistence under non-growing/dormant condition. The arrest of cell division coincides with limited denovo protein synthesis, whereas metabolic activity and survival can be maintained over a long period of time (Ercan et al, 2015; Erkus et al, 2016). This non-growing state can be induced by adverse circumstances, e.g., starvation, lethal stress or inhibitory compounds (Oliver et al, 1995; Keren et al, 2004; Magajna and Schraft, 2015), as commonly found in industrial processes, such as bioreactor metabolite production (Förberg et al, 1983), wastewater treatment (Witzig et al, 2002), and food processes (Millet and Lonvaud-Funel, 2000). The study of persisting metabolic activity in nongrowing cells is of relevance for food fermentation processes, and the ability to steer the activity of such cells can strongly contribute to process control
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