Label-free, single-cell-precise, and monomeric-unit-resolved monitoring of biopolymer fermentation by ramanomics

Rapid yet high-information-content monitoring of cellular metabolism during fermentation is highly desirable for process optimization. While single-cell Raman spectroscopy can rapidly distinguish intracellular biopolymer classes, finer-resolution discrimination of monomeric units remains challenging. In this study, we demonstrated that the ramanome can classify polyhydroxyalkanoate (PHA)-producing Halomonas bluephagenesis cells that synthesize either polyhydroxybutyrate or poly(3-hydroxybutyrate-co-4-hydroxybutyrate) with 99.75% accuracy and simultaneously quantify total intracellular PHA and its constituent monomeric units (3-hydroxybutyrate and 4-hydroxybutyrate) at the single-cell level (median absolute deviation <3.8%). Such information enabled timely, data-driven selection of the optimal harvest time and revealed precursor competition between nucleic acid and PHA biosynthesis. Moreover, when monitoring PHA production in an industrial-scale 5000 l-fermenter, our method achieved a 12-min turnaround time, representing a more than100-fold acceleration over gas chromatography. Furthermore, tests on protein production by Saccharomyces cerevisiae and on lipid synthesis in Rhodococcus opacus supported its versatility. Thus, ramanomics is a valuable approach for process control and strain evaluation in biomanufacturing.