Under well-defined laboratory conditions, we grew R. pomeroyi DSS-3 and A. macleodii MIT1002 in batch cultures on a monosaccharide (glucose) and organic acid (acetate), provided either individually or in combination, and all at the same carbon equivalent. This batch culturing approach mimicked bacterial DOC assimilation in short-lived substrate ‘hot spots’, such as those formed by high phytoplankton extracellular release at peak photon availability. Measurements were made of bacterial metabolite uptake, respiration, and biomass accumulation through a growth cycle. Insights into bacterial core metabolism came from gene and protein expression measured at intervals during growth. Curated genome-scale models (flux balance analysis; FBA) were used to explore the metabolic foundation of CO2 production for insights into determinants of BGE and bCUE.

Samples were collected for proteomic analysis during the exponential growth phase from liquid cultures. Proteomic samples were pelleted by centrifugation, frozen at –80 ̊C, and analyzed at the Bioinorganic Chemistry Laboratory at the Woods Hole Oceanographic Institution (WHOI).

Protein extracts from the biological triplicates were analyzed by liquid chromatography-mass spectrometry (LC-MS) (Michrom Advance HPLC coupled to a Thermo Scientific Fusion Orbitrap mass spectrometer with a Thermo Flex source). Each sample was concentrated onto a trap column (0.2 x 10 mm ID, 5 µm particle size, 120 Å pore size, C18 Reprosil-Gold, Dr. Maisch GmbH) and rinsed with 100 µL 0.1% formic acid, 2% acetonitrile (ACN), 97.9% water before gradient elution through a reverse phase C18 column (0.1 x 500 mm ID, 3 µm particle size, 120 Å pore size, C18 Reprosil-Gold, Dr. Maisch GmbH) at a flow rate of 250 nL/min. The chromatography consisted of a nonlinear 190 min gradient from 5% to 95% buffer B, where A was 0.1% formic acid in water and B was 0.1% formic acid in ACN (all solvents were Fisher Optima grade). The mass spectrometer was set to perform MS scans on the orbitrap (240000 resolution at 200 m/z) with a scan range of 380 m/z to 1580 m/z. MS/MS was performed on the ion trap using data-dependent settings (top speed, dynamic exclusion 15 seconds, excluding unassigned and singly charged ions, precursor mass tolerance of ±3ppm, with a maximum injection time of 50 ms).

Curated genome-scale models (flux balance analysis; FBA) were used to explore the metabolic foundation of CO2 production for insights into determinants of bacterial growth efficiency (BGE) and bacterial carbon use efficiency (bCUE).