Method development and quantification of all B vitamins and selected biosynthetic precursors in winter and spring samples from the North Sea and de novo synthesized by Vibrio campbellii

Abstract

The simultaneous analysis of B vitamins and biosynthetic precursors in seawater is of great interest as it gives a better insight into the fluctuations of their availability for marine organisms and thus allows us to better understand the nature of interactions between prototrophic and auxotrophic organisms. The analysis of these micronutrients is challenging, as they are usually present in the marine environment in minuscule quantities only. Additionally, some of them are light- and heat-sensitive which complicates their handling. To meet these circumstances, the presented sample work-up method for seawater started with a freeze-drying step for volume reduction of the sample. This shortened the duration of the subsequent solid phase extraction and resulted in an increase in recovery. When comparing three solid phase extraction materials (C₁₈, HLB, PPL), PPL was found to be the most suitable. This method was applied for the quantification of all eleven B vitamins and six precursors in waters from the southern North Sea taken in winter and spring. Quantification was performed by LC-MS. The winter samples showed higher concentrations for nearly all analytes, in contrast to the uptake of most nutrients at the beginning of a potential algal bloom in spring and low analyte concentrations in springtime waters. In addition, the marine bacterium Vibrio campbellii, prototrophic for most B vitamins, was cultivated without the addition of B vitamins in order to measure them intra- and extracellularly during growth. In most cases, intracellular concentrations per cell decreased towards the end of the exponential growth phase, while extracellular concentrations increased. Extracellular B vitamin and precursor concentration measurements show that V. campbellii is in exchange with its environment and thereby possibly enables growth of B vitamin auxotrophs in the environment.