Raman spectroscopy - An innovative and versatile tool to follow the respirational activity and carbonate biomineralization of important cave bacteria

Raman gas spectrometry is introduced as a unique tool for the investigation of the respiratory activity that is indicative for growth of bacteria involved in biomineralization. Growth of these bacteria cannot be monitored using conventional turbidity-based optical density measurements due to concomitant mineral formation in the medium. The respiratory activity of carbonate-precipitating Arthrobacter sulfonivorans, isolated from the recently discovered Herrenberg Cave, was investigated during its lifecycle by means of innovative cavity-enhanced Raman gas analysis. This method allowed rapid and nonconsumptive online quantification of CO2 and O2 in situ in the headspace of the bacterial culture. Carbon dioxide production rates of A. sulfonivorans showed two maxima due to its pleomorphic growth lifecycle. In contrast, only one maximum was observed in control organism Pseudomonas fluorescens with a one-stage lifecycle. Further insight into the biomineralization process over time was provided by a combination of Raman macro- and microspectroscopy. With the help of this spatially resolved chemical imaging of the different types of calcium carbonate minerals, it was elucidated that the surface of the A. sulfonivorans bacterial cells served as nuclei for biomineralization of initially spherical vaterite precipitates. These vaterite biominerals continued growing as chemically stable rock-forming calcite crystals with rough edges. Thus, the utilization of innovative Raman multigas spectroscopy, combined with Raman mineral analysis, provided novel insights into microbial-mediated biomineralization and, therefore, provides a powerful methodology in the field of environmental sciences