Probing the Kinetic Anabolism of Poly-Beta-Hydroxybutyrate in Cupriavidus necator H16 Using Single-Cell Raman Spectroscopy.

Zhanhua Tao,Yong-Qing Li,Pengfei Zhang,Lixin Peng,Guiwen Wang

doi:10.3390/s16081257

Abstract

Poly-beta-hydroxybutyrate (PHB) can be formed in large amounts in Cupriavidus necator and is important for the industrial production of biodegradable plastics. In this investigation, laser tweezers Raman spectroscopy (LTRS) was used to characterize dynamic changes in PHB content—as well as in the contents of other common biomolecule—in C. necator during batch growth at both the population and single-cell levels. PHB accumulation began in the early stages of bacterial growth, and the maximum PHB production rate occurred in the early and middle exponential phases. The active biosynthesis of DNA, RNA, and proteins occurred in the lag and early exponential phases, whereas the levels of these molecules decreased continuously during the remaining fermentation process until the minimum values were reached. The PHB content inside single cells was relatively homogenous in the middle stage of fermentation; during the late growth stage, the variation in PHB levels between cells increased. In addition, bacterial cells in various growth phases could be clearly discriminated when principle component analysis was performed on the spectral data. These results suggest that LTRS is a valuable single-cell analysis tool that can provide more comprehensive information about the physiological state of a growing microbial population.

Highlights

Plastic materials play extremely important roles in contemporary life because of their desirable properties
The present study demonstrated the use of laser tweezers Raman spectroscopy (LTRS) for monitoring dynamic changes in the The present study demonstrated the use of LTRS for monitoring dynamic changes in the contents contents of PHB and of other common biomolecules inside C. necator H16 cells during batch growth of PHB and of other common biomolecules inside C. necator H16 cells during batch growth at both at both the population and single-cell levels
PHB accumulation began in the early stage of the batch the population and single-cell levels

Summary

Introduction

Plastic materials play extremely important roles in contemporary life because of their desirable properties. Pollution caused by petroleum-based plastic waste has generated much interest in the development of more environmentally friendly substitutes, such as biodegradable polymer materials. Poly-beta-hydroxybutyrate (PHB)—a member of the polyhydroxyalkanoate family that is produced by microbial fermentation—is an attractive substitute for conventional petrochemical plastics, and has similar material properties to various thermoplastics [1]. Its excellent biodegradability, biocompatibility, piezoelectricity, and optical activity make it a widely used material in the fields of medicine, agriculture, and food production [2,3,4]. PHB is usually synthesized and accumulated in certain microorganisms as an intracellular carbon and energy storage material during times when there is a growth-limiting factor in the presence of excess carbon. The accumulated PHB is degraded and re-utilized once balanced growth conditions become available

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Conclusion