Correlation of Carotenoid Accumulation with Aggregation and Biofilm Development in <named-content content-type="genus-species" type="simple">Rhodococcus</named-content> sp. SD-74

Aggregation of bacterial populations substantially influences their characteristic properties and functions compared with the planktonic counterpart. It is also involved in the initial stages of biofilm development. Many studies have revealed important roles of bacterial aggregation in microbial production and biodegradation. Nevertheless, mechanistic understanding of bacterial aggregation in vivo and at the molecular level is far from complete. Here, we present a noninvasive, label-free Raman microspectroscopic approach to investigate the aggregation and biofilm development of the biotechnologically important Rhodococcus sp. SD-74. We found that the concentration of intracellular carotenoids increases more than 3-fold within 1 week as the biofilm develops. Raman imaging experiments confirmed that the carotenoid accumulation occurs throughout the Rhodococcus sp. SD-74 biofilm. The correlation between the carotenoid Raman intensities and biofilm development found in the present study provides a new means for quantitative, molecular-level assessment of the level of biofilm development, which is not possible with dye staining assay or electron microscopy. Moreover, our results suggest that microbial production of carotenoids in pigmented bacteria such as Rhodococcus sp. SD-74 may potentially be controlled via bacterial aggregation and biofilm formation. C properties and functions of bacteria are often realized and/or considerably altered when bacterial cells form aggregates rather than exist in the free-floating, planktonic state. In wastewater treatment, for example, the efficiency of the conventional activated sludge process hinges upon bioflocculation and subsequent solid−liquid separation. Besides aggregates formed in culture medium, another type of bacterial aggregation can be found in biofilms, which have recently attracted keen attention because of their pivotal importance in medicine, chemical industry, and environmental science. Biofilms are structured communities of bacterial cells that adhere to a surface or an interface, embedded in a sea of extracellular polymeric substances. Aggregation of sessile cells on a surface is involved in the initial stages of biofilm formation and dictates whether the microcolonies will evolve into a rigid biofilm structure or revert to planktonic cells. If the initial aggregation can be suppressed physically or chemically, it will be possible to prevent bacterial cells from further developing hard-to-remove slime. Despite a number of biological studies attempting to analyze bacterial aggregation, the molecular-level understanding of the underlying mechanisms remains largely unclear. To achieve this goal, we require not only molecular specificity but also space specificity, in which conventional biological approaches are lacking. Here, we present a Raman microspectroscopic approach to study the aggregation and biofilm development of the bacterium Rhodococcus sp. SD-74. Having nondestructive and label-free characteristics in addition to chemical and space specificities, Raman microspectroscopy and imaging offer an ideal tool for investigating microorganisms in vivo and from a molecular viewpoint. We have recently constructed a highsensitivity confocal Raman microspectrometer and used the developed apparatus to reveal dynamic changes in molecular concentration and distribution during the yeast cell cycle with the help of multivariate data analysis. Although there has been some pioneering Raman work on biofilms relevant to wastewater treatment, the application of Raman microspectroscopy and particularly spectral imaging to bacterial aggregation and biofilm development is still in its infancy. In this work, we show that the concentration of intracellular carotenoids increases substantially (more than 3-fold within 1 week) as Rhodococcus sp. SD-74 forms aggregates and develops biofilm. Many Rhodococcus strains hold promise for bioremediation because they are capable of alkane degradation, in which bacterial aggregation has been suggested to be involved. We found a positive correlation between the concentration and biofilm development only for carotenoids; other major constituents of the Rhodococcus sp. SD-74 biofilm, Received: April 21, 2013 Accepted: June 26, 2013 Published: June 26, 2013 Article