Environ Sci Pollut Res DOI 10.1007/s11356-015-4593-5
ALTERATION AND ELEMENT MOBILITY AT THE MICROBE-MINERAL INTERFACE
Classification and identification of pigmented cocci bacteria relevant to the soil environment via Raman spectroscopy Vinay Kumar 1,3 & Bernd Kampe 1,3 & Petra Rösch 1,3 & Jürgen Popp 1,2,3
Received: 14 October 2014 / Accepted: 23 April 2015 # Springer-Verlag Berlin Heidelberg 2015
Abstract A soil habitat consists of a significant number of bacteria that cannot be cultivated by conventional means, thereby posing obvious difficulties in their classification and identification. This difficulty necessitates the need for advanced techniques wherein a well-compiled biomolecular database consisting of the already cultivable bacteria can be used as a reference in an attempt to link the noncultivable bacteria to their closest phylogenetic groups. Raman spectroscopy has been successfully applied to taxonomic studies of many systems like bacteria, fungi, and plants relying on spectral differences contributed by the variation in their overall biomolecular composition. However, these spectral differences can be obscured due to Raman signatures from photosensitive microbial pigments like carotenoids that show enormous variation in signal intensity hindering taxonomic investigations. In this study, we have applied laser-induced photobleaching to expel the carotenoid signatures from pigmented cocci bacteria. Using this method, we have investigated 12 species of pigmented bacteria abundant in soil habitats belonging to three genera mainly Micrococcus, Deinococcus, and Kocuria based on their Raman spectra with the assistance of a chemometric tool known as the radial kernel support vector machine Responsible editor: Philippe Garrigues * Jürgen Popp
[email protected] 1
Institute of Physical Chemistry and Abbe Center of Photonics, Friedrich Schiller University Jena, Helmholtzweg 4, D-07743 Jena, Germany
2
Leibniz Institute of Photonic Technology, Albert-Einstein-Straße 9, D-07745 Jena, Germany
3
InfectoGnostics, Forschungscampus Jena, Philosophenweg 7, D-07743 Jena, Germany
(SVM). Our results demonstrate the potential of Raman spectroscopy as a minimally invasive taxonomic tool to identify pigmented cocci soil bacteria at a single-cell level. Keywords Raman spectroscopy . Pigmented soil bacteria . Carotenoids . Photobleaching . Bacterial identification and classification . Radial kernel SVM
Introduction A soil environment constitutes a vast number of genetically diverse microorganisms which play key roles in processes like nitrogen fixation, carbon cycling, etc. However, a major percentage of these microbes remain to be identified and studied. A majority of these microbes have not been obtained in pure culture hence making the identification and also characterization of their physiological properties very difficult (Whitman et al. 1998; Van Der Heijden et al. 2008). Currently, culture dependent and independent complex genomic and metagenomic analysis methods such as small subunit ribosomal RNA (16S rRNA) sequencing and shotgun sequencing have been applied for the identification and classification of microbes from various environments like soil, seawater, etc (Janda and Abbott 2007; Rinke et al. 2013; Mignard and Flandrois 2006). Also, in the past few years, the research community has seen the successful use of spectroscopic techniques in the field of taxonomy in various systems like bacteria, fungi, and plants (Rösch P et al. 2005; Baranski et al. 2006; Walter et al. 2011). Spectroscopic techniques use the signatures of biomolecules constituted in the microbes and rely on comparison to well-established biomolecular databases acquired from the already cultivable microbes (Meisel et al. 2014; Kusić et al. 2014; Silge et al. 2014). These databases can then be used as a reference in an effort to link the
Environ Sci Pollut Res
uncultivable microbes from environments of high biodiversity like soil to their closest phylogenetic groups. In this study, we establish such a database by analyzing some of the commonly found cultivable pigmented bacteria in soil via Raman spectroscopy and also evaluate its use as a taxonomic tool for independent identification of these bacteria as an initiative approach toward the analysis of uncultivable bacteria from soil by comparison to these databases in our further studies. This approach will fit into the culture-independent method category of analyzing uncultivable soil bacteria amongst many other methods that have been devised to address this problem (Pham and Kim 2012). Spectroscopic techniques provide less specificity when compared to the sequencing technology but offer the huge advantage of performing rapid minimally invasive analysis at a single-cell resolution. Other spectroscopic techniques like mass spectrometry and infrared spectroscopy have been used for taxonomic studies (Kim et al. 2012; Baranska et al. 2005). However, mass spectrometry and infrared spectroscopy face obstacles in the form of requiring a large amount of cells for successful analysis and require extensive sample preparation procedures. In contrast to these techniques, Raman spectroscopy allows for noninvasive analysis of single cells either in fixed or live condition without any necessity for any vigorous sample preparation steps (Harz et al. 2009). Raman spectroscopic analysis of biological samples is based on probing the small differences in their overall biomolecular composition appearing in the Raman spectral fingerprint which are highly specific to the biomolecular composition of the cells. Raman spectral fingerprints of cells differ from one another in the form of minute variations in the signatures of biomolecules like lipids, proteins, DNA, and carbohydrates which make up the cell. However, these spectral differences can be obscured due to the presence of Raman signatures from photosensitive microbial pigments like carotenoids that show enormous variation in signal intensity hindering taxonomic investigations (Maquelin et al. 2009). In this study, we have applied laserinduced photobleaching to expel the carotenoid signatures from the bacteria under study. This method helped to obtain Raman spectral fingerprints that are reproducible at the strain and species level making it ideal for taxonomic studies. Using this methodology, we were able to identify and discriminate between 12 different species of pigmented cocci soil bacteria belonging to three genera mainly Micrococcus, Deinococcus, and Kocuria that are highly abundant in soil (Ventura et al. 2007; Janssen 2006). Our results demonstrate the potential of Raman spectroscopy as a minimally invasive taxonomic tool to identify pigmented cocci soil bacteria. This technique can be applicable as a supplement to the current advanced tools used in the taxonomic studies of noncultivable soil bacteria.
Materials and methods Pigmented bacterial species: The following are species of pigmented bacteria: Genus Micrococcus Micrococcus luteus (DSM 348), Micrococcus flavus (DSM 30824), Micrococcus endophyticus (DSM 17945), Micrococcus yunnanensis (DSM 21948) Genus Kocuria Kocuria rosea (DSM 20447), Kocuria turfanensis (DSM 22143), Kocuria polaris (DSM 14382), Kocuria himachalensis (DSM 44905), Kocuria salsicia (DSM 24776), Kocuria kristinae (DSM 20027) Genus Deinococcus Deinococcus radiodurans (DSM 46620) and Deinococcus proteolyticus (DSM 20540); all these bacterial cultures were obtained from the German Collection of Microorganisms and Cell Cultures (Leibniz Institute DSMZ, Germany). Sample preparation: All the bacterial cultures were inoculated into a suitable growth media. Trypticase soy yeast agar medium was used for all the species barring three, M. luteus was cultured on nutrient agar media, K. rosea and D. proteolyticus was cultured on corynebacterium agar media. All the cultures were incubated for 72 h and at a temperature of 30 °C. A single colony of cells growing on the petri dishes is picked using a sterilized loop and transferred into distilled water contained in a small Eppendorf vial. The cells are washed twice using distilled water by centrifuging the samples at 12,500 rpm for 3 min to remove any residual media which would hinder the Raman spectra signatures. The pellet thus obtained is again suspended into distilled water and vortexed vigorously at 2500 rpm for 2 min in order to achieve separation of the cluster of cells so as to be able to analyze single cells. Five microliter of the sample thus obtained is now transferred to a nickel foil and allowed to air dry prior to Raman analysis. Nickel foil is used because of its extremely low or negligible background signal and its reflective property, and it has no bands interfering with the Raman spectra of bacteria. Raman instrumentation: A micro-Raman setup (Bio Particle Explorer; rap.ID Particle Systems GmbH, Berlin, Germany) was used for this study. This system suits ideally for bacterial single-cell measurements and is also equipped with an automatic scan mode for automatic measurements of the cells. Single-cell Raman spectra were acquired with an excitation light of 532 nm generated from a solid-state frequency-doubled Nd:YAG laser (LCM-S-111-NNP25; Laser-export Co. Ltd.). An MPLFLN-BD 100× Olympus objective focused the excitation light onto the sample with a lateral resolution of
