Nejvíce citovaný článek - PubMed ID 22163676
Raman microspectroscopy of individual algal cells: sensing unsaturation of storage lipids in vivo
The high-altitude pre-Andean region of the Atacama Desert is characterized by its stark volcanic rock formations and unique hydrothermal gypsum outcrops (gypcrete) that it hosts. This study delves into the biomolecular composition of the endolithic phototrophic microbes that thrive within these gypcretes. Using advanced Raman spectroscopy techniques, including Raman imaging (complemented by microscopic and 3D microscopic observations), herein we unveil new insights into the adaptive strategies of these gypsum-inhabiting algae. Our Raman imaging results provide a detailed chemical map of carotenoids associated with microbial colonization. This map reveals a significant gradient in pigment content, highlighting a critical survival mechanism for algae and cyanobacteria in this polyextreme environment. Intriguingly, we detected signals for carotenoids not only in the algae-colonized layer, but also deeper within the gypsum matrix - indicating pigment migration following cell disruption. In addition, we conducted an in-depth analysis of individual algal cells from the Trebouxiaceae family, noting their color variations from green to orange, plus describing the spectral differences in detail. This investigation identified in-vivo pigments (carotenoids, chlorophyll) and lipids at the cellular level, offering a comprehensive view of the molecular adaptations enabling life in one of the Earth's most extreme habitats.
- Klíčová slova
- Astrobiology, Biomarkers, Extremophiles, Geomicrobiology, Photopigments, Raman imaging,
- MeSH
- extrémní prostředí MeSH
- fyziologická adaptace MeSH
- karotenoidy * metabolismus MeSH
- pouštní klima * MeSH
- Ramanova spektroskopie * MeSH
- sinice metabolismus genetika MeSH
- síran vápenatý * MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- karotenoidy * MeSH
- síran vápenatý * MeSH
Rapid and accurate identification of pathogens causing infections is one of the biggest challenges in medicine. Timely identification of causative agents and their antimicrobial resistance profile can significantly improve the management of infection, lower costs for healthcare, mitigate ever-growing antimicrobial resistance and in many cases, save lives. Raman spectroscopy was shown to be a useful-quick, non-invasive, and non-destructive -tool for identifying microbes from solid and liquid media. Modifications of Raman spectroscopy and/or pretreatment of samples allow single-cell analyses and identification of microbes from various samples. It was shown that those non-culture-based approaches could also detect antimicrobial resistance. Moreover, recent studies suggest that a combination of Raman spectroscopy with optical tweezers has the potential to identify microbes directly from human body fluids. This review aims to summarize recent advances in non-culture-based approaches of identification of microbes and their virulence factors, including antimicrobial resistance, using methods based on Raman spectroscopy in the context of possible use in the future point-of-care diagnostic process.
- Klíčová slova
- Raman spectroscopy, Raman tweezers, antimicrobial resistance, diagnostics, identification of microorganisms, magnetic beads, microfluidic devices,
- MeSH
- analýza jednotlivých buněk MeSH
- antiinfekční látky * MeSH
- faktory virulence MeSH
- lidé MeSH
- Ramanova spektroskopie * metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
- Názvy látek
- antiinfekční látky * MeSH
- faktory virulence MeSH
Oleogenic yeasts are characterized by the ability to accumulate increased amounts of lipids under certain conditions. These microbial lipids differ in their fatty acid composition, which allows them to be widely used in the biotechnology industry. The interest of biotechnologists is closely linked to the rising prices of fossil fuels in recent years. Their negative environmental impact is caused by significantly increased demand for biodiesel. The composition of microbial lipids is very similar to vegetable oils, which provides great potential for use in the production of biodiesel. In addition, some oleogenic microorganisms are capable of producing lipids with a high proportion of unsaturated fatty acids. The presented paper's main aim was to study the production of lipids and lipid substances by yeasts of the genus Metschnikowia, to cultivate crude waste animal fat to study its utilization by yeasts, and to apply the idea of circular economy in the biotechnology of Metschnikowia yeasts. The work focuses on the influence of various stress factors in the cultivation process, such as reduced temperature or nutritional stress through the use of various waste substrates, together with manipulating the ratio of carbon and nitrogen sources in the medium. Yeast production properties were monitored by several instrumental techniques, including gas chromatography and Raman spectroscopy. The amount of lipids and in particular the fatty acid composition varied depending on the strains studied and the culture conditions used. The ability of yeast to produce significant amounts of unsaturated fatty acids was also demonstrated in the work. The most suitable substrate for lipid production was a medium containing glycerol, where the amount of accumulated lipids in the yeast M. pulcherrima 1232 was up to 36%. In our work, the crude animal fat was used for the production of high-value lipids, which to the best of our knowledge is a new result. Moreover, quantitative screening of lipase enzyme activity cultivated on animal fat substrate on selected yeasts of the genus Metschnikowia was performed. We found that for the yeast utilizing glycerol, animal fat seems to be an excellent source of carbon. Therefore, the yeast conversion of crude processed animal fat to value-added products is a valuable process for the biotechnology and food industry.
- Klíčová slova
- Metschnikowia, Raman spectroscopy, animal fat, lipids, stress factors, yeasts,
- Publikační typ
- časopisecké články MeSH
Raman spectroscopy is a universal method designed for the analysis of a wide range of physical, chemical and biological systems or various surfaces. This technique is suitable to monitor various components of cells, tissues or microorganisms. The advantages include very fast non-contact and non-destructive analysis and no or minimal need for sample treatment. The yeasts Metschnikowia can be considered as industrially usable producers of pulcherrimin or single-cell lipids, depending on cultivation conditions and external stress. In the present study, Raman spectroscopy was used as an effective tool to identify both pulcherrimin and lipids in single yeast cells. The analysis of pulcherrimin is very demanding; so far, there is no optimal procedure to analyze or identify this pigment. Based on results, the strong dependence of pulcherrimin production on the ferric ion concentration was found with the highest yield in media containing 0.1 g/L iron. Further, production of lipids in Metschnikowia cells was studied at different temperatures and C:N ratios, using Raman spectroscopy to follow fatty acids composition, under different regimes, by monitoring the iodine number. The results of Raman spectroscopy were comparable with the fatty acid analysis obtained by gas chromatography. This study therefore supported use of Raman spectroscopy for biotechnological applications as a simple tool in the identification and analysis both the pulcherrimin and microbial lipids. This method provides a quick and relatively accurate estimation of targeted metabolites with minimal sample modification and allows to monitor metabolic changes over time of cultivation.
- Klíčová slova
- Metschnikowia, Raman spectroscopy, lipids, pulcherrimin, yeasts,
- Publikační typ
- časopisecké články MeSH
Photosynthetic energy conversion and the resulting photoautotrophic growth of green algae can only occur in daylight, but DNA replication, nuclear and cellular divisions occur often during the night. With such a light/dark regime, an algal culture becomes synchronized. In this study, using synchronized cultures of the green alga Desmodesmus quadricauda, the dynamics of starch, lipid, polyphosphate, and guanine pools were investigated during the cell cycle by two independent methodologies; conventional biochemical analyzes of cell suspensions and confocal Raman microscopy of single algal cells. Raman microscopy reports not only on mean concentrations, but also on the distribution of pools within cells. This is more sensitive in detecting lipids than biochemical analysis, but both methods-as well as conventional fluorescence microscopy-were comparable in detecting polyphosphates. Discrepancies in the detection of starch by Raman microscopy are discussed. The power of Raman microscopy was proven to be particularly valuable in the detection of guanine, which was traceable by its unique vibrational signature. Guanine microcrystals occurred specifically at around the time of DNA replication and prior to nuclear division. Interestingly, guanine crystals co-localized with polyphosphates in the vicinity of nuclei around the time of nuclear division.
- Klíčová slova
- Desmodesmus quadricauda, cell cycle, confocal Raman microscopy, guanine, lipids, microalgae, polyphosphate, starch,
- MeSH
- buněčná stěna chemie MeSH
- buněčný cyklus * MeSH
- časové faktory MeSH
- Chlorophyta cytologie růst a vývoj MeSH
- guanin analýza MeSH
- lipidová tělíska metabolismus MeSH
- lipidy analýza MeSH
- mikroskopie * MeSH
- polyfosfáty analýza MeSH
- Ramanova spektroskopie * MeSH
- škrob analýza MeSH
- velikost buňky MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- srovnávací studie MeSH
- Názvy látek
- guanin MeSH
- lipidy MeSH
- polyfosfáty MeSH
- škrob MeSH
Analyzing the cells in various body fluids can greatly deepen the understanding of the mechanisms governing the cellular physiology. Due to the variability of physiological and metabolic states, it is important to be able to perform such studies on individual cells. Therefore, we developed an optofluidic system in which we precisely manipulated and monitored individual cells of Escherichia coli. We tested optical micromanipulation in a microfluidic chamber chip by transferring individual bacteria into the chambers. We then subjected the cells in the chambers to antibiotic cefotaxime and we observed the changes by using time-lapse microscopy. Separately, we used laser tweezers Raman spectroscopy (LTRS) in a different micro-chamber chip to manipulate and analyze individual cefotaxime-treated E. coli cells. Additionally, we performed conventional Raman micro-spectroscopic measurements of E. coli cells in a micro-chamber. We found observable changes in the cellular morphology (cell elongation) and in Raman spectra, which were consistent with other recently published observations. The principal component analysis (PCA) of Raman data distinguished between the cefotaxime treated cells and control. We tested the capabilities of the optofluidic system and found it to be a reliable and versatile solution for this class of microbiological experiments.
- Klíčová slova
- E. coli, Raman micro-spectroscopy, antibiotics, optical tweezers, opto-fluidics,
- MeSH
- analýza hlavních komponent MeSH
- antibakteriální látky škodlivé účinky MeSH
- Escherichia coli účinky léků růst a vývoj MeSH
- laboratoř na čipu * MeSH
- mikromanipulace metody MeSH
- optická pinzeta * MeSH
- Ramanova spektroskopie MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- antibakteriální látky MeSH
Clinical treatment of the infections caused by various staphylococcal species differ depending on the actual cause of infection. Therefore, it is necessary to develop a fast and reliable method for identification of staphylococci. Raman spectroscopy is an optical method used in multiple scientific fields. Recent studies showed that the method has a potential for use in microbiological research, too. Our work here shows a possibility to identify staphylococci by Raman spectroscopy. We present a method that enables almost 100% successful identification of 16 of the clinically most important staphylococcal species directly from bacterial colonies grown on a Mueller-Hinton agar plate. We obtained characteristic Raman spectra of 277 staphylococcal strains belonging to 16 species from a 24-hour culture of each strain grown on the Mueller-Hinton agar plate using the Raman instrument. The results show that it is possible to distinguish among the tested species using Raman spectroscopy and therefore it has a great potential for use in routine clinical diagnostics.
- MeSH
- agar MeSH
- analýza hlavních komponent MeSH
- časové faktory MeSH
- diagnostické testy rutinní MeSH
- fluorescence MeSH
- odběr biologického vzorku MeSH
- Ramanova spektroskopie metody MeSH
- Staphylococcus izolace a purifikace MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- agar MeSH
We report herein on the application of Raman spectroscopy to the rapid quantitative analysis of polyhydroxyalkanoates (PHAs), biodegradable polyesters accumulated by various bacteria. This theme was exemplified for quantitative detection of the most common member of PHAs, poly(3-hydroxybutyrate) (PHB) in Cupriavidus necator H16. We have identified the relevant spectral region (800-1800 cm-1) incorporating the Raman emission lines exploited for the calibration of PHB (PHB line at 1736 cm-1) and for the selection of the two internal standards (DNA at 786 cm-1 and Amide I at 1662 cm-1). In order to obtain quantitative data for calibration of intracellular content of PHB in bacterial cells reference samples containing PHB amounts-determined by gas chromatography-from 12% to 90% (w/w) were used. Consequently, analytical results based on this calibration can be used for fast and reliable determination of intracellular PHB content during biotechnological production of PHB since the whole procedure-from bacteria sampling, centrifugation, and sample preparation to Raman analysis-can take about 12 min. In contrast, gas chromatography analysis takes approximately 8 h.
- Klíčová slova
- Cupriavidus necator H16, Raman spectroscopy, polyhydroxyalkanoates,
- Publikační typ
- časopisecké články MeSH
Raman spectroscopy has a broad range of applications across numerous scientific fields, including microbiology. Our work here monitors the influence of culture media on the Raman spectra of clinically important microorganisms (Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis and Candida albicans). Choosing an adequate medium may enhance the reproducibility of the method as well as simplifying the data processing and the evaluation. We tested four different media per organism depending on the nutritional requirements and clinical usage directly on a Petri dish. Some of the media have a significant influence on the microbial fingerprint (Roosvelt-Park Institute Medium, CHROMagar) and should not be used for the acquisition of Raman spectra. It was found that the most suitable medium for microbiological experiments regarding these organisms was Mueller-Hinton agar.
- Klíčová slova
- Raman spectroscopy, bacteria, culture media, yeasts,
- MeSH
- Bacteria * chemie účinky léků metabolismus MeSH
- kultivační média farmakologie MeSH
- kvasinky * chemie účinky léků metabolismus MeSH
- Ramanova spektroskopie metody MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- kultivační média MeSH
Colonies of Candida parapsilosis on culture plates were probed directly in situ using Raman spectroscopy for rapid identification of specific strains separated by a given time intervals (up to months apart). To classify the Raman spectra, data analysis was performed using the approach of principal component analysis (PCA). The analysis of the data sets generated during the scans of individual colonies reveals that despite the inhomogeneity of the biological samples unambiguous associations to individual strains (two biofilm-positive and two biofilm-negative) could be made.
