Quantitative spectroscopy analysis of prokaryotic cells: vegetative cells and spores

Multiwavelength ultraviolet/visible (UV-Vis) spectra of microorganisms and cell suspensions contain quantitative information on properties such as number, size, shape, chemical composition, and internal structure of the suspended particles. These properties are essential for the identification and classification of microorganisms and cells. The complexity of microorganisms in terms of their chemical composition and internal structure make the interpretation of their spectral signature a difficult task. In this paper, a model is proposed for the quantitative interpretation of spectral patterns resulting from transmission measurements of prokaryotic microorganism suspensions. It is also demonstrated that different organisms give rise to spectral differences that may be used for their identification and classification. The proposed interpretation model is based on light scattering theory, spectral deconvolution techniques, and on the approximation of the frequency dependent optical properties of the basic constituents of living organisms. The quantitative deconvolution in terms of the interpretation model yields critical information necessary for the detection and identification of microorganisms, such as size, dry mass, dipicolinic acid concentration, nucleotide concentration, and an average representation of the internal scattering elements of the organisms. E. coli, P. agglomerans, B. subtilis spores, and vegetative cells and spores of Bacillus globigii are used as case studies. It is concluded that spectroscopy techniques coupled with effective interpretation models are applicable to a wide range of cell types found in diverse environments.     

Progress in developing a new detection method for the harmful algal bloom species,Karenia brevis,through multiwavelength spectroscopy

Multiwavelength spectroscopy is a rapid analytical technique that can be applied to detect, identify, and quantify microorganisms such as Karenia brevis, the species known for frequent red-tide blooms in Florida's coastal waters. This research will report on a model-based interpretation of UV–vis spectra of K. brevis. The spectroscopy models are based on light scattering and absorption theories, and the approximation of the frequency-dependant optical properties of the basic constituents of living organisms. Absorption and scattering properties of K. brevis, such as cell size/shape, internal structure, and chemical composition, are shown to predict the spectral features observed in the measured spectra. The parameters for the interpretation model were based upon both reported literature values, and experimental values obtained from live cultures and pigment standards. Measured and mathematically derived spectra were compared to determine the adequacy of the model, contribute new spectral information, and to establish the proposed spectral interpretation approach as a new detection method for K. brevis.     

The effects of diet and caloric restriction on adipose tissue fatty acid signatures of tufted puffin (<Emphasis Type="Italic">Fratercula cirrhata</Emphasis>) nestlings

Fatty acid (FA) signature analysis is a powerful tool to investigate foraging ecology and food web dynamics in marine ecosystems. However, use of FA signatures to qualitatively or quantitatively infer diets is potentially complicated by effects of nutritional state on lipid metabolism. Estimation of diets using the quantitative fatty acid signature analysis (QFASA) model requires the use of calibration coefficients to account for predator metabolism of individual FAs. We conducted a captive feeding experiment to determine the effects of a 50% reduction in food intake on growth rate and adipose tissue FA signatures of tufted puffin (Fratercula cirrhata) nestlings, a species that routinely experiences food restriction during growth. FA signatures of chicks fed low- and high-calorie diets both exhibited a change in composition in response to the dietary shift with the direction of change in the composition of individual FAs matching the direction of change in the dietary FAs. Despite a growth rate in the restricted nestlings that was 38% of those in the well-fed group, rates of FA turnover were not different between high and low-calorie treatments, and turnover was close to, but not entirely complete, after 27 days on both high-calorie and restricted diets. FA signatures of tufted puffin nestlings were significantly affected by caloric restriction, but these effects were much less pronounced than those of dietary turnover, and calibration coefficients of puffins fed low and high-calorie diets were highly correlated. Our results demonstrate that changes in physiological state can affect FA metabolism, but future research is required to better understand whether the size of these effects is sufficient to substantially alter diet estimation using the QFASA model.     

Development of growth medium from meat processing waste products and assessment of its properties

New growth medium based on inedible substrate - meat processing waste products of slaughterhouses - was developed. New medium was characterized by physical, chemical, and biological properties using exacting and non-exacting microorganisms, as well as by periodical cultivation of Corynebacterium diphtheriae strain and obtaining the preparation of its antigens. The experimental medium provided the growth of chosen test-strains with typical properties. From biomass obtained during the periodic cultivation of model toxigenic strain of C. diphtheriae on liquid experimental growth medium, preparation with antigenic properties was extracted. It has been shown that biologic characteristics of experimental growth medium did not differ from those of meat-peptone medium that allows to use it for cultivation of bacteria from various taxonomic groups.     

Fingerprint‐to‐CH stretch continuously tunable high spectral resolution stimulated Raman scattering microscope

Stimulated Raman scattering (SRS) microscopy is a label‐free method generating images based on chemical contrast within samples, and has already shown its great potential for high‐sensitivity and fast imaging of biological specimens. The capability of SRS to collect molecular vibrational signatures in bio‐samples, coupled with the availability of powerful statistical analysis methods, allows quantitative chemical imaging of live cells with sub‐cellular resolution. This application has substantially driven the development of new SRS microscopy platforms. Indeed, in recent years, there has been a constant effort on devising configurations able to rapidly collect Raman spectra from samples over a wide vibrational spectral range, as needed for quantitative analysis by using chemometric methods. In this paper, an SRS microscope which exploits spectral shaping by a narrowband and rapidly tunable acousto‐optical tunable filter (AOTF) is presented. This microscope enables spectral scanning from the Raman fingerprint region to the Carbon‐Hydrogen (CH)‐stretch region without any modification of the optical setup. Moreover, it features also a high enough spectral resolution to allow resolving Raman peaks in the crowded fingerprint region. Finally, application of the developed SRS microscope to broadband hyperspectral imaging of biological samples over a large spectral range from 800 to 3600 cm^?1, is demonstrated.     

Applications of light scattering in microbiology

Applications of the three principal light scattering techniques of turbidimetry, differential light scattering, and quasi-elastic light scattering to systems of microorganisms are reviewed. The relation between the three techniques is demonstrated and it is shown how these techniques can yield basic structural, optical, and even hydrodynamic properties for a wide range of microorganisms, with particular emphasis on changes in such properties. Such applications include antibiotic susceptibility testing, the effects of inhibitors on trypanosome motility, spore structure, virus self-assembly, and bacterial motility on the surface of fermentation reactors.     

Analysis of Methods for Growth Detection in the Search for Extraterrestrial Life

In the search for life on other planets, experiments designed to detect the growth of microorganisms may prove to be definitive when coupled with chemical characterization and metabolic experiments. If organisms are not abundant, growth provides the only means for obtaining a large mass of biological material suitable for chemical compositional analyses and metabolic assays. Several methods of monitoring growth are described. Of these, optical monitoring in a unique system free of soil particles is advanced as the most appropriate. Theoretical problems related to the formulation of culture media are discussed, and several possible solutions are proposed. The sampling system, the type of monitoring, the size and placement of inoculum, and the medium volume and composition are contingent upon one another and must be integrated without sacrifice to the biological demands.     

Effects of degraded optical conditions on behavioural responses to alarm cues in a freshwater fish

Prey organisms often use multiple sensory cues to gain reliable information about imminent predation threat. In this study we test if a freshwater fish increases the reliance on supplementary cues when the reliability of the primary cue is reduced. Fish commonly use vision to evaluate predation threat, but may also use chemical cues from predators or injured conspecifics. Environmental changes, such as increasing turbidity or water colour, may compromise the use of vision through changes in the optical properties of water. In an experiment we tested if changes in optical conditions have any effects on how crucian carp respond to chemical predator cues. In turbidity treatments we added either clay or algae, and in a brown water colour treatment we added water with a high humic content. We found that carp reduced activity in response to predator cues, but only in the turbidity treatments (clay, algae), whereas the response in the brown water treatment was intermediate, and not significantly different from, clear and turbid water treatments. The increased reliance on chemical cues indicates that crucian carp can compensate for the reduced information content from vision in waters where optical conditions are degraded. The lower effect in brown water may be due to the reduction in light intensity, changes in the spectral composition (reduction of UV light) or to a change in chemical properties of the cue in humic waters.     

Impact of elemental uptake in the root chemistry of wetland plants

Plants play a key role in the accumulation of metals in contaminated environment. Ephemeral plants, such as cyperus vaginatus, from the family Cyperaceae have been used in constructed wetlands to alter the biogeochemistry of waterlogged soils. High elemental content in wetlands often induces chemical changes in the root, stem and leaf of wetland plants. Elemental uptake and possible chemical changes in the roots of Cyperus vaginatus was investigated and compared with plants grown away from the wetland. Among the 9 heavy metals (Cr, Mn, Fe, Co, Ni, Cu, Zn, Cd, and Pb) and metalloid (As) measured, with the exception of Mn, all metals had higher content in the plant roots grown within the wetland. This was followed by plants grown near to the wetland that receive stormwater occasionally and then plants grown far from the wetland. The 3-D fluorescence spectra record showed notable differences in the chemical composition of roots grown in the three locations. The spectra combined with parallel factor analysis showed three dominant fluorescence components. Comparison of the fluorescence signatures showed a continuum of spectral properties constrained by the degree of metal contamination.     

Label-free cellular imaging by broadband coherent anti-Stokes Raman scattering microscopy

Raman microspectroscopy can provide the chemical contrast needed to characterize the complex intracellular environment and macromolecular organization in cells without exogenous labels. It has shown a remarkable ability to detect chemical changes underlying cell differentiation and pathology-related chemical changes in tissues but has not been widely adopted for imaging, largely due to low signal levels. Broadband coherent anti-Stokes Raman scattering (B-CARS) offers the same inherent chemical contrast as spontaneous Raman but with increased acquisition rates. To date, however, only spectrally resolved signals from the strong CH-related vibrations have been used for CARS imaging. Here, we obtain Raman spectral images of single cells with a spectral range of 600-3200 cm⁻¹, including signatures from weakly scattering modes as well as CH vibrations. We also show that B-CARS imaging can be used to measure spectral signatures of individual cells at least fivefold faster than spontaneous Raman microspectroscopy and can be used to generate maps of biochemical species in cells. This improved spectral range and signal intensity opens the door for more widespread use of vibrational spectroscopic imaging in biology and clinical diagnostics.     

Photodegradation of DOC in a shallow prairie wetland: evidence from seasonal changes in DOC optical properties and chemical characteristics

Wetlands across the Canadian prairies are typically shallow (<1.0 m) and exhibit high dissolved organic carbon (DOC) concentrations (>10 mg l^–1). Studies have shown that DOC in such shallow wetlands is not as reliable an indicator of ultraviolet radiation (UVR) attenuation as it is in clearwater. Changes in DOC character and composition as a result of sunlight exposure might provide a reasonable explanation for this observation. To test this, we investigated seasonal changes in DOC optical and chemical properties in a shallow prairie wetland over a 2-year period. Although DOC concentration increased at least two-fold from spring until fall, DOC specific absorption (at 350 nm) and fluorescence decreased by 30 and 32%, respectively, for the same period. In both years, seasonal decreases in DOC molecular weight and size (from measurements of tangential filtration and mass electrospray mass spectrometry) were reflected in concomitant increases in spectral slope. ¹³C NMR analysis of DOC isolated on XAD-8 resins revealed a 49% decrease in aromatic moieties when spring values were compared to those in the fall. As well, ¹³C signatures of this isolated DOC became heavier seasonally. In a short term photodegradation experiment (6 days) we noted a 47% decline in DOC specific absorption coefficients at 350 nm and a 15% increase in spectral slope when water exposed to the total light spectrum was compared to that of a dark control. Taken together, all of these observations were consistent with the occurrence of seasonal DOC photodegradation in shallow prairie wetlands and underlined the importance of this process in shaping DOC character and composition in these hydrologically dynamic systems. Our data also indicates that constant mixing and shallow depths in these wetlands were factors which enhanced DOC photodegradation. Although the high DOC concentrations of prairie wetlands should theoretically offer protection for their biota, seasonal photodegradation of DOC means that these systems may not be as protected as their high DOC concentrations suggest.     

The radioprotective properties of fungal melanin are a function of its chemical composition, stable radical presence and spatial arrangement

Melanized microorganisms are often found in environments with very high background radiation levels such as in nuclear reactor cooling pools and the destroyed reactor in Chernobyl. These findings and the laboratory observations of the resistance of melanized fungi to ionizing radiation suggest a role for this pigment in radioprotection. We hypothesized that the radioprotective properties of melanin in microorganisms result from a combination of physical shielding and quenching of cytotoxic free radicals. We have investigated the radioprotective properties of melanin by subjecting the human pathogenic fungi Cryptococcus neoformans and Histoplasma capsulatum in their melanized and non-melanized forms to sublethal and lethal doses of radiation of up to 8 kGy. The contribution of chemical composition, free radical presence, spatial arrangement, and Compton scattering to the radioprotective properties of melanin was investigated by high-performance liquid chromatography, electron spin resonance, transmission electron microscopy, and autoradiographic techniques. Melanin protected fungi against ionizing radiation and its radioprotective properties were a function of its chemical composition, free radical quenching, and spherical spatial arrangement.     

Temporal variations in phytoplankton, particulates, and colored dissolved organic material based on optical properties during a Long Island brown tide compared to an adjacent embayment

Since 1985, the coastal embayments of Long Island, New York, have been plagued with recurrent blooms, aptly called brown tides, of the pelagophyte Aureococcus anophagefferens. The distinct ocean color observed during these blooms suggests that optical methods can be used as a tool to study, detect, and track brown tides. Thus, the goal of our project was to compare the optical properties and pigment composition during bloom and non-bloom conditions and assess temporal variations in the phytoplankton and other constituents in the seawater associated with bloom development. From 17 May to 8 June 2000, we measured a time series of particle size distributions and concentrations as well as size-fractioned algal pigments and optical properties in two Long Island embayments where brown tides are known to occur. During our study, A. anophagefferens represented an insignificant contribution to the algal community in West Neck Bay (WNB), whereas a bloom developed in Quantuck Bay (QB). Initially, temperature and salinity were similar at the two locations; however, bulk optical properties, chlorophyll, and particle concentrations were nearly a factor of 2 greater at QB. Bulk optical properties remained constant at WNB, yet increased exponentially at QB as the bloom developed. The composition of particulates, including phytoplankton, varied little at QB, and the optical properties suggested the dominance of A. anophagefferens (confirmed by microscopy). The largest temporal variations were observed in the colored dissolved organic material (CDOM); the colloidal (0.2–0.7 μm) fraction, exhibiting a strong protein-like signal, increased dramatically at the height of the bloom. At WNB particle sizes and algal composition varied despite the invariant bulk optical properties; CDOM variations were minimal. Overall, the optical properties in the two bays demonstrated that at QB temporal variations were dominated by biomass and colloidal protein changes, whereas shifts in the algal community occurred at WNB. This study demonstrates the utility of in situ optical observations to resolve temporal changes in the ecological conditions associated with algal bloom development.     

Brazilian Kefir-Fermented Sheep’s Milk,a Source of Antimicrobial and Antioxidant Peptides

Fermented milks are a source of bioactive peptides and may be considered as functional foods. Among these, sheep’s milk fermented with kefir has not been widely studied and its most relevant properties need to be more thoroughly characterized. This research study is set out to investigate and evaluate the antioxidant and antimicrobial properties of peptides from fermented sheep’s milk in Brazil when produced by using kefir. For this, the chemical and microbiological composition of the sheep’s milk before and after the fermentation was evaluated. The changes in the fermented milk and the peptides extracted before the fermentation and in the fermented milk during its shelf life were verified. The antimicrobial and antioxidant activities of the peptides from the fermented milk were evaluated and identified according to the literature. The physicochemical properties and mineral profile of the fermented milk were like those of fresh milk. The peptide extract presented antimicrobial activity and it was detected that 13 of the 46 peptides were able to inhibit the growth of pathogenic microorganisms. A high antioxidant activity was observed in the peptides extracted from fermented milk (3.125 mg/mL) on the 28th day of storage. Two fractions displayed efficient radical scavenging properties by DPPH and ABTS methods. At least 11 peptides distributed in the different fractions were identified by tandem mass spectrometry. This sheep’s milk fermented by Brazilian kefir grains, which has antioxidant and antimicrobial activities and probiotic microorganisms, is a good candidate for further investigation as a source for bioactive peptides. The fermentation process was thus a means by which to produce potential bioactive peptides.     

Microbial degradation of water miscible metal working fluids

Water miscible metal working fluids (MWF) are prone to contamination by bacteria and fungi. In the present work it was investigated which components of a model MWF emulsion were most readily degraded by microorganisms and which are relatively resistant to biodegradation. The microbial community colonising an MWF emulsion, during its lifetime under in-use conditions was analysed up to species level. Shifts in the composition of microbial community were related to chemical changes in the MWF emulsion over time.     

Estimation of leaf nitrogen content from spectral characteristics of rice canopy

Ground-based remotely sensed reflectance spectra of hyperspectral resolution were monitored during the growing period of rice under various nitrogen application rates. It was found that reflectance spectrum of rice canopy changed in both wavelength and reflectance as the plants developed. Fifteen characteristic wavebands were identified from the apparent peaks and valleys of spectral reflectance curves, in accordance with the results of the first-order differentiation, measured over the growing season of rice. The bandwidths and center wavelengths of these characteristic wavebands were different among nitrogen treatments. The simplified features by connecting these 15 characteristic wavelengths may be considered as spectral signatures of rice canopy, but spectral signatures varied with developmental age and nitrogen application rates. Among these characteristic wavebands, the changes of the wavelength in band 11 showed a positive linear relationship with application rates of nitrogen fertilizer, while it was a negative linear relationship in band 5. Mean reflectance of wavelengths in bands 1, 2, 3, 5, 11, and 15 was significantly correlated with application rates. Reflectance of these six wavelengths changed nonlinearly after transplanting and could be used in combination to distinguish rice plants subjected to different nitrogen application rates. From the correlation analyses, there are a variety of correlation coefficients for spectral reflectance to leaf nitrogen content in the range of 350-2400 nm. Reflectance of most wavelengths exhibited an inverse correlation with leaf nitrogen content, with the largest negative value (r = -0.581) located at about 1376 nm. Changes in reflectance at 1376 nm to leaf nitrogen content during the growing period were closely related and were best fitted to a nonlinear function. This relationship may be used to estimate and to monitor nitrogen content of rice leaves during rice growth. Reflectance of red light minimum and near-infrared peak and leaf nitrogen content were correlated nonlinearly.     

Coexistence of photosynthetic microorganisms with growth rates depending on the spectral quality of light

A new equation for the growth rate of photosynthetic microorganisms is derived. It is based on a series formulation of the mechanism of photosynthesis and it has the form of a functional of the spectral qualities of light. Using this functional to model their growth it is shown that populations of photosynthetic microorganisms can coexist in the same homogeneous environment even though they compete for the available light.     

羧酶体的组成、结构、功能和检测及其在脱氮细菌中的意义

羧酶体(Carboxysome)是一种具有CO2浓缩功能的"类细胞器",它存在于自养型脱氮细菌中,可增强细菌的自养生长能力。硝化细菌、厌氧氨氧化细菌和部分反硝化细菌都是重要的自养型脱氮细菌,探明其羧酶体的组成、结构和功能,将有助于揭示自养型脱氮菌的生长规律,进而强化生物脱氮过程。基于文献阅读和相关研究,本文对自养型细菌中羧酶体在组成、结构、功能和检测等方面的研究进展进行综述,以期为自养生物脱氮过程的深入理解和优化改进提供参考。     

Pigmentation and spectral absorbance in the deep-sea arctic amphipods Eurythenes gryllus and Anonyx sp.

As for many deep-sea animals, the red colouration of the two amphipods Eurythenes gryllus and Anonyx sp. has an important function providing camouflage, as the attenuation of the red wavelengths in seawater is higher than other colours within the visible range. Variation in colouration between different stages of colour intensity (related to size) is evident in both species. The red colour is caused by carotenoids, and the carotenoid composition was identified and quantified using spectral optical density signatures, high-performance liquid chromatography (HPLC) and liquid chromatography–mass spectrometry (LC–MS). The carotenoid astaxanthin was identified as the major carotenoid in both amphipods, both in pure and in esterified forms. In addition, minor amounts of lutein-like, canthaxanthin-like and several unidentified carotenoids were found in E. gryllus, while diatoxanthin, β,β-carotene and canthaxanthin-like carotenoids were detected in Anonyx sp. Generally, both species displayed an increase in the amount of carotenoids as a function of colour intensity and size. Shifts in λ_max in the OD (Optical density; dimensionless, acronym absorbance) spectra were evident in both species between the different colour stages in both the in vivo and the in vitro material, probably caused by changes in pigment composition. Similar shifts in λ_max were observed between the in vivo and in vitro pigment raw extracts in general, most likely caused by pigment-binding proteins. The differences in pigment composition and wavelength shifts suggest large intra- and inter-specific differences between the two species. Probable reasons for changes in pigment composition could be related to diet, season, moulting patterns, metabolic pathways and reproduction.     

Structural heterogeneity of wheat arabinoxylans revealed by Raman spectroscopy

A set of arabinoxylan samples differing in their arabinose composition and various samples of arabino-xylo-oligosaccharide samples were analysed by Raman spectroscopy. Specific signatures for arabinose substitution were found in several spectral regions, that is, 400-600, 800-950 and 1030-1100 cm(-1). A linear relationship was observed between the peak ratio 855/895 cm(-1) of the second derivative spectra and the A/X ratio determined by chemical analysis. Moreover, spectral changes were observed in the 400-600 cm(-1) region assigned to the coupled vibrations mode in the skeleton: while the intensity of the band at 570 cm(-1) increased with the degree of substitution, that at 494 cm(-1) decreased. Similarly, a linear relationship was observed between the peak intensity ratio 570/494 cm(-1) calculated on the second derivative spectra and the composition data. Analysis of Raman spectra of arabino-xylo-oligosaccharides allowed to identify specific spectral features of disubstitution.