Discrimination of grass pollen of different species by FTIR spectroscopy of individual pollen grains.

Fourier-transform infrared (FTIR) spectroscopy enables the chemical characterization and identification of pollen samples, leading to a wide range of applications, such as paleoecology and allergology. This is of particular interest in the identification of grass (Poaceae) species since they have pollen grains of very similar morphology. Unfortunately, the correct identification of FTIR microspectroscopy spectra of single pollen grains is hindered by strong spectral contributions from Mie scattering. Embedding of pollen samples in paraffin helps to retrieve infrared spectra without scattering artifacts. In this study, pollen samples from 10 different populations of five grass species (Anthoxanthum odoratum, Bromus inermis, Hordeum bulbosum, Lolium perenne, and Poa alpina) were embedded in paraffin, and their single grain spectra were obtained by FTIR microspectroscopy. Spectra were subjected to different preprocessing in order to suppress paraffin influence on spectral classification. It is shown that decomposition by non-negative matrix factorization (NMF) and extended multiplicative signal correction (EMSC) that utilizes a paraffin constituent spectrum, respectively, leads to good success rates for the classification of spectra with respect to species by a partial least square discriminant analysis (PLS-DA) model in full cross-validation for several species. PLS-DA, artificial neural network, and random forest classifiers were applied on the EMSC-corrected spectra using an independent validation to assign spectra from unknown populations to the species. Variation within and between species, together with the differences in classification results, is in agreement with the systematics within the Poaceae family. The results illustrate the great potential of FTIR microspectroscopy for automated classification and identification of grass pollen, possibly together with other, complementary methods for single pollen chemical characterization.

Analysis of xanthyletin and secondary metabolites from Pseudomonas stutzeri ST1302 and Klebsiella pneumoniae ST2501 against Pythium insidiosum.

BACKGROUND: Pythium insidiosum is a member of the oomycetes class of aquatic fungus-like microorganisms. It can infect humans and animals through skin wounds and the eyes, causing pythiosis, an infectious disease with high morbidity and mortality rates. Antifungal agents are ineffective as pythiosis treatments because ergosterol, the target site of most antifungal agents, is not found in the P. insidiosum cytoplasmic membrane. The best choice for treatment is surgical removal of the infected organ. While natural plant products or secretory substances from bacterial flora have exhibited in vitro anti-P. insidiosum activity, their mechanism of action remains unknown. Therefore, this study hypothesized that the mechanism of action could be related to changes in P. insidiosum biochemical composition (such as lipid, carbohydrate, protein or nucleic acid) following exposure to the inhibitory substances. The biochemical composition of P. insidiosum was investigated by Synchrotron radiation-based Fourier-transform infrared (FTIR) microspectroscopy. RESULTS: Fraction No.6 from the crude extract of P. stutzeri ST1302, fraction No.1 from the crude extract of K. pneumoniae ST2501 and xanthyletin were used as anti-P. insidiosum substances, with MFCs at 3.125, 1.57-1.91, 0.003 mg/ml, respectively. The synchrotron FTIR results show that the deconvoluted peak distributions in the amide I, amide II, and mixed regions were significantly different between the treatment and control groups. CONCLUSIONS: Xanthyletin and the secondary metabolites from P. stutzeri ST1302 and K. pneumoniae ST2501 exerted anti-P. insidiosum activity that clearly changed the proteins in P. insidiosum. Further study, including proteomics analysis and in vivo susceptibility testing, should be undertaken to develop a better understanding of the mechanism of anti-P. insidiosum activity.

Assessing an effective feeding strategy to optimize crude glycerol utilization as sustainable carbon source for lipid accumulation in oleaginous yeasts.

BACKGROUND: Microbial lipids can represent a valuable alternative feedstock for biodiesel production in the context of a viable bio-based economy. This production can be driven by cultivating some oleaginous microorganisms on crude-glycerol, a 10% (w/w) by-product produced during the transesterification process from oils into biodiesel. Despite attractive, the perspective is still economically unsustainable, mainly because impurities in crude glycerol can negatively affect microbial performances. In this view, the selection of the best cell factory, together with the development of a robust and effective production process are primary requirements. RESULTS: The present work compared crude versus pure glycerol as carbon sources for lipid production by three different oleaginous yeasts: Rhodosporidium toruloides (DSM 4444), Lipomyces starkeyi (DSM 70295) and Cryptococcus curvatus (DSM 70022). An efficient yet simple feeding strategy for avoiding the lag phase caused by growth on crude glycerol was developed, leading to high biomass and lipid production for all the tested yeasts. Flow-cytometry and fourier transform infrared (FTIR) microspectroscopy, supported by principal component analysis (PCA), were used as non-invasive and quick techniques to monitor, compare and analyze the lipid production over time. Gas chromatography (GC) analysis completed the quali-quantitative description. Under these operative conditions, the highest lipid content (up to 60.9% wt/wt) was measured in R. toruloides, while L. starkeyi showed the fastest glycerol consumption rate (1.05 g L(-1) h(-1)). Being productivity the most industrially relevant feature to be pursued, under the presented optimized conditions R. toruloides showed the best lipid productivity (0.13 and 0.15 g L(-1) h(-1) on pure and crude glycerol, respectively). CONCLUSIONS: Here we demonstrated that the development of an efficient feeding strategy is sufficient in preventing the inhibitory effect of crude glycerol, and robust enough to ensure high lipid accumulation by three different oleaginous yeasts. Single cell and in situ analyses allowed depicting and comparing the transition between growth and lipid accumulation occurring differently for the three different yeasts. These data provide novel information that can be exploited for screening the best cell factory, moving towards a sustainable microbial biodiesel production.

Specificity of Fourier Transform Infrared (FTIR) Microspectroscopy to Estimate Depth-Wise Proteoglycan Content in Normal and Osteoarthritic Human Articular Cartilage.

BACKGROUND: Fourier transform infrared (FTIR) microspectroscopy is a promising method for estimating the depth-wise composition of articular cartilage. The aim was to compare the specificity of two earlier introduced, presumably proteoglycan (PG)-specific FTIR parameters (i.e., absorption in the carbohydrate region with and without normalization with Amide I absorption) to estimate the reference PG content of normal and osteoarthritic human articular cartilage. This study is a direct continuation of our earlier studies, from which the presented data are reanalyzed. DESIGN: Earlier, FTIR microspectroscopy, digital densitometry, histological analyses, and polarized light microscopy were conducted in vitro for articular cartilage samples of human patellae (n = 72). In the present study, earlier data were combined and statistically reanalyzed in a depth-wise manner to clarify the specificity of FTIR parameters introduced for the estimation of PG content of articular cartilage. Digital densitometry for Safranin O-stained samples was used to indicate reference PG content. RESULTS: Direct absorption of the carbohydrate region estimated well the PG content in the middle and deep zones of articular cartilage and appeared to be superior compared to carbohydrate values normalized with Amide I absorption. However, in the superficial zone, the specificity of both FTIR-derived PG parameters was limited. CONCLUSIONS: Limitations of current FTIR-based PG parameters in the superficial zone of articular cartilage should be recognized and carefully taken into account in future studies using FTIR microspectroscopy for PG content estimation. Further research is needed to improve the specificity of FTIR parameters for the estimation of PG content of articular cartilage.