Rapid detection of microbiota cell type diversity using machine-learned classification of flow cytometry data.

The study of complex microbial communities typically entails high-throughput sequencing and downstream bioinformatics analyses. Here we expand and accelerate microbiota analysis by enabling cell type diversity quantification from multidimensional flow cytometry data using a supervised machine learning algorithm of standard cell type recognition (CellCognize). As a proof-of-concept, we trained neural networks with 32 microbial cell and bead standards. The resulting classifiers were extensively validated in silico on known microbiota, showing on average 80% prediction accuracy. Furthermore, the classifiers could detect shifts in microbial communities of unknown composition upon chemical amendment, comparable to results from 16S-rRNA-amplicon analysis. CellCognize was also able to quantify population growth and estimate total community biomass productivity, providing estimates similar to those from 14C-substrate incorporation. CellCognize complements current sequencing-based methods by enabling rapid routine cell diversity analysis. The pipeline is suitable to optimize cell recognition for recurring microbiota types, such as in human health or engineered systems.

Quantification of Residual Perfume by Py-GC-MS in Fragrance Encapsulate Polymeric Materials Intended for Biodegradation Tests.

Perfume encapsulates are widely used in commercial products to control the kinetic release of odorant molecules, increase storage stability and/or improve deposition on different substrates. In most of the cases, they consist of core-shell polymeric microcapsules that contain fragrance molecules. A current challenge is to design and produce polymeric materials for encapsulation that are both resistant and non-persistent. The selection of such eco-friendly formulations is linked to a deep understanding of the polymeric material used for encapsulation and its biodegradation profile. To collect this information, pure samples of capsule shells are needed. In this article we present an innovative quantification method for residual volatiles based on pyrolysis-GC-MS to enable validation of sample quality prior to further testing. The presented analytical method also led to the development of a robust and comprehensive purification protocol for polymers from commercial samples. Standard techniques are not suited for this kind of measurement due to the non-covalent embedding of volatiles in the 3D structure of the polymers. We demonstrated the confounding impact of residual volatiles on the estimated biodegradability of fragrance encapsulates.

Prediction of response factors for gas chromatography with flame ionization detection: Algorithm improvement, extension to silylated compounds, and application to the quantification of metabolites.

We previously showed that the relative response factors of volatile compounds were predictable from either combustion enthalpies or their molecular formulae only . We now extend this prediction to silylated derivatives by adding an increment in the ab initio calculation of combustion enthalpies. The accuracy of the experimental relative response factors database was also improved and its population increased to 490 values. In particular, more brominated compounds were measured, and their prediction accuracy was improved by adding a correction factor in the algorithm. The correlation coefficient between predicted and measured values increased from 0.936 to 0.972, leading to a mean prediction accuracy of ± 6%. Thus, 93% of the relative response factors values were predicted with an accuracy of better than ± 10%. The capabilities of the extended algorithm are exemplified by (i) the quick and accurate quantification of hydroxylated metabolites resulting from a biodegradation test after silylation and prediction of their relative response factors, without having the reference substances available; and (ii) the rapid purity determinations of volatile compounds. This study confirms that Gas chromatography with a flame ionization detector and using predicted relative response factors is one of the few techniques that enables quantification of volatile compounds without calibrating the instrument with the pure reference substance.

High throughput screening of perfumery raw materials for antimicrobial properties.

A microdilution protocol was developed and automated using a liquid handling station, allowing the determination of minimum inhibitory concentrations (MIC) of hydrophobic raw materials commonly used in the perfume industry (essential oils and synthetic chemicals). Tests were performed in 96-well microtiter plates against standard bacterial test strains and skin isolates involved in underarm malodor. The comparison with data previously reported in the literature indicated that the protocol was suitable, yielding MIC values that were in general agreement with those derived from manual test methods. For the majority of active test compounds, results showed a pronounced difference in susceptibility pattern between the Gram-positive and Gram-negative test strains used in this study. For a group of acyclic aliphatic aldehydes, a structure-activity relationship depending on the chain length was found.

An assessment of biodegradability of quaternary carbon-containing fragrance compounds: comparison of experimental OECD screening test results and in silico prediction data.

An assessment of biodegradability was carried out for fragrance substances containing quaternary carbons by using data obtained from Organisation for Economic Co-operation and Development (OECD) 301F screening tests for ready biodegradation and from Biowin and Catalogic prediction models. Despite an expected challenging profile, a relatively high percentage of common-use fragrance substances showed significant biodegradation under the stringent conditions applied in the OECD 301F test. Among 27 test compounds, 37% met the pass level criteria after 28 d, while another 26% indicated partial breakdown (≥20% biodegradation). For several compounds for which structural analogs were available, the authors found that structures that were rendered less water soluble by either the presence of an acetate ester or the absence of oxygen tended to degrade to a lesser extent compared to the primary alcohols or oxygenated counterparts under the test conditions applied. Difficulties were encountered when attempting to correlate experimental with in silico data. Whereas the Biowin model combinations currently recommended by regulatory agencies did not allow for a reliable discrimination between readily and nonbiodegradable compounds, only a comparably small proportion of the chemicals studied (30% and 63% depending on the model) fell within the applicability domain of Catalogic, a factor that critically reduced its predictive power. According to these results, currently neither Biowin nor Catalogic accurately reflects the potential for biodegradation of fragrance compounds containing quaternary carbons.

Examining chemical compound biodegradation at low concentrations through bacterial cell proliferation.

We show proof of principle for assessing compound biodegradation at 1-2 mg C per L by measuring microbial community growth over time with direct cell counting by flow cytometry. The concept is based on the assumption that the microbial community will increase in cell number through incorporation of carbon from the added test compound into new cells in the absence of (as much as possible) other assimilable carbon. We show on pure cultures of the bacterium Pseudomonas azelaica that specific population growth can be measured with as low as 0.1 mg 2-hydroxybiphenyl per L, whereas in mixed community 1 mg 2-hydroxybiphenyl per L still supported growth. Growth was also detected with a set of fragrance compounds dosed at 1-2 mg C per L into diluted activated sludge and freshwater lake communities at starting densities of 10(4) cells per ml. Yield approximations from the observed community growth was to some extent in agreement with standard OECD biodegradation test results for all, except one of the examined compounds.

Caloramator viterbensis sp. nov., a novel thermophilic, glycerol-fermenting bacterium isolated from a hot spring in Italy.

A moderately thermophilic, anaerobic bacterium, strain JW/MS-VS5T, was isolated from a mixed sediment/water sample of a hot spring at Bagnaccio (near Viterbo, Italy). The cells of this organism were straight to slightly curved rods, 0.4-0.6 x 2.03.0 microm in dimension. Cells occurred singly and stained Gram-positive. The temperature range for growth at pH(25C) 6.0 was 33-64 degrees C, the optimum being 58 degrees C. The pH(25C) range for growth was from 5.0 to 7.8, the optimum being 6.0-6.5. The substrates utilized included glycerol, glucose, fructose, mannose, galactose, sucrose, cellobiose, lactose, starch and yeast extract. Acetate and 1,3-propanediol were the only detectable organic products of glycerol fermentation; significant amounts of H2 were produced during growth. The strain was unable to grow autotrophically in the presence of H2 and CO2. The main products of glucose fermentation were CO2, H2, acetate and ethanol. Single amino acids, including serine, glutamine, threonine, leucine, methionine, aspartate, valine and histidine (but not arginine), served as carbon sources. Growth was completely inhibited by ampicillin, chloramphenicol, erythromycin, rifampicin and kanamycin at 100 microg ml(-1) and was retarded by streptomycin and tetracycline. The G+C content of the DNA was 32 mol% (HPLC). According to 16S rDNA sequence analysis, the isolate is located within the Gram-type positive Bacillus-Clostridium branch of the phylogenetic tree. On the basis of physiological properties and phylogenetic analysis, it is proposed that strain JW/MS-VS5T (the only, and type, strain) (= DSM 13723T = ATCC PTA 584T), constitutes the new species Caloramator viterbensis.