Headspace Of Cultures Research Articles

Life on the fringe: microbial adaptation to growth on carbon monoxide.

Microbial adaptation to extreme conditions takes many forms, including specialized metabolism which may be crucial to survival in adverse conditions. Here, we analyze the diversity and environmental importance of systems allowing microbial carbon monoxide (CO) metabolism. CO is a toxic gas that can poison most organisms because of its tight binding to metalloproteins. Microbial CO uptake was first noted by Kluyver and Schnellen in 1947, and since then many microbes using CO via oxidation have emerged. Many strains use molecular oxygen as the electron acceptor for aerobic oxidation of CO using Mo-containing CO oxidoreductase enzymes named CO dehydrogenase. Anaerobic carboxydotrophs oxidize CO using CooS enzymes that contain Ni/Fe catalytic centers and are unrelated to CO dehydrogenase. Though rare on Earth in free form, CO is an important intermediate compound in anaerobic carbon cycling, as it can be coupled to acetogenesis, methanogenesis, hydrogenogenesis, and metal reduction. Many microbial species—both bacteria and archaea—have been shown to use CO to conserve energy or fix cell carbon or both. Microbial CO formation is also very common. Carboxydotrophs thus glean energy and fix carbon from a “metabolic leftover” that is not consumed by, and is toxic to, most microorganisms. Surprisingly, many species are able to thrive under culture headspaces sometimes exceeding 1 atmosphere of CO. It appears that carboxydotrophs are adapted to provide a metabolic “currency exchange” system in microbial communities in which CO arising either abiotically or biogenically is converted to CO 2 and H 2 that feed major metabolic pathways for energy conservation or carbon fixation. Solventogenic CO metabolism has been exploited to construct very large gas fermentation plants converting CO-rich industrial flue emissions into biofuels and chemical feedstocks, creating renewable energy while mitigating global warming. The use of thermostable CO dehydrogenase enzymes to construct sensitive CO gas sensors is also in progress.

Profiling of headspace volatiles from Escherichia coli cultures using silicone-based sorptive media and thermal desorption GC-MS.

Headspace sorptive extraction technique using silicone based sorptive media coated stir bars is used for the first time here to extract, identify, and quantify heavy volatile organic compounds present in Escherichia coli culture headspace. Detection of infection presence is largely accomplished in laboratories through physical sampling and subsequent growth of cultures for biochemical testing. The use of volatile biomarkers released from pathogens as indicators for pathogenic presence can vastly reduce the time needed whilst improving the success rates for infection detection. To validate this, by using a contactless headspace sorptive extraction technique, the volatile compounds released from E. coli, grown in vitro, have been extracted and identified. Two different sorptive media for extracting these headspace volatiles were compared in this study and the identified volatiles were quantified. The large phase volume and wider retention of this sorptive technique compared to traditional sampling approach enabled preconcentration and collection of wider range of volatiles towards developing an extensive database of such heavy volatiles associated with E. coli. This supplements the existing data of potential bacterial markers and use of internal standards in these tests allows semi-quantitative estimation of these compounds towards the development and optimization of novel pathogen sensing devices.

The volatile molecular profiles of seven Streptococcus pneumoniae serotypes

In this study, the volatile molecule profile of Streptococcus pneumoniae serotypes was evaluated using solid phase microextraction (SPME) and two dimensional gas chromatography time-of-flight mass spectrometry (GC × GC-TOFMS). Here, seven serotypes (6B, 14, 15, 18C, 19F, 9V, and 23F) were analyzed in an isogenic background. We identified 13 core molecules associated with all seven serotypes, and seven molecules that were differentially produced between serotypes. Serotype 14 was found to have the most distinct volatile profile, and could be discriminated from the other six serotypes in aggregate with an area under the curve (AUC) of 89%. This study suggests that molecules from S. pneumoniae culture headspace show potential for rapid serotype identification.

TD/GC\u2013MS analysis of volatile markers emitted from mono- and co-cultures of Enterobacter cloacae and Pseudomonas aeruginosa in artificial sputum

IntroductionInfections such as ventilator-associated pneumonia (VAP) can be caused by one or more pathogens. Current methods for identifying these pathogenic microbes often require invasive sampling, and can be time consuming, due to the requirement for prolonged cultural enrichment along with selective and differential plating steps. This results in delays in diagnosis which in such critically ill patients can have potentially life-threatening consequences. Therefore, a non-invasive and timely diagnostic method is required. Detection of microbial volatile organic compounds (VOCs) in exhaled breath is proposed as an alternative method for identifying these pathogens and may distinguish between mono- and poly-microbial infections.ObjectivesTo investigate volatile metabolites that discriminate between bacterial mono- and co-cultures.MethodsVAP-associated pathogens Enterobacter cloacae and Pseudomonas aeruginosa were cultured individually and together in artificial sputum medium for 24 h and their headspace was analysed for potential discriminatory VOCs by thermal desorption gas chromatography–mass spectrometry.ResultsOf the 70 VOCs putatively identified, 23 were found to significantly increase during bacterial culture (i.e. likely to be released during metabolism) and 13 decreased (i.e. likely consumed during metabolism). The other VOCs showed no transformation (similar concentrations observed as in the medium). Bacteria-specific VOCs including 2-methyl-1-propanol, 2-phenylethanol, and 3-methyl-1-butanol were observed in the headspace of axenic cultures of E. cloacae, and methyl 2-ethylhexanoate in the headspace of P. aeruginosa cultures which is novel to this investigation. Previously reported VOCs 1-undecene and pyrrole were also detected. The metabolites 2-methylbutyl acetate and methyl 2-methylbutyrate, which are reported to exhibit antimicrobial activity, were elevated in co-culture only.ConclusionThe observed VOCs were able to differentiate axenic and co-cultures. Validation of these markers in exhaled breath specimens could prove useful for timely pathogen identification and infection type diagnosis.

Practices of shake-flask culture and advances in monitoring CO2 and O2.

About 85years have passed since the shaking culture was devised. Since then, various monitoring devices have been developed to measure culture parameters. O2 consumed and CO2 produced by the respiration of cells in shaking cultures are of paramount importance due to their presence in both the culture broth and headspace of shake flask. Monitoring in situ conditions during shake-flask culture is useful for analysing the behaviour of O2 and CO2, which interact according to Henry's law, and is more convenient than conventional sampling that requires interruption of shaking. In situ monitoring devices for shake-flask cultures are classified as direct or the recently developed bypass type. It is important to understand the characteristics of each type along with their unintended effect on shake-flask cultures, in order to improve the existing devices and culture conditions. Technical developments in the bypass monitoring devices are strongly desired in the future. It is also necessary to understand the mechanism underlying conventional shake-flask culture. The existing shaking culture methodology can be expanded into next-generation shake-flask cultures constituting a novel culture environment through a judicious selection of monitoring devices depending on the intended purpose of shake-flask culture. Construction and sharing the databases compatible with the various types of the monitoring devices and measurement instruments adapted for shaking culture can provide a valuable resource for broadening the application of cells with shake-flask culture.

Development of an adaptable headspace sampling method for metabolic profiling of the fungal volatome.

Pulmonary aspergillosis can cause serious complications in people with a suppressed immune system. Volatile metabolites emitted by Aspergillus spp. have shown promise for early detection of pathogenicity. However, volatile profiles require further research, as effective headspace analysis methods are required for extended chemical coverage of the volatome; in terms of both very volatile and semi-volatile compounds. In this study, we describe a novel adaptable sampling method in which fungal headspace samples can be sampled continuously throughout a defined time period using both active (pumped) and passive (diffusive) methods, with the capability for samples to be stored for later off-line analysis. For this method we utilise thermal desorption-gas chromatography-mass spectrometry to generate volatile metabolic profiles using Aspergillus fumigatus as the model organism. Several known fungal-specific volatiles associated with secondary metabolite biosynthesis (including α-pinene, camphene, limonene, and several sesquiterpenes) were identified. A comparison between the wild-type A. fumigatus with a phosphopantetheinyl transferase null mutant strain (ΔpptA) that is compromised in secondary metabolite synthesis, revealed reduced production of sesquiterpenes. We also showed the lack of terpene compounds production during the early growth phase, whilst pyrazines were identified in both early and late growth phases. We have demonstrated that the fungal volatome is dynamic and it is therefore critically necessary to sample the headspace across several time periods using a combination of active and passive sampling techniques to analyse and understand this dynamism.

Fe(III) Reducing Microorganisms from Iron Ore Caves Demonstrate Fermentative Fe(III) Reduction and Promote Cave Formation

ABSTRACTThe banded iron formations (BIF) of Brazil are composed of silica and Fe(III) oxide lamina, and are largely covered by a rock cap of BIF fragments in a goethite matrix (canga). Despite both BIF and canga being highly resistant to erosion and poorly soluble, >3,000 iron ore caves (IOCs) have formed at their interface. Fe(III) reducing microorganisms (FeRM) can reduce the Fe(III) oxides present in the BIF and canga, which could account for the observed speleogenesis. Here, we show that IOCs contain a variety of microbial taxa with member species capable of dissimilatory Fe(III) reduction, including the Chloroflexi, Acidobacteria and the Alpha- Beta- and Gammaproteobacteria; however, Fe(III) reducing enrichment cultures from IOCs indicate the predominance of Firmicutes and Enterobacteriaceae, despite varying the carbon/electron donor, Fe(III) type, and pH. We used model-based inference to evaluate multiple candidate hypotheses that accounted for the variation in medium chemistry and culture composition. Model selection indicated that none of the tested variables account for the dominance of the Firmicutes in these cultures. The addition of H2 to the headspace of the enrichment cultures enhanced Fe(III) reduction, while addition of N2 resulted in diminished Fe(III) reduction, indicating that these Enterobacteriaceae and Firmicutes were reducing Fe(III) during fermentative growth. These results suggest that fermentative reduction of Fe(III) may play a larger role in iron-rich environments than expected. Our findings also demonstrate that FeRM are present within the IOCs, and that their reductive dissolution of Fe(III) oxides, combined with mass transport of solubilized Fe(II) by groundwater, could contribute to IOC formation.

Emissions of volatile odorous metabolites by Clostridium perfringens - in vitro study using two broth cultures

An in vitro study was carried out to investigate the volatile odorous metabolites produced by necrotic enteritis inducing Clostridium perfringens (Cp) type A field strain (EHE-NE18) cultured in two broth media (thioglycollate broth, brain heart infusion broth) at three levels (0, 103 cfu/mL, 106 cfu/mL) using 2 × 3 factorial arrangement of treatments, with each replicated three times. The culture media with and without Cp was freshly prepared on the day of measurement and the culture headspace concentration of odorants was measured using selected ion-flow tube mass spectrometry (SIFT-MS). The results showed that media type and Cp level affected the concentration of a range of volatile odorous metabolites including sulfur compounds, alcohols, ketones, amines, and carboxylic acids. Thus, Cp contributes to the production of a wide range of odorous metabolites that can impart noxious smell during infection.

Evaluation of peroxidative stress of cancer cells in vitro by real-time quantification of volatile aldehydes in culture headspace.

Peroxidation of lipids in cellular membranes results in the release of volatile organic compounds (VOCs), including saturated aldehydes. The real-time quantification of trace VOCs produced by cancer cells during peroxidative stress presents a new challenge to non-invasive clinical diagnostics, which as described here, we have met with some success. A combination of selected ion flow tube mass spectrometry (SIFT-MS), a technique that allows rapid, reliable quantification of VOCs in humid air and liquid headspace, and electrochemistry to generate reactive oxygen species (ROS) in vitro has been used. Thus, VOCs present in the headspace of CALU-1 cancer cell line cultures exposed to ROS have been monitored and quantified in real time using SIFT-MS. The CALU-1 lung cancer cells were cultured in 3D collagen to mimic in vivo tissue. Real-time SIFT-MS analyses focused on the volatile aldehydes: propanal, butanal, pentanal, hexanal, heptanal and malondialdehyde (propanedial), that are expected to be products of cellular membrane peroxidation. All six aldehydes were identified in the culture headspace, each reaching peak concentrations during the time of exposure to ROS and eventually reducing as the reactants were depleted in the culture. Pentanal and hexanal were the most abundant, reaching concentrations of a few hundred parts-per-billion by volume, ppbv, in the culture headspace. The results of these experiments demonstrate that peroxidation of cancer cells in vitro can be monitored and evaluated by direct real-time analysis of the volatile aldehydes produced. The combination of adopted methodology potentially has value for the study of other types of VOCs that may be produced by cellular damage.

Metabolomics strategy for the mapping of volatile exometabolome from Saccharomyces spp. widely used in the food industry based on comprehensive two-dimensional gas chromatography.

Saccharomyces spp. are widely used in the food and beverages industries. Their cellular excreted metabolites are important for general quality of products and can contribute to product differentiation. This exploratory study presents a metabolomics strategy for the comprehensive mapping of cellular metabolites of two yeast species, Saccharomyces cerevisiae and S. pastorianus (both collected in an industrial context) through a multidimensional chromatography platform. Solid-phase microextraction was used as a sample preparation method. The yeast viability, a specific technological quality parameter, was also assessed. This untargeted analysis allowed the putative identification of 525 analytes, distributed over 14 chemical families, the origin of which may be explained through the pathways network associated with yeasts metabolism. The expression of the different metabolic pathways was similar for both species, event that seems to be yeast genus dependent. Nevertheless, these species showed different growth rates, which led to statistically different metabolites content. This was the first in-depth approach that characterizes the headspace content of S. cerevisiae and S. pastorianus species cultures. The combination of a sample preparation method capable of providing released volatile metabolites directly from yeast culture headspace with comprehensive two-dimensional gas chromatography was successful in uncovering a specific metabolomic pattern for each species.

Stable isotope profiles reveal active production of VOCs from human-associated microbes

Volatile organic compounds (VOCs) measured from exhaled breath have great promise for the diagnosis of bacterial infections. However, determining human or microbial origin of VOCs detected in breath remains a great challenge. For example, the microbial fermentation product 2,3-butanedione was recently found in the breath of Cystic Fibrosis (CF) patients; parallel culture-independent metagenomic sequencing of the same samples revealed that Streptococcus and Rothia spp. have the genetic capacity to produce 2,3-butanedione. To investigate whether the genetic capacity found in metagenomes translates to bacterial production of a VOC of interest such as 2,3-butanedione, we fed stable isotopes to three bacterial strains isolated from patients: two gram-positive bacteria, Rothia mucilaginosa and Streptococcus salivarius, and a dominant opportunistic gram-negative pathogen, Pseudomonas aeruginosa. Culture headspaces were collected and analyzed using a gas chromatographic system to quantify the abundance of VOCs of interest; mass spectroscopy was used to determine whether the stable isotope label had been incorporated. Our results show that R. mucilaginosa and S. salivarius consumed D-Glucose-13C6 to produce labeled 2,3-butanedione. R. mucilaginosa and S. salivarius also produced labeled acetaldehyde and ethanol when grown with 2H2O. Additionally, we find that P. aeruginosa growth and dimethyl sulfide production are increased when exposed to lactic acid in culture. These results highlight the importance VOCs produced by P. aeruginosa, R. mucilaginosa, and S. salivarius as nutrients and signals in microbial communities, and as potential biomarkers in a CF infection.

Selected ion flow tube study of the reactions of H3 O+ and NO+ with a series of primary alcohols in the presence of water vapour in support of selected ion flow tube mass spectrometry.

Alcohols are often present in foods and other biological media, including exhaled breath, urine and cell culture headspace. For their analysis by selected ion flow tube mass spectrometry (SIFT-MS), the ion chemistry initiated by the reactions of the reagent ions H3 O+ and NO+ with alcohol molecules in the presence of water molecules needs to be understood and quantitatively described. The reactions of H3 O+ and NO+ ions have been studied with the primary alcohols, methanol, ethanol, 1-propanol, 1-butanol, 1-pentanol and 1-hexanol, under the conditions used for SIFT-MS analyses (1Torr He; 0.1Torr air sample; 300K) and over a range of sample gas humidity from 1% to 5.5%. The H3 O+ reactions led to the formation of protonated alcohol molecules MH+ and their hydrates MH+ (H2 O)1,2,3 and (MH+ -H2 O) fragment ions. The NO+ reactions were observed to proceed mainly via hydride ion transfer, resulting in the formation of [M-H]+ product ions. Formation of the NO+ M adduct ions was also observed due to ligand switching between the NO+ (H2 O)1,2 hydrated reagent ions and M, and via direct NO+ /M association in the case of ethanol. The variation in the percentages of the hydrated product ions with the air sample humidity is reported. This detailed study has provided the kinetics data, including the secondary hydrated ion product distributions, for the reactions of a number of volatile primary alcohols with the SIFT-MS reagent ions H3 O+ and NO+ , which allows their analyses by SIFT-MS in humid air and also helps in the interpretation of proton transfer reaction (PTR)-MS data. Copyright © 2016 John Wiley & Sons, Ltd.

Volatile-Mediated Effects Predominate in Paraburkholderia phytofirmans Growth Promotion and Salt Stress Tolerance of Arabidopsis thaliana.

Abiotic stress has a growing impact on plant growth and agricultural activity worldwide. Specific plant growth promoting rhizobacteria have been reported to stimulate growth and tolerance to abiotic stress in plants, and molecular mechanisms like phytohormone synthesis and 1-aminocyclopropane-1-carboxylate deamination are usual candidates proposed to mediate these bacterial effects. Paraburkholderia phytofirmans PsJN is able to promote growth of several plant hosts, and improve their tolerance to chilling, drought and salinity. This work investigated bacterial determinants involved in PsJN stimulation of growth and salinity tolerance in Arabidopsis thaliana, showing bacteria enable plants to survive long-term salinity treatment, accumulating less sodium within leaf tissues relative to non-inoculated controls. Inactivation of specific bacterial genes encoding ACC deaminase, auxin catabolism, N-acyl-homoserine-lactone production, and flagellin synthesis showed these functions have little influence on bacterial induction of salinity tolerance. Volatile organic compound emission from strain PsJN was shown to reproduce the effects of direct bacterial inoculation of roots, increasing plant growth rate and tolerance to salinity evaluated both in vitro and in soil. Furthermore, early exposure to VOCs from P. phytofirmans was sufficient to stimulate long-term effects observed in Arabidopsis growth in the presence and absence of salinity. Organic compounds were analyzed in the headspace of PsJN cultures, showing production of 2-undecanone, 7-hexanol, 3-methylbutanol and dimethyl disulfide. Exposure of A. thaliana to different quantities of these molecules showed that they are able to influence growth in a wide range of added amounts. Exposure to a blend of the first three compounds was found to mimic the effects of PsJN on both general growth promotion and salinity tolerance. To our knowledge, this is the first report on volatile compound-mediated induction of plant abiotic stress tolerance by a Paraburkholderia species.

High Frequency of Thermodesulfovibrio spp. and Anaerolineaceae in Association with Methanoculleus spp. in a Long-Term Incubation of n-Alkanes-Degrading Methanogenic Enrichment Culture.

In the present study, the microbial community and functional gene composition of a long-term active alkane-degrading methanogenic culture was established after two successive enrichment culture transfers and incubated for a total period of 1750 days. Molecular analysis was conducted after the second transfer (incubated for 750 days) for both the active alkanes-degrading methanogenic enrichment cultures (T2-AE) and the background control (T2-BC). A net increase of methane as the end product was detected in the headspace of the enrichment cultures amended with long-chain n-alkanes and intermediate metabolites, including octadecanoate, hexadecanoate, isocaprylate, butyrate, isobutyrate, propionate, acetate, and formate were measured in the liquid cultures. The composition of microbial community shifted through the successive transfers over time of incubation. Sequences of bacterial and archaeal 16S rRNA gene (16S rDNA) and mcrA functional gene indicated that bacterial sequences affiliated to Thermodesulfovibrio spp. and Anaerolineaceae and archaeal sequences falling within the genus Methanoculleus were the most frequently encountered and thus represented the dominant members performing the anaerobic degradation of long-chain n-alkanes and methanogenesis. In addition, the presence of assA functional genes encoding the alkylsuccinate synthase α subunit indicated that fumarate addition mechanism could be considered as a possible initial activation step of n-alkanes in the present study. The succession pattern of microbial communities indicates that Thermodesulfovibrio spp. could be a generalist participating in the metabolism of intermediates, while Anaerolineaceae plays a key role in the initial activation of long-chain n-alkane biodegradation.

Microalgae Synthesize Hydrocarbons from Long-Chain Fatty Acids via a Light-Dependent Pathway.

Microalgae are considered a promising platform for the production of lipid-based biofuels. While oil accumulation pathways are intensively researched, the possible existence of a microalgal pathways converting fatty acids into alka(e)nes has received little attention. Here, we provide evidence that such a pathway occurs in several microalgal species from the green and the red lineages. In Chlamydomonas reinhardtii (Chlorophyceae), a C17 alkene, n-heptadecene, was detected in the cell pellet and the headspace of liquid cultures. The Chlamydomonas alkene was identified as 7-heptadecene, an isomer likely formed by decarboxylation of cis-vaccenic acid. Accordingly, incubation of intact Chlamydomonas cells with per-deuterated D31-16:0 (palmitic) acid yielded D31-18:0 (stearic) acid, D29-18:1 (oleic and cis-vaccenic) acids, and D29-heptadecene. These findings showed that loss of the carboxyl group of a C18 monounsaturated fatty acid lead to heptadecene formation. Amount of 7-heptadecene varied with growth phase and temperature and was strictly dependent on light but was not affected by an inhibitor of photosystem II. Cell fractionation showed that approximately 80% of the alkene is localized in the chloroplast. Heptadecane, pentadecane, as well as 7- and 8-heptadecene were detected in Chlorella variabilis NC64A (Trebouxiophyceae) and several Nannochloropsis species (Eustigmatophyceae). In contrast, Ostreococcus tauri (Mamiellophyceae) and the diatom Phaeodactylum tricornutum produced C21 hexaene, without detectable C15-C19 hydrocarbons. Interestingly, no homologs of known hydrocarbon biosynthesis genes were found in the Nannochloropsis, Chlorella, or Chlamydomonas genomes. This work thus demonstrates that microalgae have the ability to convert C16 and C18 fatty acids into alka(e)nes by a new, light-dependent pathway.

Evaluation of ferric oxide and ferric citrate for their effects on fermentation, production of sulfide and methane, and abundance of select microbial populations using in vitro rumen cultures

This study systematically evaluated the effect of ferric iron on sulfate reduction to sulfide, feed digestion and fermentation, methane production, and populations of select ruminal microbes using in vitro rumen cultures. Ferric oxide (Fe2O3) and ferric citrate (C6H5FeO7) at six concentrations (0, 25, 50, 100, 150, and 200mg/L as Fe3+) were tested. Ferric iron decreased production of both H2S gas in culture headspace (up to 71.9%) and aqueous sulfide (up to 80.8%), without adversely affecting other fermentation parameters, with ferric citrate being more effective than ferric oxide. Total archaeal population was increased by ferric citrate, but methane production was not affected significantly. The population of sulfate reducing bacteria was affected differently by ferric oxide than by ferric citrate. The results of this study could guide future in vivo studies to develop effective solutions to abate sulfur-associated polioencephalomalacia in cattle fed high-sulfur diet such as dried distiller’s grains with solubles.

A Study on VOCs Released by Lung Cancer Cell Line Using GCMS-SPME

The gas chromatography mass spectrometry (GCMS) with combination of Solid Phase Micro-extraction (SPME) was used to study the volatile organic compounds (VOCs) which emitted by the in-vitro cultured human cells and compared with documented volatile biomarker of lung cancer. For this purpose, the lung cancer cell (A549) and non-cancerous lung cell (WI38VA13) were cultured in identical growth medium, concurrently. The VOCs in the headspace of the cell cultures and the blank growth media (reference sample) were collected directly from the culture flask using SPME for 15minutes. The results show that two different volatile metabolites were screened out between A549 cells and Wi38VA13 cells. A549 cell found to emit 2 noticeable VOC which are decane and heneicosane. While for WI38VA13, the VOCs released were 1-Heptanol and heptadecane. The acquired VOCs were compared with the previous studies. The findings in this work conclude that the specific VOC of cells can be act as their odour signature and can be used to provide non-invasively screening of lung cancer using gas array sensor devices.

Detection of signature volatiles for cariogenic microorganisms.

The development of a breath test by the identification of volatile organic compounds (VOCs) emitted by cariogenic bacteria is a promising approach for caries risk assessment and early caries detection. The aim of the present study was to investigate the volatile profiles of three major cariogenic bacteria and to assess whether the obtained signatures were species-specific. Therefore, the headspaces above cultures of Streptococcus mutans, Lactobacillus salivarius and Propionibacterium acidifaciens were analysed after 24 and 48 h of cultivation using gas chromatography and mass spectrometry. A volatile database was queried for the obtained VOC profiles. Sixty-four compounds were detected within the analysed culture headspaces and were absent (36) or at least only present in minor amounts (28) in the control headspace. For S. mutans 18, for L. salivarius three and for P. acidifaciens five compounds were found to be unique signature VOCs. Database matching revealed that the identified signatures of all bacteria were unique. Furthermore, 13 of the 64 detected substances have not been previously reported to be emitted by bacteria or fungi. Specific VOC signatures were found in all the investigated bacteria cultures. The obtained results encourage further research to investigate the transferability to in vivo conditions towards the development of a breath test.

Investigation of cell culture volatilomes using solid phase micro extraction: Options and pitfalls exemplified with adenocarcinoma cell lines.

Three strategies to sample volatile organic compounds (VOC) from lung cancer cell lines cultured in vitro were compared. Headspace solid phase microextraction was applied in situ to culture flasks and alternatively to subsamples of headspace gas or to nutrient solution subsamples followed by gas chromatography-mass spectrometry. The direct quantification of 55 VOC in the headspace of cell cultures was validated and is discussed with respect to reproducibility and system-related interferences. The role of the VOC background from culture media and usually employed polystyrene culture vessels is examined and was seen to invoke potentially misleading conclusions. The commercial A549 and two further adenocarcinoma cell lines displayed largely similar VOC profiles with distinct differences regarding certain individual substances. There is evidence for the inappropriateness of the standard cell culturing methods in the search for volatile cancer markers.

Dynamic collection and analysis of volatile organic compounds from the headspace of cell cultures

Exhaled breath has proven to be a valuable source of information about human bodies. Subtle differences between volatile organic compounds (VOCs) formed endogenously can be detected and become a base for a potential monitoring tool for health and disease. Until now, there has been a lack of biological and mechanistic knowledge of the processes involved in the production of relevant VOCs. Among the possible sources of health-related and disease-related VOCs are microorganisms found in the respiratory tract and in the gut. Other VOCs in the body are produced by cells that are influenced by the disease, for instance, due to metabolic disorders and/or inflammation. To gain insight into the in vivo production of VOCs by human cells and thus the exhaled breath composition, in vitro experiments involving relevant cells should be studied because they may provide valuable information on the production of VOCs by the affected cells. To this aim we developed and validated a system for dynamically (continuously) collecting headspace air in vitro using a Caco-2 cell line. The system allows the application of different cell lines as well as different experimental setups, including varying exposure times and treatment options while preserving cell viability. Significant correlation (p ⩽ 0.0001) between collection outputs within each studied group confirmed high reproducibility of the collection system. An example of such an application is presented here. We studied the influence of oxidative stress on the VOC composition of the headspace air of Caco-2 cells.By comparing the VOC composition of air flushed through empty culture flasks (n = 35), flasks with culture medium (n = 35), flasks with medium and cells (n = 20), flasks with medium and an oxidative stressor (H2O2) (n = 20), and flasks with medium, stressor, and cells (n = 20), we were able to separate the effects from the stressor on the cells from all other interactions. Measurements were performed with gas chromatography time-of-flight mass spectrometry. Multivariate data analysis allowed detection of significant altered compounds in the compared groups.We found a significant change (p ⩽ 0.001) of the composition of VOCs due to the stressing of the Caco-2 cells by H2O2. A total of ten VOCs showed either increased or decreased abundance in the headspace of the cell cultures due to the presence of the H2O2 stressor.