Production Of Microbial Volatile Organic Compounds Research Articles

Dimethylpolysulfides production as the major mechanism behind wheat fungal pathogen biocontrol, by Arthrobacter and Microbacterium actinomycetes.

Volatile organic compounds (VOCs) produced by plant-associated microorganisms could serve as natural biocontrol compounds. In this work, we investigate the potential of wheat rhizosphere Microbacterium and Arthrobacter actinomycetes to inhibit the growth of major wheat phytopathogenic fungi, Fusarium graminearum and Zymoseptoria tritici via production of antifungal VOCs. A correlative analysis between fungal-growth inhibition versus bacterial volatilomic profiles identified four dimethylpolysulfide (DMPS) VOCs as the main active compounds behind their biocontrol potential. Subsequent inhibition assays reveal that DMTriS (dimethyltrisulfide) exhibits the strongest inhibition effects, then dimethyldisulfide (DMDiS). Further investigation focuses on the mechanisms behind F. graminearum-growth inhibition by the most active strain Microbacterium JM188 in dual culture. Surprisingly, nine interaction-induced VOCs, including two fungal sesquiterpenes, were exclusively detected in dual cultures, suggesting a complex interplay between microbial VOC production and sensing. More importantly, all JM188 VOCs, including antifungal DMPS, were less abundant in dual cultures, suggesting an uptake of bacterial VOCs by the fungus. Quantification of pure DMPS after confrontation with F. graminearum confirmed complete and partial uptake by the fungus of DMTriS and DMDiS VOCs, respectively, suggesting a potential link between the fungal uptake level of bacterial VOCs and their toxicity. Finally, we demonstrated that F. graminearum growth inhibition leads to a complete depletion of DON (deoxynivalenol) carcinogenic mycotoxins, highlighting a significant modulation of fungal metabolism. Collectively, these results pinpoint DMPS as broad-range fungal-inhibiting VOCs produced by rhizosphere Microbacterium and Arthrobacter, and emphasize the extensive VOC-mediated interplay between bacterial biocontrol agents and fungal pathogens.IMPORTANCEAs the management of wheat fungal diseases becomes increasingly challenging, the use of bacterial agents with biocontrol potential against the two major wheat phytopathogens, Fusarium graminearum and Zymoseptoria tritici, may prove to be an interesting alternative to conventional pest management. Here, we have shown that dimethylpolysulfide volatiles are ubiquitously and predominantly produced by wheat-associated Microbacterium and Arthrobacter actinomycetes, displaying antifungal activity against both pathogens. By limiting pathogen growth and DON virulence factor production, the use of such DMPS-producing strains as soil biocontrol inoculants could limit the supply of pathogen inocula in soil and plant residues, providing an attractive alternative to dimethyldisulfide fumigant, which has many non-targeted toxicities. Notably, this study demonstrates the importance of bacterial volatile organic compound uptake by inhibited F. graminearum, providing new insights for the study of volatiles-mediated toxicity mechanisms within bacteria-fungus signaling crosstalk.

Rapid and Online Detection of Foodborne Bacteria via a Novel Ultraviolet Photoionization Time-of-Flight Mass Spectrometry.

Foodborne bacteria are widespread contaminated sources of food; hence, the real-time monitoring of pathogenic bacteria in food production is important for the food industry. In this study, a novel rapid detection method based on microbial volatile organic compounds (MVOCs) emitted from foodborne bacteria was established by using ultraviolet photoionization time-of-flight mass spectrometry (UVP-TOF-MS). The results showed obvious differences of MVOCs among the five species of bacteria, and the characteristic MVOCs for each bacterium were selected by a feature selection algorithm. Online monitoring of MVOCs during bacterial growth displayed distinct metabolomic patterns of the five species. MVOCs were most abundant and varied among species during the logarithmic phase. Finally, MVOC production by bacteria in different food matrixes was explored. The machine learning models for bacteria cultured in different matrixes showed a good classification performance for the five species with an accuracy of over 0.95. This work based on MVOC analysis by online UVP-TOF-MS achieved effective rapid detection of bacteria and showed its great application potential in the food industry for bacterial monitoring.

Electrophysiological and Behavioral Responses of Virgin Female Bactrocera tryoni to Microbial Volatiles from Enterobacteriaceae.

The Queensland fruit fly (Bactrocera tryoni) is a major polyphagous pest widespread in Australia and several Pacific Islands. Bacteria present on the host plant phyllosphere supply proteins, essential for egg development and female sexual maturity. We investigated the role of microbial volatile organic compounds (MVOCs) emitted by Enterobacteriaceae commonly found on the host plant and in the fly gut in attracting virgin females. Bacteria were cultured on artificial media and natural fruits, at various pH, and MVOCs were collected using different headspace volatile absorbent materials. The olfactory responses of virgin females to bacterial MVOCs were assessed via electrophysiology and behavioral assays. The production of MVOCs was strongly influenced qualitatively by the bacterial strain and the type of media, and it semi-quantitatively varied with pH and time. MVOCs emitted by Klebsiella oxytoca invoked the strongest antennal response and were the most attractive. Among the identified compounds triggering an olfactory response, D-limonene and 2-nonanone were both significantly behaviorally attractive, whereas phenol, nonanal, isoamyl alcohol, and some pyrazines appeared to be repulsive. This study deepens our understanding of the chemical ecology between fruit flies and their bacterial symbionts and paves the way for novel synthetic lures based on specifically MVOCs targeting virgin females.

Influence of bacterial and alveolar cell co-culture on microbial VOC production using HS-GC/MS.

Volatile organic compounds (VOCs) found in exhaled breath continue to garner interest as an alternative diagnostic tool in pulmonary infections yet, their clinical integration remains a challenge with difficulties in translating identified biomarkers. Alterations in bacterial metabolism secondary to host nutritional availability may explain this but is often inadequately modelled in vitro. The influence of more clinically relevant nutrients on VOC production for two common respiratory pathogens was investigated. VOCs from Staphylococcus aureus (S.aureus) and Pseudomonas aeruginosa (P.aeruginosa) cultured with and without human alveolar A549 epithelial cells were analyzed using headspace extraction coupled with gas chromatography-mass spectrometry. Untargeted and targeted analyses were performed, volatile molecules identified from published data, and the differences in VOC production evaluated. Principal component analysis (PCA) could differentiate alveolar cells from either S. aureus or P. aeruginosa when cultured in isolation based on PC1 (p = 0.0017 and 0.0498, respectively). However, this separation was lost for S. aureus (p = 0.31) but not for P. aeruginosa (p = 0.028) when they were cultured with alveolar cells. S. aureus cultured with alveolar cells led to higher concentrations of two candidate biomarkers, 3-methyl-1-butanol (p = 0.001) and 3-methylbutanal (p = 0.002) when compared to S. aureus, alone. P. aeruginosa metabolism resulted in less generation of pathogen-associated VOCs when co-cultured with alveolar cells compared to culturing in isolation. VOC biomarkers previously considered indicative of bacterial presence are influenced by the local nutritional environment and this should be considered when evaluating their biochemical origin.

Identification of MVOCs Produced by Coniophora puteana and Poria placenta Growing on WPC Boards by Using Subtraction Mass Spectra.

Volatile fungal metabolites are responsible for various odors and may contribute to a “sick building syndrome” (SBS) with a negative effect on the heath of building. The authors have attempted to fill the research gaps by analyzing microbial volatile organic compounds (MVOCs) originating from representatives of the Basidiomycetes class that grow on wood-polymer composite (WPC) boards. WPCs have been analyzed as a material exposed to biodeterioration. Indoor air quality (IAQ) is affected by the increased use of WPCs inside buildings, and is becoming a highly relevant research issue. The emission profiles of MVOCs at various stages of WPC decay have been demonstrated in detail for Coniophora puteana and Poria placenta, and used to set the European industrial standards for wood-decay fungi. Differences in the production of MVOCs among these species of fungi have been detected using the thermal desorption-gas chromatography/mass spectrometry (TD-GC/MS) method. This study identifies the production of alcohols, aldehydes, ketones, carboxylic acids and other compounds during one month of fungal growth. The identified level of metabolites indicates a relation between the level of air pollution and condition of the WPC material, which may become part of IAQ quantification in the future. The study points to the species-specific compounds for representatives of brown and white-rot fungi and the compounds responsible for their odor. In this study, 1-Octen-3-ol was indicated as a marker for their active growth, which is also associated with SBS. The proposed experimental set-up and data analysis are a simple and convenient way to obtain emission profiles of MVOCs from microbes growing on different materials.

Effects of Growth Parameters on the Analysis of Aspergillus flavus Volatile Metabolites

Aspergillus flavus produces dangerous secondary metabolites known as aflatoxins, which are toxic and carcinogenic, and their contamination of agricultural products results in health issues and economic hardships in the U.S. and around the world. Early identification of aflatoxigenic isolates of A. flavus is the key in the management of these fungi. An emerging detection method for specific fungi identification involves the analysis of microbial volatile organic compounds (MVOCs) released by the fungi. Complicating this approach is the understanding that many factors influence metabolic production, including growth parameters, such as growth media, temperature, spore counts and oxidation stress. In addition, analytical and data analysis methods can also influence the results. Several growth and analysis methods were evaluated and optimized in order to better understand the effect of the methods on fungi MVOC signatures. The results indicate that carboxen/polydimethylsiloxane (CAR/PDMS) has the best extraction efficiency for the MVOCs emitted by A. flavus. Both chemical defined agar (CDA) and chemical defined liquid (CDL) are suitable growth media for MVOC emission studies. The highest MVOC production was found at 30 °C. Log transformation was considered one of the best data pretreatment methods when analyzing MVOC data and resulted in the best principal component analysis (PCA) clustering in the experiments with different growth media. This study aims to elucidate fungal volatile organic compounds (VOCs) differences due to variations in growth parameters as a first step in the development of an analytical method for the monitoring of aflatoxigenic A. flavus contamination in crop storage facilities.

Identification of volatile markers for indoor fungal growth and chemotaxonomic classification of Aspergillus species

Microbial volatile organic compounds (MVOCs) were collected in water-damaged buildings to evaluate their use as possible indicators of indoor fungal growth. Fungal species isolated from contaminated buildings were screened for MVOC production on malt extract agar by means of headspace solid-phase microextraction followed by gas chromatography-mass spectrometry (GC–MS) analysis. Some sesquiterpenes, specifically derived from fungal growth, were detected in the sampled environments and the corresponding fungal producers were identified. Statistical analysis of the detected MVOC profiles allowed the identification of species-specific MVOCs or MVOC patterns for Aspergillus versicolor group, Aspergillus ustus, and Eurotium amstelodami. In addition, Chaetomium spp. and Epicoccum spp. were clearly differentiated by their volatile production from a group of 76 fungal strains belonging to different genera. These results are useful in the chemotaxonomic discrimination of fungal species, in aid to the classical morphological and molecular identification techniques.

Influence of environmental conditions on production of volatiles by Trichoderma atroviride in relation with the sick building syndrome

A Trichoderma atroviride strain was isolated from a water-damaged office and the production of microbial volatile organic compounds (MVOCs) was investigated by means of headspace solid phase microextraction GC–MS. Different growth conditions (substrate, temperature, relative humidity) were selected, resembling indoor parameters, to elucidate a possible relationship between MVOCs, produced by Trichoderma atroviride, and the Sick Building Syndrome. In general, the range of MVOCs and the emitted quantities were larger on malt extract agar (MEA) than on wallpaper and plasterboard. Particular attention was dedicated to the volatile marker 6-pentyl-2-pyrone, a compound produced in high quantities on MEA, and its mucosal irritation potency was shown in a slug mucosal irritation assay. Some compounds characteristic for growth on specific building materials were detected, e.g. 2-ethylcyclopentanone, menthone, iso-menthone and trans- p-menth-2-en-7-ol on plasterboard and 4-heptanone and 1-octen-3-ol on wallpaper. Relative humidity and substrate had a more important effect on MVOC production than temperature.

Determination of volatile metabolites originating from mould growth on wall paper and synthetic media

Microbial volatile organic compounds (MVOCs) emitted from the mould species Penicillium expansum, P. chrysogenum, Aspergillus versicolor, A. fumigatus, A. niger and Cladosporium cladosporoides were analyzed by means of solid phase microextraction (SPME) and GCMS. The mould species were cultivated on the synthetic agar dichloran chloramphenicol (DG 18) and on wet wall paper. The production of MVOCs was monitored over several weeks to detect changes in the emission rates between the initial stage and later periods of growth. The cultivation on the synthetic agar resulted in MVOC patterns with a wide variety of signals. In contrast, the growth on wet wall paper led to changed MVOC patterns with less signals. The emission rates were drastically reduced. Components emitted by all six fungi species on wall paper were 2-pentanol and 2-pentanone. 1-Octen-3-ol was emitted by five fungi species. 2-Pentanol was only detected in considerable amounts during the first days of growth whereas 1-octen-3-ol had a more constant emission rate over the whole period of growth. On the basis of our studies some MVOCs could be proposed as specific for single fungi on wall paper, e.g. 1,3-dimethoxybenzene for A. versicolor and 2,4-pentandione for A. fumigatus.

Use of headspace SPME-GC-MS for the analysis of the volatiles produced by indoor molds grown on different substrates

An automated headspace solid phase microextraction method followed by GC-MS analysis was used to evaluate and compare the in vitro production of microbial volatile organic compounds (MVOCs) on malt extract agar, plasterboard and wallpaper. Five fungal strains were isolated from the walls of water-damaged houses and identified. In addition, four other common molds were studied. In general, MVOC production was the highest on malt extract agar. On this synthetic medium, molds typically produced 2-methylpropanol, 2-methylbutanol and 3-methylbutanol. On wallpaper, mainly 2-ethylhexanol, methyl 2-ethylhexanoate and compounds of the C8-complex such as 1-octene-3-ol, 3-octanone, 3-octanol and 1,3-octadiene were detected. The detection of 2-ethylhexanol and methyl 2-ethylhexanoate indicates an enhanced degradation of the substrate by most fungi. For growth on plasterboard, no typical metabolites were detected. Despite these metabolite differences on malt extract agar, wallpaper and plasterboard, some molds also produced specific compounds independently of the used substrate, such as trichodiene from Fusarium sporotrichioides and aristolochene from Penicillium roqueforti. Therefore, these metabolites can be used as markers for the identification and maybe also mycotoxin production of these molds. All five investigated Penicillium spp. in this study were able to produce two specific diterpenes, which were not produced by the other species studied. These two compounds, which remain unidentified until now, therefore seem specific for Penicillium spp. and are potentially interesting for the monitoring of this fungal genus. Further experiments will be performed with other Penicillium spp. to study the possibility that these two compounds are specific for this group of molds.

Emission patterns and emission rates of MVOC and the possibility for predicting hidden mold damage?

Laboratory trials were performed in order to search for the variety of the production of microbial volatile organic compounds (MVOC), which could be used as indicators for hidden mold damage. Concerning MVOC production the experiments showed a dependency on the mold genus/species, the different strains used and the building materials used as substrate. It could be proved that the production of certain MVOC is not consistent at all times. On the whole low emission rates in terms of microg/h/m2 of the MVOC were found. Extrapolating the emissions rates from the laboratory trails to an indoor air situation results in concentrations below the analytical detection limit in most cases. According to these results only heavy or very large fungal contaminations might be detected by this method in indoor air. The studies were performed at the Institute of Hygiene and Environmental Medicine, Charite, Germany. Microorganisms like bacteria and molds produce a huge variety of substances, and a part of them are released into the environment. Some compounds like, e.g. alcohols or ketones are volatile, therefore found in the air and called MVOC. Those compounds were considered helpful to track especially hidden mold damage. The study presented here showed, that the emission pattern varies from genus to genus and sometimes even from fungal strain to fungal strain. The results concerning the emission rates from different infested building materials proved, that the concentrations produced are much too low to be detected in indoor air, especially considering the dilution because of ventilation. Therefore, we conclude that MVOC should not be used as predictors for mold damage in indoor environments.

Relevance of airborne fungi and their secondary metabolites for environmental, occupational and indoor hygiene.

Airborne fungal contaminants are increasingly gaining importance in view of health hazards caused by the spores themselves or by microbial metabolites. In addition to the risk for infection, the allergenic and toxigenic properties, as well as the inflammatory effects are discussed in this review as possible health impacts of bioaerosols. A major problem is the lack of threshold values for pathogenic and non-pathogenic fungi, both in the workplace and in outdoor air. While the relevance of mycotoxins has been intensely studied in connection with contamination of food and feed, the possible respiratory uptake of mycotoxins from the air has so far not been sufficiently taken into account. Toxic secondary metabolites are expected to be present in airborne spores, and may thus occur in airborne dust and bioaerosols. Potential health risks cannot be estimated reliably unless exposure to mycotoxins is determined qualitatively and quantitatively. Microbial volatile organic compounds (MVOC) have been suggested to affect human health, causing lethargy, headache, and irritation of the eyes and mucous membranes of the nose and throat. The production of MVOC by fungi has been discussed in connection with domestic indoor microbial pollution, but the relevance of fungal metabolites in working environments remains insufficiently studied.