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The study of the effect of temperature on the accumulation of primary and secondary metabolites by lichens Flavocetraria cucullata (Bellardi) Kärnefelt & Thell and Cetraria laevigata Rass was carried out. Lichen samples were taken out from under the snow (-20 °C) together with the soil substrate and transferred to the climatic chamber. Then the temperature in the climatic chamber was sequentially increased to +10 and +20 °C. The lichen exposure was carried out for 30 days for each temperature regime. The analysis of primary metabolites was performed by gas chromatography-mass spectrometry. It was shown that in lichens F. cucullata and C. laevigata at temperatures of +10 and +20 °C, an increase in the content of mannitol, ribitol, sucrose, and hydroxyproline was observed, as well as a decrease in the content of unsaturated fatty acids as compared to the initial samples. At the same time, the content of glycerol and arabitol in the thalli of the initial lichens (-20 °C) was higher than after exposure at +10 and +20 °C, which, apparently, is associated with the cryoprotective properties of these compounds. The content of secondary metabolites in lichens was determined by high performance liquid chromatography. It was shown that the content of usnic, allo-protolichesterinic, and protolichesterinic acids in F. cucullata increased after exposure in a climatic chamber, which may be associated with a general activation of metabolic processes with an increase in temperature. At the same time, the content of fumarprotocetraric acid in C. laevigata lichens decreased at temperatures of +10 and +20 °C compared to the initial samples, which may be associated with its protective properties under the action of low-temperature stress.

Liamocins synthesized by Aureobasidium spp. are glycolipids composed of a single mannitol or arabitol headgroup linked to either three, four or even six 3,5-dihydroxydecanoic ester tail-groups. The...

The accumulation of low molecular weight cytoprotective compounds (osmolytes) and changes in the membrane lipids composition are of key importance for the adaptation to stress impacts. However, the reason behind the wide variety of osmolytes present in the cell remains unclear. We suggest that specific functions of osmolytes can be revealed by studying the adaptation mechanisms of the mycelial fungus Emericellopsis alkalina (Hypocreales, Ascomycota) that is resistant to both alkaline pH values and high sodium chloride concentrations. It has been established that the fungus uses different osmolytes to adapt to ambient pH and NaCl concentration. Arabitol was predominant osmolyte in alkaline conditions, while mannitol prevailed in acidic conditions. On the salt-free medium mannitol was the main osmolyte; under optimal conditions (pH 10.2; 0.4 M NaCl) arabitol and mannitol were both predominant. Higher NaCl concentrations (1.0–1.5 M) resulted in the accumulation of low molecular weight polyol - erythritol, which amounted up to 12–14%, w/w. On the contrary, changes in the composition of membrane lipids were limited under pH and NaCl impacts; only higher NaCl concentrations led to the increase in the degree of unsaturation of membrane lipids. Results obtained indicated the key role of the osmolytes in the adaptation to the ambient pH and osmotic impacts.

So far, it has been still unknown how liamocins are biosynthesized, regulated, transported and secreted. In this study, a highly reducing polyketide synthase (HR-PKS), a mannitol-1-phosphate dehydrogenase (MPDH), a mannitol dehydrogenase (MtDH), an arabitol dehydrogenase (ArDH) and an esterase (Est1) were found to be closely related to core biosynthesis of extracellular liamocins in Aureobasidium melanogenum 6-1-2. The HR-PKS was responsible for biosynthesis of 3,5-dihydroxydecanoic acid. The MPDH and MtDH were implicated in mannitol biosynthesis and the ArDH was involved in arabitol biosynthesis. The Est1 catalyzed ester bond formation of them. A phosphopantetheine transferase (PPTase) activated the HR-PKS and a transcriptional activator Ga11 activated expression of the PKS1 gene. Therefore, deletion of the PKS1 gene, all the three genes encoding MPDH, MtDH and ArDH, the EST1, the gene responsible for PPTase and the gene for Ga11 made all the disruptants (Δpks13, Δpta13, Δest1, Δp12 and Δg11) totally lose the ability to produce any liamocins. A GLTP gene encoding a glycolipid transporter and a MDR1 gene encoding an ABC transporter took part in transport and secretion of the produced liamocins into medium. Removal of the GLTP gene and the MDR1 gene resulted in a Δgltp1 mutant and a Δmdr16 mutant, respectively, that lost the partial ability to secrete liamocins, but which cells were swollen and intracellular lipid accumulation was greatly enhanced. Hydrolysis of liamocins released 3,5-dihydroxydecanoic acid, mannitol, arabitol and acetic acid. We proposed a core biosynthesis pathway, regulation, transport and secretion of liamocins in A. melanogenum.

Previously, we showed for the first time that alkaliphilic fungi, in contrast to alkalitolerant fungi, accumulated trehalose under extremely alkaline conditions, and we have proposed its key role in alkaliphilia. We propose that high levels of trehalose in the mycelium of alkaliphiles may promote adaptation not only to alkaline conditions, but also to other stressors. Therefore, we studied changes in the composition of osmolytes, and storage and membrane lipids under the action of cold (CS), heat (HS) and osmotic (OS) shocks in the obligate alkaliphilic micromycete Sodiomyces tronii. During adaptation to CS, an increase in the degree of unsaturation of phospholipids was observed while the composition of osmolytes, membrane and storage lipids remained the same. Under HS conditions, a twofold increase in the level of trehalose and an increase in the proportion of phosphatidylethanolamines were observed against the background of a decrease in the proportion of phosphatidic acids. OS was accompanied by a decrease in the amount of membrane lipids, while their ratio remained unchanged, and an increase in the level of polyols (arabitol and mannitol) in the fungal mycelium, which suggests their role for adaptation to OS. Thus, the observed consistency of the composition of membrane lipids suggests that trehalose can participate in adaptation not only to extremely alkaline conditions, but also to other stressors – HS, CS and OS. Taken together, the data obtained indicate the adaptability of the fungus to the action of various stressors, which can point to polyextremotolerance.

Size-segregated aerosol samples collected from a rural site (Rajim: 20˚59′N and 81˚55′E) in eastern central India during the winter of 2011 were analyzed for anhydrosugars, sugar alcohols and primary sugars to better understand their potential sources and formation. Anhydrosugars showed the predominance of levoglucosan, whereas sugar alcohol showed the predominance of mannitol, with glucose as a major primary sugar in the sub-micron (Da 1.1 μm). Levoglucosan gave a bimodal size distribution pattern with a major peak at 0.43–0.65 μm in the sub-micron mode during period 2 and period 3, suggesting its origin from biomass burning, whereas during period 1 the major peak was found at 4.4–5.8 μm in super-micron mode that may be due to the mixing of biomass burning derived particles with soil dust particles. Sugar alcohols (arabitol and mannitol) and primary sugars (glucose and trehalose) showed bimodal size distribution trend with a major peak at 4.4–5.8 μm in super-micron mode during all periods, perhaps due to fungal activity and soil dust re-suspension, respectively. The potential ingress of biomass burning derived particles into the lungs was calculated to be high whereas fungal spores and soil particles were more likely found in the upper respiratory tract. Principal component analysis and correlation analysis showed that biomass burning was a major source in the sub-micron mode whereas fungal spores and vegetative soil were dominant sources for quantified sugars in super-micron mode.

Liamocins are biosurfactants produced by the fungus Aureobasidium pullulans. A. pullulans belongs to the black yeasts and is known for its ability to produce pullulan and melanin. However, the production of liamocins has not been investigated intensively. Initially, HPLC methods for the quantification of liamocin and the identification of liamocin congeners were established. Eleven congeners could be detected, differing in the polyol head groups arabitol or mannitol. In addition, headless molecules, so-called exophilins, were also identified. The HPLC method reported here allows quick and reliable quantification of all identified congeners, an often-overlooked prerequisite for the investigation of valuable product formation. Liamocin synthesis was optimized during cultivation in lab-scale fermenters. While the pH can be kept constant, the best strategy for liamocin synthesis consists of a growth phase at neutral pH and a subsequent production phase induced by a manual pH shift to pH 3.5. Finally, combining increased nitrogen availability with a pulsed fed-batch fermentation, cell growth, and liamocin titers could be enhanced. Here, the maximal titers of above 10 g/L that were reached are the highest reported to date for liamocin synthesis using A. pullulans in lab-scale fermenters.

Trehalose, mannitol and arabitol are the main saccharides of extant fungal metabolism, but their occurrence and distribution in geological materials have rarely been considered. Here, we identify these sugars in Miocene lignites and for the first time in Late Cretaceous mudstones and coals. The co-occurrence of trehalose, mannitol and arabitol in the sedimentary rocks investigated suggests their fungal origin, because these three saccharides are major compounds present in most modern fungi, including the very common mycorrhizal and wood-rotting groups. Therefore, we conclude that these sugars should be treated as new fungal biomarkers (biomolecules) present in geological rocks. Trehalose and mannitol are major compounds in total extracts of the samples and a sum of their concentration reaches 4.6 μg/g of sample. The arabitol concentrations do not exceed 0.5 μg/g, but in contrast to trehalose, the concentration correlates well with mannitol (R2 = 0.94), suggesting that they have the same, translocatory role in fungi. Based on the trehalose vs. mannitol and arabitol distributions in Cretaceous samples and their comparison with data for modern fungi, we preliminarily conclude that the coal seams from the Rakowice Male (SW Poland) section were formed during warmer climatic periods than the overlying sediments. Furthermore, no DNA could be isolated from the samples of lignites and overlying sediments, whereas it was abundant in the control samples of maple, birch and oak wood degraded by fungi. This indicates an absence of recent fungi responsible for decay in lignites and implies that the saccharide origin is connected with ancient fungi. Other sugar alcohols and acids like D-pinitol, quinic acid and shikimic acid, were found for the first time in sedimentary rocks, and their source is inferred to be from higher plants, most likely conifers. The preservation of mono- and disaccharides of fungal origins in pre-Palaeogene strata implies that compounds previously thought as unstable can survive for tens to hundreds of millions of years without structural changes in immature rocks unaffected by secondary processes.

The daily evolution of seventeen sugar compounds (seven saccharides, seven alcohol-saccharides and three anhydrosaccharides) in atmospheric aerosol samples collected between 9 March 2016 and 14 March 2017 was studied in Leon (Spain). The main links between the concentration of sugar compounds and various chemical species, pollen, fungal spores and meteorological conditions were investigated. The results showed that, in spring, when high levels of metabolic activity of the plants occur and temperatures increase, glucose, sucrose, 2-methyl-erithritol, mannitol, arabitol and inositol, are significantly correlated with airborne pollen concentrations. Between spring and autumn, Alternaria air concentrations are significantly correlated with temperatures, arabitol and sorbitol + adonitol concentrations. Furthermore, during rainy days, Alternaria is also correlated with mannitol. In autumn, lower temperatures cause an increase in the concentrations of levoglucosan, mannosan and galactosan, probably due to the increased use of domestic heating devices. These anhydrosugars and arabinose, fructose and glucose, are significantly correlated with K, NO3−, EC, OC, Cu, Zn, Se, Pb, V and Ni, while mannosan also correlates with As, showing that these anhydrosaccharides can be emitted from different anthropogenic sources. Precipitation causes an increase in glucose and sucrose concentrations, due to the break of pollen particles that produce hundreds of fine size particles. Besides, precipitation causes an increase in arabitol concentrations, due to the release and growth of fungi.

The presence of anhydrosugars and sugar alcohols in airborne articulate matter &lt, 10 µm (PM10) samples collected between December 2018 and June 2019 was studied for two urban environments in Coimbra. Anhydrosugars were used to estimate the biomass burning contribution, and sugar alcohols were investigated regarding biological sources. Anhydrosugars contributed more than sugar alcohols to the total sugars, mainly levoglucosan. Higher levoglucosan concentrations were linked with the use of biomass-fueled heating appliances, mainly during cold periods. A significant contribution from biomass burning smoke was registered, accounting for 20% to 23% of the PM10 mass in the colder period. Xylitol presented higher concentrations in the colder period and was well correlated with levoglucosan, indicating a common origin. Mannitol and arabitol were well correlated with each other but did not present any kind of correlation with anhydrosugars or xylitol, suggesting a natural source. A quantitative estimation based on the concentration of ambient tracers (mannitol) was evaluated, and the results reveal that, for the two sites, the fungal spore relative contribution to PM10 (roadside site: 2.7% to 2.8%, urban background: 1.9% to 2.7%) and OC mass (roadside site: 6.2% to 8.1%, urban background: 3.9% to 7.5%) was significant and always higher in the warmer period.

Methanol is inexpensive, is easy to transport, and can be produced both from renewable and from fossil resources without mobilizing arable lands. As such, it is regarded as a potential carbon source to transition toward a greener industrial chemistry. Metabolic engineering of bacteria and yeast able to efficiently consume methanol is expected to provide cell factories that will transform methanol into higher-value chemicals in the so-called methanol economy. Toward that goal, the study of natural methylotrophs such as Bacillus methanolicus is critical to understand the origin of their efficient methylotrophy. This knowledge will then be leveraged to transform such natural strains into new cell factories or to design methylotrophic capability in other strains already used by the industry., Bacillus methanolicus MGA3 is a thermotolerant and relatively fast-growing methylotroph able to secrete large quantities of glutamate and lysine. These natural characteristics make B. methanolicus a good candidate to become a new industrial chassis organism, especially in a methanol-based economy. Intriguingly, the only substrates known to support B. methanolicus growth as sole sources of carbon and energy are methanol, mannitol, and, to a lesser extent, glucose and arabitol. Because fluxomics provides the most direct readout of the cellular phenotype, we hypothesized that comparing methylotrophic and nonmethylotrophic metabolic states at the flux level would yield new insights into MGA3 metabolism. In this study, we designed and performed a 13C metabolic flux analysis (13C-MFA) of the facultative methylotroph B. methanolicus MGA3 growing on methanol, mannitol, and arabitol to compare the associated metabolic states. On methanol, results showed a greater flux in the ribulose monophosphate (RuMP) pathway than in the tricarboxylic acid (TCA) cycle, thus validating previous findings on the methylotrophy of B. methanolicus. New insights related to the utilization of cyclic RuMP versus linear dissimilation pathways and between the RuMP variants were generated. Importantly, we demonstrated that the linear detoxification pathways and the malic enzyme shared with the pentose phosphate pathway have an important role in cofactor regeneration. Finally, we identified, for the first time, the metabolic pathway used to assimilate arabitol. Overall, those data provide a better understanding of this strain under various environmental conditions. IMPORTANCE Methanol is inexpensive, is easy to transport, and can be produced both from renewable and from fossil resources without mobilizing arable lands. As such, it is regarded as a potential carbon source to transition toward a greener industrial chemistry. Metabolic engineering of bacteria and yeast able to efficiently consume methanol is expected to provide cell factories that will transform methanol into higher-value chemicals in the so-called methanol economy. Toward that goal, the study of natural methylotrophs such as Bacillus methanolicus is critical to understand the origin of their efficient methylotrophy. This knowledge will then be leveraged to transform such natural strains into new cell factories or to design methylotrophic capability in other strains already used by the industry.

A new yeast wild-type Yarrowia lipolytica isolate presented efficient growth on glycerol. During flask cultures, nitrogen limitation led to the secretion of sugar-alcohols as the major metabolites of the process (mannitol, arabitol and erythritol), whereas insignificant quantities of citrate were synthesized. Although in some instances high initial glycerol concentrations were employed (&asymp, 150 g/L), remarkable glycerol assimilation and polyol secretion was observed. Total polyols &asymp, 52 g/L (conversion yield on glycerol consumed = 0.43 g/g) was recorded in the flask experiments. The sugar-alcohol production bioprocess was successfully simulated with the aid of a modified Velhlust&ndash, Aggelis model that fitted very well with the experimental data, while optimized parameter values seemed to be quite consistent. In bioreactor trials, a noticeable metabolic shift towards citric acid production was observed, while simultaneously insignificant polyol quantities were produced. In fed-batch bioreactor experiments, a total citric acid quantity &asymp, 102 g/L was recorded&mdash, one of the highest in the literature for wild-type Y. lipolytica strains. This metabolic transition was due to higher oxygen saturation into the medium that occurred in the bioreactor experiments compared with the flasks. Cellular lipids produced in the bioreactor trial contained higher concentrations of unsaturated fatty acids compared with those produced in flasks.

Rice straw was substituted for sawdust at five different ratios of 0, 20%, 40%, 60%, and 80% (Control, RS20, RS40, RS60 and RS80, respectively) to obtain five kinds of Lentinula edodes. The effects of adding cropped rice straw to substrate formulas on the proximate composition and non-volatile taste compounds in mushrooms were investigated. The control group had the highest level of MY and BE among the five formulations. The protein levels in mushrooms decreased with the addition of rice straw and the ash levels increased. We found that trehalose, mannitol, and arabitol were the main soluble sugars in the five kinds of mushrooms. The contents of total free amino acids varied from 16.29 to 24.59 mg/g and the highest level of free amino acids was found in mushrooms cultivated from RS20 and RS40. Moreover, the addition of rice straw improved the contents of monosodium glutamate (MSG)-like amino acids in mushrooms. The 5′-Nucleotide levels ranged from 1.66 to 4.48 mg/g and equivalent umami concentration (EUC) value increased with the addition of rice straw. Our results suggest that rice straw is a potential substitute for sawdust to cultivate L. edodes with more non-volatile taste compounds.

Penicillium roqueforti is a major cause of fungal food spoilage. Its conidia are the main dispersal structures of this fungus and therefore the main cause of food contamination. These stress resistant asexual spores can be killed by preservation methods such as heat treatment. Here, the effects of cultivation time and temperature on thermal resistance of P. roqueforti conidia were studied. To this end, cultures were grown for 3, 5, 7 and 10 days at 25 °C or for 7 days at 15, 25 and 30 °C. Conidia of 3- and 10-day-old cultures that had been grown at 25 °C had D56-values of 1.99 ± 0.15 min and 5.31 ± 1.04 min, respectively. The effect of cultivation temperature was most pronounced between P. roqueforti conidia cultured for 7 days at 15 °C and 30 °C, where D56-values of 1.12 ± 0.05 min and 4.19 ± 0.11 min were found, respectively. Notably, D56-values were not higher when increasing both cultivation time and temperature by growing for 10 days at 30 °C. A correlation was found between heat resistance of conidia and levels of trehalose and arabitol, while this was not found for glycerol, mannitol and erythritol. RNA-sequencing showed that the expression profiles of conidia of 3- to 10-day-old cultures that had been grown at 25 °C were distinct from conidia that had been formed at 15 °C and 30 °C for 7 days. Only 33 genes were upregulated at both prolonged incubation time and increased growth temperature. Their encoded proteins as well as trehalose and arabitol may form the core of heat resistance of P. roqueforti conidia.

Liamocins are a class of biosurfactants with growing interest. However, methods for identification and quantification of liamocins on the molecular level are lagging behind. Therefore, we developed a chromatographic separation based on supercritical fluid chromatography (SFC) for liamocins and structurally related exophilins. The different congeners could be separated on a charge modulated hydroxyethyl amide functionalized silica-based column. Coupling to high-resolution mass spectrometry (MS) revealed four exophilin species and four liamocin species with mannitol and arabitol as polyol head group in a sample of the yeast-like fungus Aureobasidium pullulans (A. pullulans). In contrast to a recently published reversed phase high-performance liquid chromatography (HPLC) method, the different subclasses (exophilins, mannitol liamocins and arabitol liamocins) were additionally separated by means of SFC. The structures were confirmed by their accurate masses and tandem mass spectrometry (MS/MS). A complementary quantification method was developed using SFC coupled to charged-aerosol detection (CAD) to overcome the disadvantages of quantification by means of MS without authentic standards. A flow compensation by varying the make-up flow was used to obtain a constant composition of the mobile phase during detection and to ensure a stable detector response. The concentrations of the individual liamocin species were determined using an external calibration with n-octyl-β-d-glycopyranoside. The total amount of these concentrations agrees with the dry weight of an aliquot of the heavy oil. The developed SFC-based method has the advantage of shorter analysis time and superior selectivity compared to the previously published LC separation. In brief, the here presented SFC hyphenations enable comprehensive analysis of liamocin biosurfactants providing identification and absolute quantification of individual congeners.

Saccharides are common constituents of soils, but their role and origin in the initial phases of pedogenesis remain unclear. Here we show the detailed composition of neutral sugars extracted from arenosols at different development stages, combined with additional lipids of diverse origins using gas chromatography-mass spectrometry (GC–MS). During the first stage (I) of development sucrose is the most abundant saccharide in the soil crust at up to 45,000 µg/g TOC. Sucrose is also the predominant compound in the second and third development stages, but its concentration decreased to the range of 1600 to 16,000 µg/g TOC. Stages II and III of soil development were characterized by a gradual increase in arabitol, mannitol and trehalose, compounds typical for fungi and lichen. Their abundances increased from several percent (compared to the major sucrose) to 10–32% for mannitol and 34–54% for trehalose. Moreover, in stage III there was a considerable increase in the contents of the saccharides: pinitol, myo-inositol, scyllo-inositol, arabinose, together with non-sugar compounds: dehydroabietic acid, p-hydroxybenzoic acid, gallic acid and sitosterol. All these latter compounds are higher plant markers, mainly derived from conifer detritus. The relationships between the ratios of trehalose/sucrose vs. (mannitol + arabitol)/sucrose and TOC vs. (mannitol + arabitol)/sucrose differentiated precisely the top soil layer of arenosols which are covered by different stages of biological soil crust. Our study shows that free sugars, supplemented by lipid biomarkers and total organic carbon contents, are good indicators of soil in the initial phase of pedogenesis.

In order to investigate the fungal spore tracers in fine particles (PM2.5), including mannitol and arabitol at an urban site in a Chinese megacity, PM2.5 samples were collected in Shanghai from May 22 to June 19, 2015. The analysis results showed that the average concentration of airborne mannitol and arabitol were 5.79 and 3.86 ng m−3, respectively. Mannitol and arabitol exhibited obvious positive correlations at ambient temperature, resulting from improving fungal spores formation rate and emission strength along with higher temperature. The concentrations of fungal spore tracers with Relative humidity-RH 70%–85% were higher than that RH>85% and RH

Alterations in the concentrations of cell cytosol carbohydrates of polyextremophilic yeasts Yarrowia lipolytica under stresses of diverse nature were observed. Under pH stress, mannitol was the main storage carbohydrate (up to 89% of the total cytosol carbohydrates), while arabitol, glucose, and inositol were present in insignificant amounts (3 to 6%). Experiments with inhibition of de novo mannitol synthesis by bis(p-nitrophenyl) disulfide revealed that the cytoprotective effect of mannitol was most noticeable in the cells grown under acidic conditions (pH 4.0), while the role of catalase and superoxide dismutase, the enzymes of the first line of antioxidant protection, increased under alkaline conditions (pH 9.0). The constitutively high mannitol content in Y. lipolytica cells was hypothesized to be a part of the core mechanism of stress resistance in this yeast species.

In a 54 m3 large-scale penicillin fermentor, the cells experience substrate gradient cycles at the timescales of global mixing time about 20–40 s. Here, we used an intermittent feeding regime (IFR) and a two-compartment reactor (TCR) to mimic these substrate gradients at laboratory-scale continuous cultures. The IFR was applied to simulate substrate dynamics experienced by the cells at full scale at timescales of tens of seconds to minutes (30 s, 3 min and 6 min), while the TCR was designed to simulate substrate gradients at an applied mean residence time ((Formula presented.)) of 6 min. A biological systems analysis of the response of an industrial high-yielding P. chrysogenum strain has been performed in these continuous cultures. Compared to an undisturbed continuous feeding regime in a single reactor, the penicillin productivity (qPenG) was reduced in all scale-down simulators. The dynamic metabolomics data indicated that in the IFRs, the cells accumulated high levels of the central metabolites during the feast phase to actively cope with external substrate deprivation during the famine phase. In contrast, in the TCR system, the storage pool (e.g. mannitol and arabitol) constituted a large contribution of carbon supply in the non-feed compartment. Further, transcript analysis revealed that all scale-down simulators gave different expression levels of the glucose/hexose transporter genes and the penicillin gene clusters. The results showed that qPenG did not correlate well with exposure to the substrate regimes (excess, limitation and starvation), but there was a clear inverse relation between qPenG and the intracellular glucose level.

Comparative composition of lipids and cytosol soluble carbohydrates at different ambient pH values was studied for two obligately alkaliphilic fungi (Sodiomyces magadii and S. alkalinus) and for two alkalitolerant ones (Acrostalagmus luteoalbus and Chordomyces antarcticus). The differences and common patterns were revealed in responses to pH stress for the fungi with different types of adaptation to ambient pH. While trehalose was one of the major cytosol carbohydrates in alkaliphilic fungi under optimal growth conditions (pH 10.2), pH decrease to 7.0 resulted in doubling its content. In alkalitolerant fungi trehalose was a minor component and its level did not change significantly at different pH. In alkalitolerant fungi, arabitol and mannitol were the major carbohydrate components, with their highest ratio observed under alkaline conditions and the lowest one, under neutral and acidic conditions. In alkaliphiles, significant levels of arabitol were revealed only under alkaline conditions, which indicated importance of trehalose and arabitol for alkaliphily. Decreased pH resulted in the doubling of the proportion of phosphatidic acids among the membrane lipids, which was accompanied by a decrease in the fractions of phosphatidylcholines and sterols. Alkalitolerant fungi also exhibited a decrease in sterol level at decreased pH, but against the background of increased proportion of one of phospholipids. Decreased unsaturation degree in the fatty acids of the major phospholipids was a common response to decreased ambient pH.

Mutants of Yarrowia lipolytica with high erythritol production were generated through an atmospheric and room temperature plasma (ARTP) mutation system. Among these mutants, Y. lipolytica M53 exhibited the highest erythritol yield. In a batch culture, M53 produced 64.8 g/L erythritol from 100 g/L glycerol. The yields of byproducts (e.g. mannitol, arabitol, and α-ketoglutaric acid) were low, and the mechanisms underlying these changes were examined by measuring enzyme activities in the pentose phosphate pathway. Up to 145.2 g/L erythritol was produced by M53 from 200 g/L of glycerol, and erythritol accumulation was promoted by 3.7 mg/L of Cu2+, 10.15 mg/L of Mn2+, and 30.37 g/L of NaCl. Fed-batch cultivation of M53 in a 5-L fermentor produced 169.3 g/L erythritol with low levels of byproducts within 168 h. This finding confirmed the potential of M53 as an erythritol producer on a commercial scale.

Alkaliphily, the ability of an organism to thrive optimally at high ambient pH, has been well-documented in several lineages: archaea, bacteria and fungi. The molecular mechanics of such adaptation has been extensively addressed in alkaliphilic bacteria and alkalitolerant fungi. In this study, we consider an additional property that may have enabled fungi to prosper at alkaline pH: altered contents of membrane lipids and cytoprotectant molecules. In the alkaliphilic Sodiomyces tronii, we showed that at its optimal growth pH 9.2, the fungus accumulates abundant cytosolic trehalose (4–10% dry weight) and phosphatidic acids in the membrane lipids, properties not normally observed in neutrophilic species. At a very high pH 10.2, the major carbohydrate, glucose, was rapidly substituted by mannitol and arabitol. Conversely, lowering the pH to 5.4–7.0 had major implications both on the content of carbohydrates and membrane lipids. It was shown that trehalose dominated at pH 5.4. Fractions of sphingolipids and sterols of plasma membranes rapidly elevated possibly indicating the formation of membrane structures called rafts. Overall, our results reveals complex dynamics of the contents of membrane lipids and cytoplasmic sugars in alkaliphilic S. tronii, suggesting their adaptive functionality against pH stress.

A growing number of studies are using specific primary sugar species, such as sugar alcohols or primary saccharides, as marker compounds to characterize and apportion primary biogenic organic aerosols (PBOAs) in the atmosphere. To better understand their annual cycles, as well as their spatiotemporal abundance in terms of concentrations and sources, we conducted a large study focusing on three major atmospheric primary sugar compounds (i.e., arabitol, mannitol, and glucose) measured in various environmental conditions for about 5300 filter samples collected at 28 sites in France. Our results show significant atmospheric concentrations of polyols (defined here as the sum of arabitol and mannitol) and glucose at each sampling location, highlighting their ubiquity. Results also confirm that polyols and glucose are mainly associated with the coarse rather than the fine aerosol mode. At nearly all sites, atmospheric concentrations of polyols and glucose display a well-marked seasonal pattern, with maximum concentrations from late spring to early autumn, followed by an abrupt decrease in late autumn, and a minimum concentration during wintertime. Such seasonal patterns support biogenic emissions associated with higher biological metabolic activities (sporulation, growth, etc.) during warmer periods. Results from a previous comprehensive study using positive matrix factorization (PMF) based on an extended aerosol chemical composition dataset of up to 130 species for 16 of the same sample series have also been used in the present work. The polyols-to-PMPBOA ratio is 0.024±0.010 on average for all sites, with no clear distinction between traffic, urban, or rural typology. Overall, even if the exact origin of the PBOA source is still under investigation, it appears to be an important source of particulate matter (PM), especially during summertime. Results also show that PBOAs are significant sources of total organic matter (OM) in PM10 (13±4 % on a yearly average, and up to 40 % in some environments in summer) at most of the investigated sites. The mean PBOA chemical profile is clearly dominated by contribution from OM (78±9 % of the mass of the PBOA PMF on average), and only a minor contribution from the dust class (3±4 %), suggesting that ambient polyols are most likely associated with biological particle emissions (e.g., active spore discharge) rather than soil dust resuspension.

Liamocins are polyol lipids produced by the fungus Aureobasidium pullulans, and have selective antibacterial activity against Streptococcus species. Liamocins produced by A. pullulans strain NRRL 50380 on sucrose medium have a d-mannitol head group ester-linked to 3,5-dihydroxydecanoate acyl chains, three or four of which are joined together by 1,5-polyester bonds (liamocins Man-A1 and Man-B1), and similar 3'-O-acetylated analogs (Man-A2 and Man-B2). However, other types of liamocins are produced depending on the choice of strain and growth conditions. In the current study, growth on different polyols, but not sugars, resulted in considerable structural variation, including liamocins with d-galactitol (dulcitol), d-sorbitol (glucitol), d- and l-arabitol, d-xylitol, l-threitol and glycerol head groups. The head groups of liamocins produced on arabitol were shown to be entirely composed of d-arabitol. These liamocin variants were structurally characterized by NMR and MS, and tested for antibacterial activity. The new liamocin variants also had selective activity against Streptococcus. Liamocin structural variants are novel antibacterials against Streptococcus sp. that merit further investigation.

Lipid production by oleaginous yeasts is optimal at high carbon-to-nitrogen ratios. In the current study, nitrogen and carbon consumption by Lipomyces starkeyi were directly measured in defined minimal media with nitrogen content and agitation rates as variables. Shake flask cultures with an initial C:N ratio of 72:1 cultivated at 200rpm resulted in a lipid output of 10g/L, content of 55%, yield of 0.170g/g, and productivity of 0.06g/L/h. All of these values decreased by ≈50-60% when the agitation rate was raised to 300rpm or when the C:N ratio was lowered to 24:1, demonstrating the importance of these parameters. Under all conditions, L. starkeyi cultures tolerated acidified media (pH≈2.6) without difficulty, and produced considerable amounts of alcohols; including ethanol, mannitol, arabitol, and 2,3-butanediol. L. starkeyi also produced lipids from a corn stover hydrolysate, showing its potential to produce biofuels from renewable agricultural feedstocks.

Saccharomycopsis fibuligera KJJ81 isolated from nuruk is an amylolytic yeast that is widely used as a microbial starter in various fermented foods. Volatile and nonvolatile metabolites of S. fibuligera KJJ81 were investigated according to different carbon sources and cultivation times using a nontargeted metabolomic approach. Partial-least-squares discriminant analysis was applied to determine the major metabolites, which were found to be closely related to the clustering and discrimination of S. fibuligera KJJ81 samples. Some volatile metabolites derived from phenylalanine, such as 2-phenylethanol, 2-phenylethyl acetate, and ethyl phenylacetate, were predominantly found in cultivation medium containing glucose (YPD medium). In addition, the level of 2-phenylethanol increased continuously with the cultivation time. In terms of nonvolatile metabolites, carbohydrates (mannose, arabitol, and mannitol), fatty acids (palmitic acid and stearic acid), organic acids (oxalic acid and succinic acid), and amino acids (isoleucine, serine, alanine, glutamic acid, glycine, proline, phenylalanine, and threonine) were the main contributors to S. fibuligera KJJ81 samples cultivated in YPD medium according to cultivation time. These results show that the formation of volatile and nonvolatile metabolites of S. fibuligera KJJ81 can be significantly affected by both the carbon sources and the cultivation time.

Background & Objective:Crude glycerol (Glol), used as substrate for screening eleven natural Yarrowia lipolytica strains in shake-flask experiments. Aim of this study was to assess the ability of the screened strains to produce biomass (dry cell weight; X), lipid (L), citric acid (Cit), mannitol (Man), arabitol (Ara) and erythritol (Ery), compounds presenting pharmaceutical and biotechnological interest, in glycerol-based nitrogen-limited media, in which initial glycerol concentration had been adjusted to 40 g/L.Methods:Citric acid may find use in biomedical engineering (i.e. drug delivery, tissue engineering, bioimaging, orthopedics, medical device coating, wound dressings). Polyols are considered as compounds with non-cariogenic and less calorigenic properties as also with low insulin-mediated response. Microbial lipids containing polyunsaturated fatty acids (PUFA) are medically and dietetically important (selective pharmaceutical and anticancer properties, aid fetal brain development, the sight function of the eye, hormonal balance and the cardio-vascular system, prevent reasons leading to type-2 diabetes, present healing and anti-inflammatory effects).Results:All strains presented satisfactory microbial growth (Xmax=5.34-6.26 g/L) and almost complete substrate uptake. The principal metabolic product was citric acid (Citmax=8.5-31.7 g/L). Production of cellular lipid reached the values of 0.33-0.84 g/L. Polyols were also synthesized as strain dependent compounds (Manmax=2.8-6.1 g/L, Aramax ~2.0 g/L, Erymax= 0.5-3.8 g/L). The selected Y. lipolytica strain ACA-DC 5029 presented satisfactory growth along with synthesis of citric acid and polyols, thus, was further grown on media presenting an increased concentration of Glol~75 g/L. Biomass, lipid and citric acid production presented significant enhancement (Xmax=11.80 g/L, Lmax=1.26 g/L, Citmax=30.8 g/L), but conversion yield of citric acid produced per glycerol consumed was decreased compared to screening trials. Erythritol secretion (Erymax=15.6 g/L) was highly favored, suggesting a shift of yeast metabolism from citric acid accumulation towards erythritol production. Maximum endopolysaccharides (IPS) concentration was 4.04 g/L with yield in dry weight 34.2 % w/w.Conclusion:Y. lipolytica strain ACA-YC 5029 can be considered as a satisfactory candidate grown in high concentrations of crude glycerol to produce added-value compounds that interest pharmaceutical and biotechnology industries.

Successful commercialization of microbial biocontrol agents, such as Metarhizium spp., is often constrained by poor drying survival and shelf life. Here, we hypothesized that culture age would influence endogenous arabitol, erythritol, mannitol and trehalose contents in M. brunneum mycelium and that elevated levels of these compounds would improve drying survival and shelf life of encapsulated mycelium coupled with enhanced fungal virulence against T. molitor larvae. We found that culture age significantly influenced endogenous arabitol and mannitol contents in mycelium with highest concentrations of 0.6 +/- 0.2 and 2.1 +/- 0.2 A mu g/mg after 72 h, respectively. Drying survival of encapsulated mycelium was independent of culture age and polyol content with 41.1 +/- 4.4 to 55.0 +/- 6.2%. Best shelf life was determined for biomass harvested after 72 h at all investigated storage temperatures with maximum values of 59.5 +/- 3.3% at 5 A degrees C followed by 54.5 +/- 1.6% at 18 A degrees C and 19.4 +/- 1.3% at 25 A degrees C after 6 months. Finally, high fungal virulence against T. molitor larvae of 83.3 +/- 7.6 to 98.0 +/- 1.8% was maintained during storage of encapsulated mycelium for 12 months with larval mortalities being independent of culture age and polyol content. In conclusion, our findings indicate beneficial effects of endogenous polyols in improving shelf life of encapsulated mycelium and this may spur the successful development of microbial biocontrol agents in the future.

Tasty components in Lentinula edodes pileus and stipe at different growth stages were studied. Mannitol, trehalose, arabitol, and glucose were the main soluble polyols and sugars, whereas succinic acid, malic acid, and citric acid were the main organic acids. Mannitol contents were the highest in the pileus and increased at mature growth stages, although arabitol contents were the highest in the stipe and peaked at stage 5. Succinic acid contents peaked at stage 5 in the pileus and stipe during mature growth stages. Threonine (sweet taste) values were the highest among all the detected amino acids, followed by glutamic acid (MSG-like taste). MSG-like 5'-nucleotide contents could account for nearly 50% of the total 5'-nucleotides. Equivalent umami concentration (EUC) values of stage 5 exhibited higher levels during mature growth stages. Tasty components in the stipe were rich and EUC values were high, which might be useful for further processing and byproduct development of L. edodes.

Biosurfactants have several desirable characteristics in the industrial sector: detergency, antimicrobial effects, skin hydration, and emulsibility. Several yeast glycolipids are currently being utilized in these capacities: sophorolipids, ustilagic acid, and mannosylerythritol lipids (MELs). An emerging class of glycolipids, termed polyol esters of fatty acids (PEFA), have recently been reported for Rhodotorula babjevae, a basidiomycetous yeast species that secretes hyperacetylated congeners of PEFA (typically with 3-6 acetylation modifications). While screening Rhodotorula species for surfactant production, we identified a new environmental isolate identified as Rhodotorula taiwanensis MD1149 that dropped the surface tension of the liquid medium, indicating that it produced a potent biosurfactant. Acid depolymerization of the purified biosurfactants, followed by gas chromatography-mass spectrometry (GC-MS) analysis revealed that the biosurfactants were composed of PEFA compounds composed mainly of mannitol and arabitol esters of 3-hydroxy fatty acid, 3-methoxy fatty acid, and fatty acids with a single double bond; chain lengths were mainly C16 and C18. Liquid chromatography-mass spectrometry (LC-MS) confirmed the predicted accurate mass of these compounds. Interestingly, PEFA compounds produced by Rhodotorula taiwanensis MD1149 were more surface active due to their hypoacetylation profile (0-4 acetylation modifications) compared to Rhodotorula babjevae MD1169. These disparate surface active properties, based on acetylation, change the hydrophilic-lipophilic balance (HLB) of these compounds, and their potential utility within industrial applications.

Organic component of airborne particulate matter originates from both natural and anthropogenic sources whose contributions can be identified through the analysis of chemical markers. The validation of analytical methods for analysis of compounds used as chemical markers is of great importance especially if they must be determined in rather complex matrices. Currently, standard reference materials (SRM) with certified values for all those analytes are not available. In this paper, we report a method for the simultaneous determination of levoglucosan and xylitol as tracers for biomass burning emissions, and arabitol, mannitol and ergosterol as biomarkers for airborne fungi in SRM 1649a, by GC/MS. Their quantitative analysis in SRM 1649a was carried out using both internal standard calibration curves and standard addition method. A matrix effect was observed for all analytes, minor for levoglucosan and major for polyols and ergosterol. The results related to levoglucosan around 160 μg g−1 agreed with those reported by other authors, while no comparison was possible for xylitol (120 μg g−1), arabitol (15 μg g−1), mannitol (18 μg g−1), and ergosterol (0.5 μg g−1). The analytical method used for SRM 1649a was also applied to PM10 samples collected in Rome during four seasonal sampling campaigns. The ratios between annual analyte concentrations in PM10 samples and in SRM 1649a were of the same order of magnitude although particulate matter samples analyzed were collected in two different sites and periods.

Fungal spores constitute the most abundant fraction of biological aerosols in the atmosphere, influencing human health, the biosphere, atmospheric chemistry, and climate. However, the total abundance of fungal spores in the atmosphere is rather uncertain and likely underestimated to a large extent by traditional Colony Forming Units (CFU) assays. In this study, flow cytometry (FCM) was utilized in combination with fluorescent stains for the rapid counting of ambient fungal spores, with complementary quantification of two molecular tracers for fungal spores. The FCM results had significant positive correlation with the concentrations of the fungal tracers ( R 2 was 0.75 and 0.70 for arabitol and mannitol, respectively). During this study, total particle counts, fungal spore numbers and the fractions of fungal spores of the total particle numbers were in the range of 44,698–9,54,211 m −3 , 8224–261,154 m −3 and 1.9–46.5%, respectively, at an urban location in northern China. Meteorological conditions were shown to have complex effects on the ambient concentrations of fungal spores: the number concentrations of fungal spores exhibited significant positive correlation with relative humidity and temperature, negative correlation with wind speed and no relationship with solar radiation during the sampling period.

Airborne microorganisms, as bacteria and fungi, are ubiquitous components of the atmospheric aerosol particles. In this paper, we report a method for the simultaneous extraction, purification, separation, identification and quantification of ergosterol, mannitol and arabitol as biomarkers of fungal spores in bioaerosol particles. After sampling by a low volume sampler, filters were spiked with mannitol-13C and dehydrocholesterol as internal standards. Samples were then extracted by accelerated solvent extraction using pure ethanol. The extract was then passed through an amino cartridge and divided in two parts: the apolar fraction, released from the cartridge, was subjected to liquid liquid extraction (by n-hexane), while polar compounds, retained by the cartridge, were eluted by a mixture of methanol–water. The two fractions were joined and analyzed by HPLC equipped with two different columns in series, and coupled to a triple-quadrupole mass spectrometer with Atmospheric Pressure Chemical Ionization source. In addition, the same fractions were analyzed, after derivatization, by GC–MS. The results obtained by the two techniques were finally compared, showing good agreement between them. Last, the contents of the three biomarkers have been estimated in three atmospheric samples collected in a suburban/rural site and, using literature conversion factors, correlated to fungal biomass.

Fungal spores are the dominant biological component of air. Although ubiquitous in outdoor air, they are scarcely measured due to the inadequacy of measurement methods. The use of biomarkers as tools for the determination of fungal contribution to bioaerosol has often been suggested, and ergosterol, arabitol and mannitol have been associated to fungal spores as tracers. In the present paper, the fungal component of aerosol was studied at suburban/rural and at urban sites. Ergosterol, arabitol, and mannitol contents in airborne particulate matter, even at different sizes, were determined. Literature conversion factors and calculated conversion factors correlating ergosterol, arabitol, and mannitol masses to fungi mass were applied and compared to each other. The obtained fungal spore concentrations were different depending on the marker utilized both with the conversion factors found in literature and the calculated ones. Size-segregated marker distribution suggested different sources for the three tracers indicating ergosterol as the only reliable biomarker at our latitudes. The fungal spore concentrations were higher at the suburban/rural location and respectively inversely and directly proportional to temperature and relative humidity.

International audience; When exposed to constant low temperatures (CLTs), insects often suffer from cumulative physiological injuries that can severely compromise their fitness and survival. Yet, mortality can be considerably lowered when the cold stress period is interrupted by periodic warm interruption(s), referred to as fluctuating thermal regimes, FTRs. In this study, we have shown that FTRs strongly promoted cold tolerance of Drosophila melanogaster adults. We then assessed whether this marked phenotypic shift was associated with detectable physiological changes, such as synthesis of cryoprotectants and/or membrane remodeling. To test these hypotheses, we conducted two different time-series Omics analyzes in adult flies submitted to CLTs vs. FTRs: metabolomics (GC/MS) and lipidomics (LC/ESI/MS) targeting membrane phospholipids. We observed increasing levels in several polyhydric alcohols (arabitol, erythritol, sorbitol, mannitol, glycerol), sugars (fructose, mannose) and amino acids (serine, alanine, glutamine) in flies under CLT. Prolonged exposure to low temperature was also associated with a marked deviation of metabolic homeostasis and warm interruptions as short as 2 h were sufficient to periodically return the metabolic system to functionality. Lipidomics revealed an increased relative proportion of phosphatidylethanolamines and a shortening of fatty acyl chains in flies exposed to cold, likely to compensate for the ordering effect of low temperature on membranes. We found a remarkable correspondence in the time-course of changes between the metabolic and phospholipids networks, both suggesting a fast homeostatic regeneration during warm intervals under FTRs. In consequence, we suggest that periodic opportunities to restore system-wide homeostasis contribute to promote cold tolerance under FTRs. © 2016 Elsevier B.V.