Unlocking the genome of the non-sourdough Kazachstania humilis MAW1: insights into inhibitory factors and phenotypic properties.

BACKGROUND: Ascomycetous budding yeasts are ubiquitous environmental microorganisms important in food production and medicine. Due to recent intensive genomic research, the taxonomy of yeast is becoming more organized based on the identification of monophyletic taxa. This includes genera important to humans, such as Kazachstania. Until now, Kazachstania humilis (previously Candida humilis) was regarded as a sourdough-specific yeast. In addition, any antibacterial activity has not been associated with this species. RESULTS: Previously, we isolated a yeast strain that impaired bio-hydrogen production in a dark fermentation bioreactor and inhibited the growth of Gram-positive and Gram-negative bacteria. Here, using next generation sequencing technologies, we sequenced the genome of this strain named K. humilis MAW1. This is the first genome of a K. humilis isolate not originating from a fermented food. We used novel phylogenetic approach employing the 18 S-ITS-D1-D2 region to show the placement of the K. humilis MAW1 among other members of the Kazachstania genus. This strain was examined by global phenotypic profiling, including carbon sources utilized and the influence of stress conditions on growth. Using the well-recognized bacterial model Escherichia coli AB1157, we show that K. humilis MAW1 cultivated in an acidic medium inhibits bacterial growth by the disturbance of cell division, manifested by filament formation. To gain a greater understanding of the inhibitory effect of K. humilis MAW1, we selected 23 yeast proteins with recognized toxic activity against bacteria and used them for Blast searches of the K. humilis MAW1 genome assembly. The resulting panel of genes present in the K. humilis MAW1 genome included those encoding the 1,3-ß-glucan glycosidase and the 1,3-ß-glucan synthesis inhibitor that might disturb the bacterial cell envelope structures. CONCLUSIONS: We characterized a non-sourdough-derived strain of K. humilis, including its genome sequence and physiological aspects. The MAW1, together with other K. humilis strains, shows the new organization of the mating-type locus. The revealed here pH-dependent ability to inhibit bacterial growth has not been previously recognized in this species. Our study contributes to the building of genome sequence-based classification systems; better understanding of K.humilis as a cell factory in fermentation processes and exploring bacteria-yeast interactions in microbial communities.

PEP444c encoded within the MIR444c gene regulates microRNA444c accumulation in barley.

MicroRNAs are small, noncoding RNA molecules that regulate the expression of their target genes. The MIR444 gene family is present exclusively in monocotyledons, and microRNAs444 from this family have been shown to target certain MADS-box transcription factors in rice and barley. We identified three barley MIR444 (MIR444a/b/c) genes and comprehensively characterised their structure and the processing pattern of the primary transcripts (pri-miRNAs444). Pri-microRNAs444 undergo extensive alternative splicing, generating functional and nonfunctional pri-miRNA444 isoforms. We show that barley pri-miRNAs444 contain numerous open reading frames (ORFs) whose transcripts associate with ribosomes. Using specific antibodies, we provide evidence that selected ORFs encoding PEP444a within MIR444a and PEP444c within MIR444c are expressed in barley plants. Moreover, we demonstrate that CRISPR-associated endonuclease 9 (Cas9)-mediated mutagenesis of the PEP444c-encoding sequence results in a decreased level of PEP444 transcript in barley shoots and roots and a 5-fold reduced level of mature microRNA444c in roots. Our observations suggest that PEP444c encoded by the MIR444c gene is involved in microRNA444c biogenesis in barley.

Critical Assessment of the Chemical Space Covered by LC-HRMS Non-Targeted Analysis.

Non-targeted analysis (NTA) has emerged as a valuable approach for the comprehensive monitoring of chemicals of emerging concern (CECs) in the exposome. The NTA approach can theoretically identify compounds with diverse physicochemical properties and sources. Even though they are generic and have a wide scope, non-targeted analysis methods have been shown to have limitations in terms of their coverage of the chemical space, as the number of identified chemicals in each sample is very low (e.g., ≤5%). Investigating the chemical space that is covered by each NTA assay is crucial for understanding the limitations and challenges associated with the workflow, from the experimental methods to the data acquisition and data processing techniques. In this review, we examined recent NTA studies published between 2017 and 2023 that employed liquid chromatography-high-resolution mass spectrometry. The parameters used in each study were documented, and the reported chemicals at confidence levels 1 and 2 were retrieved. The chosen experimental setups and the quality of the reporting were critically evaluated and discussed. Our findings reveal that only around 2% of the estimated chemical space was covered by the NTA studies investigated for this review. Little to no trend was found between the experimental setup and the observed coverage due to the generic and wide scope of the NTA studies. The limited coverage of the chemical space by the reviewed NTA studies highlights the necessity for a more comprehensive approach in the experimental and data processing setups in order to enable the exploration of a broader range of chemical space, with the ultimate goal of protecting human and environmental health. Recommendations for further exploring a wider range of the chemical space are given.

The tomato cytochrome P450 CYP712G1 catalyses the double oxidation of orobanchol en route to the rhizosphere signalling strigolactone, solanacol.

Strigolactones (SLs) are rhizosphere signalling molecules and phytohormones. The biosynthetic pathway of SLs in tomato has been partially elucidated, but the structural diversity in tomato SLs predicts that additional biosynthetic steps are required. Here, root RNA-seq data and co-expression analysis were used for SL biosynthetic gene discovery. This strategy resulted in a candidate gene list containing several cytochrome P450s. Heterologous expression in Nicotiana benthamiana and yeast showed that one of these, CYP712G1, can catalyse the double oxidation of orobanchol, resulting in the formation of three didehydro-orobanchol (DDH) isomers. Virus-induced gene silencing and heterologous expression in yeast showed that one of these DDH isomers is converted to solanacol, one of the most abundant SLs in tomato root exudate. Protein modelling and substrate docking analysis suggest that hydroxy-orbanchol is the likely intermediate in the conversion from orobanchol to the DDH isomers. Phylogenetic analysis demonstrated the occurrence of CYP712G1 homologues in the Eudicots only, which fits with the reports on DDH isomers in that clade. Protein modelling and orobanchol docking of the putative tobacco CYP712G1 homologue suggest that it can convert orobanchol to similar DDH isomers as tomato.

UPLC-MS/MS analysis and biological activity of the potato cyst nematode hatching stimulant, solanoeclepin A, in the root exudate of Solanum spp.

MAIN CONCLUSION: Solanoeclepin A is a hatching stimulant for potato cyst nematode in very low (pM) concentrations. We report a highly sensitive method for the analysis of SolA in plant root exudates using UHPLC-MS/MS and show that there is considerable natural variation in SolA production in Solanum spp. corresponding with their hatching inducing activity. Potato cyst nematode (PCN) is a plant root sedentary endoparasite, specialized in the infection of solanaceous species such as potato (Solanum tuberosum) and tomato (Solanum lycopersicum). Earlier reports (Mulder et al. in Hatching agent for the potato cyst nematode, Patent application No. PCT/NL92/00126, 1996; Schenk et al. in Croat Chem Acta 72:593-606, 1999) showed that solanoeclepin A (SolA), a triterpenoid metabolite that was isolated from the root exudate of potato, induces the hatching of PCN. Its low concentration in potato root exudate has hindered progress in fully understanding its hatching inducing activity and exploitation in the control of PCN. To further investigate the role of SolA in hatching of PCN, the establishment of a highly sensitive analytical method is a prerequisite. Here we present the efficient single-step extraction and UHPLC-MS/MS based analysis for rapid determination of SolA in sub-nanomolar concentrations in tomato root exudate. This method was used to analyze SolA production in different tomato cultivars and related solanaceous species, including the trap crop Solanum sisymbriifolium. Hatching assays with PCN, Globodera pallida, with root exudates of tomato genotypes revealed a significant positive correlation between SolA concentration and hatching activity. Our results demonstrate that there is natural variation in SolA production within solanaceous species and that this has an effect on PCN hatching. The analytical method we have developed can potentially be used to support breeding for crop genotypes that induce less hatching and may therefore display reduced infection by PCN.

Tocopherols and Tocotrienols-Bioactive Dietary Compounds; What Is Certain, What Is Doubt?

Tocopherols and tocotrienols are natural compounds of plant origin, available in the nature. They are supplied in various amounts in a diet, mainly from vegetable oils, some oilseeds, and nuts. The main forms in the diet are α- and γ-tocopherol, due to the highest content in food products. Nevertheless, α-tocopherol is the main form of vitamin E with the highest tissue concentration. The α- forms of both tocopherols and tocotrienols are considered as the most metabolically active. Currently, research results indicate also a greater antioxidant potential of tocotrienols than tocopherols. Moreover, the biological role of vitamin E metabolites have received increasing interest. The aim of this review is to update the knowledge of tocopherol and tocotrienol bioactivity, with a particular focus on their bioavailability, distribution, and metabolism determinants in humans. Almost one hundred years after the start of research on α-tocopherol, its biological properties are still under investigation. For several decades, researchers' interest in the biological importance of other forms of vitamin E has also been growing. Some of the functions, for instance the antioxidant functions of α- and γ-tocopherols, have been confirmed in humans, while others, such as the relationship with metabolic disorders, are still under investigation. Some studies, which analyzed the biological role and mechanisms of tocopherols and tocotrienols over the past few years described new and even unexpected cellular and molecular properties that will be the subject of future research.

Dynamics of dark fermentation microbial communities in the light of lactate and butyrate production.

BACKGROUND: This study focuses on the processes occurring during the acidogenic step of anaerobic digestion, especially resulting from nutritional interactions between dark fermentation (DF) bacteria and lactic acid bacteria (LAB). Previously, we have confirmed that DF microbial communities (MCs) that fed on molasses are able to convert lactate and acetate to butyrate. The aims of the study were to recognize the biodiversity of DF-MCs able and unable to convert lactate and acetate to butyrate and to define the conditions for the transformation. RESULTS: MCs sampled from a DF bioreactor were grown anaerobically in mesophilic conditions on different media containing molasses or sucrose and/or lactate and acetate in five independent static batch experiments. The taxonomic composition (based on 16S_rRNA profiling) of each experimental MC was analysed in reference to its metabolites and pH of the digestive liquids. In the samples where the fermented media contained carbohydrates, the two main tendencies were observed: (i) a low pH (pH ≤ 4), lactate and ethanol as the main fermentation products, MCs dominated with Lactobacillus, Bifidobacterium, Leuconostoc and Fructobacillus was characterized by low biodiversity; (ii) pH in the range 5.0-6.0, butyrate dominated among the fermentation products, the MCs composed mainly of Clostridium (especially Clostridium_sensu_stricto_12), Lactobacillus, Bifidobacterium and Prevotella. The biodiversity increased with the ability to convert acetate and lactate to butyrate. The MC processing exclusively lactate and acetate showed the highest biodiversity and was dominated by Clostridium (especially Clostridium_sensu_stricto_12). LAB were reduced; other genera such as Terrisporobacter, Lachnoclostridium, Paraclostridium or Sutterella were found. Butyrate was the main metabolite and pH was 7. Shotgun metagenomic analysis of the selected butyrate-producing MCs independently on the substrate revealed C.tyrobutyricum as the dominant Clostridium species. Functional analysis confirmed the presence of genes encoding key enzymes of the fermentation routes. CONCLUSIONS: Batch tests revealed the dynamics of metabolic activity and composition of DF-MCs dependent on fermentation conditions. The balance between LAB and the butyrate producers and the pH values were shown to be the most relevant for the process of lactate and acetate conversion to butyrate. To close the knowledge gaps is to find signalling factors responsible for the metabolic shift of the DF-MCs towards lactate fermentation. Video Abstract.

Long-term effect of one-time nutritional education in school on nutritional knowledge of early school-aged children.

BACKGROUND: In the group of school-aged children nutritional education (NE) enables an early development of healthy eating habits, which can be transferred to the adult life. OBJECTIVE: The aim of the study was to assess the long-term effect of a one-time nutritional education, including also culinary workshops, on the level of nutritional knowledge (NK) of early school-aged children as well as to analyse the nutritional content of the children's books. MATERIAL AND METHODS: The study was conducted among second- and third-grade pupils from a primary school in Warsaw (n=76). The level of NK was determined using the same, self-administrated questionnaire at each of the three stages of the study. RESULTS: The level of NK was assumed as very good in 47% of respondents before the education, in 91% immediately after the education, and in 74% six months after it. Out of all 23.0 points (max.) on average 18.1 points were obtained at the first stage, 21.1 points at the second stage and 19.7 points at the third stage (p<0.001). Immediately after the NE and six months after it the level of NK increased significantly (comparing to the first stage) especially on these topics: the daily water requirements and the recommendations on physical activity (p<0.001 for both questions). CONCLUSIONS: The results of the study confirmed the effectiveness of a one-time nutritional education on the level of nutritional knowledge. Moreover, results show that in the research group nutritional knowledge about importance of physical activity, adequate nutrition, eating varicoloured vegetables and fruits, screen time limits and distinguishing between healthy and unhealthy products was satisfactory, even at the baseline, but in the main source of fats in a diet, the role of milk products, the effect of having too little water in a diet and the sugar content in food children's knowledge was not enough.

Evaluation of acidogenesis products' effect on biogas production performed with metagenomics and isotopic approaches.

BACKGROUND: During the acetogenic step of anaerobic digestion, the products of acidogenesis are oxidized to substrates for methanogenesis: hydrogen, carbon dioxide and acetate. Acetogenesis and methanogenesis are highly interconnected processes due to the syntrophic associations between acetogenic bacteria and hydrogenotrophic methanogens, allowing the whole process to become thermodynamically favorable. The aim of this study is to determine the influence of the dominant acidic products on the metabolic pathways of methane formation and to find a core microbiome and substrate-specific species in a mixed biogas-producing system. RESULTS: Four methane-producing microbial communities were fed with artificial media having one dominant component, respectively, lactate, butyrate, propionate and acetate, for 896 days in 3.5-L Up-flow Anaerobic Sludge Blanket (UASB) bioreactors. All the microbial communities showed moderately different methane production and utilization of the substrates. Analyses of stable carbon isotope composition of the fermentation gas and the substrates showed differences in average values of δ13C(CH4) and δ13C(CO2) revealing that acetate and lactate strongly favored the acetotrophic pathway, while butyrate and propionate favored the hydrogenotrophic pathway of methane formation. Genome-centric metagenomic analysis recovered 234 Metagenome Assembled Genomes (MAGs), including 31 archaeal and 203 bacterial species, mostly unknown and uncultivable. MAGs accounted for 54%-67% of the entire microbial community (depending on the bioreactor) and evidenced that the microbiome is extremely complex in terms of the number of species. The core microbiome was composed of Methanothrix soehngenii (the most abundant), Methanoculleus sp., unknown Bacteroidales and Spirochaetaceae. Relative abundance analysis of all the samples revealed microbes having substrate preferences. Substrate-specific species were mostly unknown and not predominant in the microbial communities. CONCLUSIONS: In this experimental system, the dominant fermentation products subjected to methanogenesis moderately modified the final effect of bioreactor performance. At the molecular level, a different contribution of acetotrophic and hydrogenotrophic pathways for methane production, a very high level of new species recovered, and a moderate variability in microbial composition depending on substrate availability were evidenced. Propionate was not a factor ceasing methane production. All these findings are relevant because lactate, acetate, propionate and butyrate are the universal products of acidogenesis, regardless of feedstock.

Patterns of genetic diversity in North Africa: Moroccan-Algerian genetic split in Juniperus thurifera subsp. africana.

Juniperus thurifera is a key element of the forest communities in arid and semi-arid areas of the western Mediterranean. Previous genetic and morphological investigations suggested that Algerian populations are genetically more similar to European than to Moroccan populations and advocated their recognition at the variety rank. We aimed to investigate the spatial genetic structure in J. thurifera to verify the distinct character of the Algerian population in terms of the genetic breaks reported among several North African taxa. We also modelled species distributions since the Eemian to recognise the impact of past climatic changes on the current pattern of diversity and predict possible changes in species distribution in the future. Species-specific microsatellites were used in the analysis of 11 populations from Algeria, Morocco and Europe. We revealed the significant genetic distinctiveness of the Algerian populations from the Moroccan and European stands that may have important taxonomic and conservation implications. The diversity pattern revealed for J. thurifera reflects the east-west genetic splits reported among some North African plant and animal taxa and suggests an impact of shared historical processes. Additionally, modelling of the distribution allowed us to identify possible glacial refugia and their impact on the modern pattern of differentiation in J. thurifera. Reduction of species occurrence, especially in the European domain, is likely according to the future projections of the species distribution.

Dynamics and Complexity of Dark Fermentation Microbial Communities Producing Hydrogen From Sugar Beet Molasses in Continuously Operating Packed Bed Reactors.

This study describes the dynamics and complexity of microbial communities producing hydrogen-rich fermentation gas from sugar-beet molasses in five packed-bed reactors (PBRs). The bioreactors constitute a part of a system producing hydrogen from the by-products of the sugar-beet industry that has been operating continuously in one of the Polish sugar factories. PBRs with different working volumes, packing materials, construction and inocula were tested. This study focused on analysis (based on 16S rRNA profiling and shotgun metagenomics sequencing) of the microbial communities selected in the PBRs under the conditions of high (>100 cm3/g COD of molasses) and low (<50 cm3/g COD of molasses) efficiencies of hydrogen production. The stability and efficiency of the hydrogen production are determined by the composition of dark fermentation microbial communities. The most striking difference between the tested samples is the ratio of hydrogen producers to lactic acid bacteria. The highest efficiency of hydrogen production (130-160 cm3/g COD of molasses) was achieved at the ratios of HPB to LAB ≈ 4:2.5 or 2.5:1 as determined by 16S rRNA sequencing or shotgun metagenomics sequencing, respectively. The most abundant Clostridium species were C. pasteurianum and C. tyrobutyricum. A multiple predominance of LAB over HPB (3:1-4:1) or clostridia over LAB (5:1-60:1) results in decreased hydrogen production. Inhibition of hydrogen production was illustrated by overproduction of short chain fatty acids and ethanol. Furthermore, concentration of ethanol might be a relevant marker or factor promoting a metabolic shift in the DF bioreactors processing carbohydrates from hydrogen-yielding toward lactic acid fermentation or solventogenic pathways. The novelty of this study is identifying a community balance between hydrogen producers and lactic acid bacteria for stable hydrogen producing systems. The balance stems from long-term selection of hydrogen-producing microbial community, operating conditions such as bioreactor construction, packing material, hydraulic retention time and substrate concentration. This finding is confirmed by additional analysis of the proportions between HPB and LAB in dark fermentation bioreactors from other studies. The results contribute to the advance of knowledge in the area of relationships and nutritional interactions especially the cross-feeding of lactate between bacteria in dark fermentation microbial communities.

Cell factories converting lactate and acetate to butyrate: Clostridium butyricum and microbial communities from dark fermentation bioreactors.

BACKGROUND: Interactions between microorganisms during specific steps of anaerobic digestion determine metabolic pathways in bioreactors and consequently the efficiency of fermentation processes. This study focuses on conversion of lactate and acetate to butyrate by bacteria of dark fermentation. The recently recognized flavin-based electron bifurcation as a mode of energy coupling by anaerobes increases our knowledge of anaerobic lactate oxidation and butyrate formation. RESULTS: Microbial communities from dark fermentation bioreactors or pure culture of Clostridium butyricum are able to convert lactate and acetate to butyrate in batch experiments. The ability of C. butyricum to transform lactate and acetate to butyrate was shown for the first time, with ethanol identified as an additional end product of this process. A search for genes encoding EtfAB complexes and their gene neighbourhood in C. butyricum and other bacteria capable of lactate and acetate conversion to butyrate as well as butyrate-producers only and the lactate oxidiser Acetobacterium woodii, revealed that the Etf complexes involved in (i) lactate oxidation and (ii) butyrate synthesis, form separate clusters. There is a more extent similarity between Etf subunits that are involved in lactate oxidation in various species (e.g. A. woodii and C. butyricum) than between the different etf gene products within the same species of butyrate producers. A scheme for the metabolic pathway of lactate and acetate transformation to butyrate in C. butyricum was constructed. CONCLUSIONS: Studies on the conversion of lactate and acetate to butyrate by microbial communities from dark fermentation bioreactors or Clostridium butyricum suggest that a phenomenon analogous to cross-feeding of lactate in gastrointestinal tract also occurs in hydrogen-yielding reactors. A scheme of lactate and acetate transformation pathway is proposed, based on the example of C. butyricum, which employs flavin-based electron bifurcation. This process utilizes electron-transferring flavoprotein (Etf) complexes specific for (i) lactate oxidation and (ii) butyrate formation. Phylogenetic analysis revealed that such complexes are encoded in the genomes of other bacteria capable of lactate and acetate conversion to butyrate. These findings contribute significantly to our understanding of the metabolic pathways and symbiotic interactions between bacteria during the acidogenic step of anaerobic digestion.

Methane-yielding microbial communities processing lactate-rich substrates: a piece of the anaerobic digestion puzzle.

BACKGROUND: Anaerobic digestion, whose final products are methane and carbon dioxide, ensures energy flow and circulation of matter in ecosystems. This naturally occurring process is used for the production of renewable energy from biomass. Lactate, a common product of acidic fermentation, is a key intermediate in anaerobic digestion of biomass in the environment and biogas plants. Effective utilization of lactate has been observed in many experimental approaches used to study anaerobic digestion. Interestingly, anaerobic lactate oxidation and lactate oxidizers as a physiological group in methane-yielding microbial communities have not received enough attention in the context of the acetogenic step of anaerobic digestion. This study focuses on metabolic transformation of lactate during the acetogenic and methanogenic steps of anaerobic digestion in methane-yielding bioreactors. RESULTS: Methane-yielding microbial communities instead of pure cultures of acetate producers were used to process artificial lactate-rich media to methane and carbon dioxide in up-flow anaerobic sludge blanket reactors. The media imitated the mixture of acidic products found in anaerobic environments/digesters where lactate fermentation dominates in acidogenesis. Effective utilization of lactate and biogas production was observed. 16S rRNA profiling was used to examine the selected methane-yielding communities. Among Archaea present in the bioreactors, the order Methanosarcinales predominated. The acetoclastic pathway of methane formation was further confirmed by analysis of the stable carbon isotope composition of methane and carbon dioxide. The domain Bacteria was represented by Bacteroidetes, Firmicutes, Proteobacteria, Synergistetes, Actinobacteria, Spirochaetes, Tenericutes, Caldithrix, Verrucomicrobia, Thermotogae, Chloroflexi, Nitrospirae, and Cyanobacteria. Available genome sequences of species and/or genera identified in the microbial communities were searched for genes encoding the lactate-oxidizing metabolic machinery homologous to those of Acetobacterium woodii and Desulfovibrio vulgaris. Furthermore, genes for enzymes of the reductive acetyl-CoA pathway were present in the microbial communities. CONCLUSIONS: The results indicate that lactate is oxidized mainly to acetate during the acetogenic step of AD and this comprises the acetotrophic pathway of methanogenesis. The genes for lactate utilization under anaerobic conditions are widespread in the domain Bacteria. Lactate oxidation to the substrates for methanogens is the most energetically attractive process in comparison to butyrate, propionate, or ethanol oxidation.

Noteworthy Facts about a Methane-Producing Microbial Community Processing Acidic Effluent from Sugar Beet Molasses Fermentation.

Anaerobic digestion is a complex process involving hydrolysis, acidogenesis, acetogenesis and methanogenesis. The separation of the hydrogen-yielding (dark fermentation) and methane-yielding steps under controlled conditions permits the production of hydrogen and methane from biomass. The characterization of microbial communities developed in bioreactors is crucial for the understanding and optimization of fermentation processes. Previously we developed an effective system for hydrogen production based on long-term continuous microbial cultures grown on sugar beet molasses. Here, the acidic effluent from molasses fermentation was used as the substrate for methanogenesis in an upflow anaerobic sludge blanket bioreactor. This study focused on the molecular analysis of the methane-yielding community processing the non-gaseous products of molasses fermentation. The substrate for methanogenesis produces conditions that favor the hydrogenotrophic pathway of methane synthesis. Methane production results from syntrophic metabolism whose key process is hydrogen transfer between bacteria and methanogenic Archaea. High-throughput 454 pyrosequencing of total DNA isolated from the methanogenic microbial community and bioinformatic sequence analysis revealed that the domain Bacteria was dominated by Firmicutes (mainly Clostridia), Bacteroidetes, δ- and γ-Proteobacteria, Cloacimonetes and Spirochaetes. In the domain Archaea, the order Methanomicrobiales was predominant, with Methanoculleus as the most abundant genus. The second and third most abundant members of the Archaeal community were representatives of the Methanomassiliicoccales and the Methanosarcinales. Analysis of the methanogenic sludge by scanning electron microscopy with Energy Dispersive X-ray Spectroscopy and X-ray diffraction showed that it was composed of small highly heterogeneous mineral-rich granules. Mineral components of methanogenic granules probably modulate syntrophic metabolism and methanogenic pathways. A rough functional analysis from shotgun data of the metagenome demonstrated that our knowledge of methanogenesis is poor and/or the enzymes responsible for methane production are highly effective, since despite reasonably good sequencing coverage, the details of the functional potential of the microbial community appeared to be incomplete.

Detailed study of polystyrene solubility using pyrolysis-gas chromatography-mass spectrometry and combination with size-exclusion chromatography.

Measuring polymer solubility accurately and precisely is challenging. This is especially true at unfavourable solvent compositions, when only very small amounts of polymer dissolve. In this paper, pyrolysis-gas chromatography-mass spectrometry (Py-GC-MS) is demonstrated to be much more informative and sensitive than conventional methods, such as ultraviolet spectroscopy. By using a programmed-temperature-vapourisation injector as the pyrolysis chamber, we demonstrate that Py-GC-MS can cover up to five orders of magnitude in dissolved polymer concentrations. For polystyrene, a detection limit of 1 ng mL(-1) is attained. Dissolution in poor solvents is demonstrated to be discriminating in terms of the analyte molecular weight. Py-GC-MS additionally can yield information on polymer composition (e.g. in case of copolymers). In combination with size-exclusion chromatography, Py-GC-MS allows us to estimate the molecular weight distributions of minute amounts of a dissolved polymer and variations therein as a function of time.

Molar mass, chemical-composition, and functionality-type distributions of poly(2-oxazoline)s revealed by a variety of separation techniques.

Detailed characterization of synthetic polymers often required multiple advanced separation technologies since the various molecular distributions present, e.g. polymer molar mass, chemical composition, functionality distributions, etc. are generally mutually dependent. The complexity of polymeric materials necessitates the use of a variety of analytical methods, either in conjunction or in integrated ("hyphenated") systems. Poly(2-oxazoline) homo- and copolymers with two different side groups rendering the systems hydrophobic, i.e. phenyl and dec-9-enyl substituents, synthesized by living cationic ring-opening polymerization, were investigated. The average chemical composition obtained by pyrolysis-gas chromatography-mass spectrometry (Py-GC-MS) corresponded well with the theoretical composition. The chemical-composition distribution was studied with gradient elution liquid chromatography (GELC) using water and tetrahydrofuran as mobile-phase components. Statistical copolymer samples - in contrast to their block copolymer analogues - revealed two well-separated peaks in GELC. By combining GELC with size-exclusion chromatography (SEC) it was confirmed that the GELC separation was not based on differences in the molar mass. A more likely explanation of the GELC results is the presence of an ionic fraction in the samples of statistical copolymers resulting from either chain-transfer reactions or termination by addition of water. This hypothesis was confirmed with capillary electrophoresis.

Mercury in Yellow-cracking Boletes Xerocomus subtomentosus mushrooms and soils from spatially diverse sites: assessment of bioconcentration potential by species and human intake.

This study investigated the Hg concentrations in Yellow-cracking Boletes Xerocomus subtomentosus mushrooms and beneath soils collected from the wild at twelve sites across Poland. This mushroom species has some potential to bioconcentrate Hg in the fruiting bodies, and the amount of Hg sequestered, depending on geographical location, can pose health risk to consumers. The values of Hg bioconcentration factor (BCF) varied for the sites between 0.80 ± 0.20 and 17 ± 12 in caps and 0.50 ± 0.10 and 7.9 ± 6.6 in stipes of fruiting bodies but decreased as soil Hg content increased from 72 ± 32 to 570 ± 130 ng/g dry weight. The specimens collected from minerals rich area of Zlotoryja contained the highest Hg concentration, which reached 630 ± 400 in caps and 420 ± 260 ng/g dw in stipes, while the lowest observed Hg concentrations at the other sites were 72 ± 32 and 57 ± 13 ng/g, for cap and stipes respectively. Available literature data on Hg in Yellow-cracking Boletes was also up-dated.

Concentrations and bioconcentration factors of minerals in yellow-cracking Bolete (Xerocomus subtomentosus) mushroom collected in Notec Forest, Poland.

UNLABELLED: Yellow-cracking Bolete (Xerocomus subtomentosus) mushrooms and soil were collected from Notec Forest--a large forested enclave in western part of Poland. Mercury was determined by cold vapour atomic absorption spectroscopy and the other elements by inductively coupled plasma atomic emission spectroscopy. K, P, and Mg were particularly abundant, with mean values of 46000, 8400, and 1100 mg/kg dry weight (dw) in caps followed by Na, Rb, Zn, and Ca with mean concentrations of 580, 350, 200, and 170 mg/kg dw, respectively. In descending order, the mean concentrations of Fe, Al, Cu, and Mn were 52, 49, 46, and 14 mg/kg dw, while the mean for the remaining elements was around 1.0 mg/kg dw or less. The elements such as Ca, Cu, Hg, K, Mg, Na, P, Rb, Zn, Ag, Cd, and Ni were accumulated (with bioconcentration factor (BCF) > 1), while Al, Ba, Fe, Mn, Sr, Co, Cr, and Pb were excluded (BCF < 1) in the fruiting bodies. The Pb and Cd content did not exceed the maximum levels set by the EU for cultivated mushrooms. Mercury in a conventional meal (300 g) portion of Yellow-cracking Bolete was far below the provisionally tolerable weekly intake of 0.004 mg/kg body weight (bw) as reevaluated recently by WHO. PRACTICAL APPLICATION: The method presented in this study allows one to determine the content of 20 elements (Ag, Al, Ba, Ca, Cd, Co, Cr, Cu, Fe, Hg, K, Mg, Mn, Na, Ni, P, Pb, Rb, Sr, and Zn) in caps and stipes of Yellow-cracking Bolete (Xerocomus subtomentosus) mushrooms and soil samples collected from Poland. This study has revealed that the total Cd, Hg, and Pb dose provided to human body due to consumption of Yellow-cracking Bolete does not pose threat to a consumer's health.

Chromatographic examination of the chemical composition and sequence distribution of copolymers from ethyl and benzyl diazoacetate.

Polymers, especially copolymers, are highly complex samples and, therefore, require various setups for their thorough characterization. In this work, one- and two-dimensional chromatographic approaches were applied to characterize two homopolymers and two dissimilar copolymers prepared by rhodium-mediated carbene polymerization using ethyl and benzyl diazoacetate as the monomers: poly-(EA-ran-BnA) and poly((EA)(b)-(BnA-ran-EA)(b)). Different strategies of synthesis suggested that poly-(EA-ran-BnA) was an approximately 1:1 random copolymer and that poly((EA)(b)-(BnA-ran-EA)(b)) was a block-type copolymer of which the (BnA-ran-EA)(b) block is dominated by BnA. The hydrodynamic volume of the investigated polymers turned out to be comparable, i.e. size exclusion chromatography (SEC) did not separate them. Temperature-gradient interaction chromatography was not feasible and one-dimensional (solvent-)gradient-elution liquid chromatography (GELC) suffered from coelution problems. Pyrolysis-gas-chromatography (Py-GC-MS) experiments allowed determining the monomer ratio. SEC with UV and refractive-index detection indicated the presence of molecular-weight-dependent chemical heterogeneity for the poly((EA)(b)-(BnA-ran-EA)(b)) copolymer. This finding was confirmed with off-line SEC//Py-GC-MS. Only a comprehensive two-dimensional GELC×SEC setup enabled the separation of high-molecular-weight material of the less-retained homopolymer from low-molecular-weight copolymeric material. In this way it was possible to detect some high-molecular-weight homopolymeric material in one of the copolymers.

Novel AlkB dioxygenases--alternative models for in silico and in vivo studies.

BACKGROUND: ALKBH proteins, the homologs of Escherichia coli AlkB dioxygenase, constitute a direct, single-protein repair system, protecting cellular DNA and RNA against the cytotoxic and mutagenic activity of alkylating agents, chemicals significantly contributing to tumor formation and used in cancer therapy. In silico analysis and in vivo studies have shown the existence of AlkB homologs in almost all organisms. Nine AlkB homologs (ALKBH1-8 and FTO) have been identified in humans. High ALKBH levels have been found to encourage tumor development, questioning the use of alkylating agents in chemotherapy. The aim of this work was to assign biological significance to multiple AlkB homologs by characterizing their activity in the repair of nucleic acids in prokaryotes and their subcellular localization in eukaryotes. METHODOLOGY AND FINDINGS: Bioinformatic analysis of protein sequence databases identified 1943 AlkB sequences with eight new AlkB subfamilies. Since Cyanobacteria and Arabidopsis thaliana contain multiple AlkB homologs, they were selected as model organisms for in vivo research. Using E. coli alkB(-) mutant and plasmids expressing cyanobacterial AlkBs, we studied the repair of methyl methanesulfonate (MMS) and chloroacetaldehyde (CAA) induced lesions in ssDNA, ssRNA, and genomic DNA. On the basis of GFP fusions, we investigated the subcellular localization of ALKBHs in A. thaliana and established its mostly nucleo-cytoplasmic distribution. Some of the ALKBH proteins were found to change their localization upon MMS treatment. CONCLUSIONS: Our in vivo studies showed highly specific activity of cyanobacterial AlkB proteins towards lesions and nucleic acid type. Subcellular localization and translocation of ALKBHs in A. thaliana indicates a possible role for these proteins in the repair of alkyl lesions. We hypothesize that the multiplicity of ALKBHs is due to their involvement in the metabolism of nucleo-protein complexes; we find their repair by ALKBH proteins to be economical and effective alternative to degradation and de novo synthesis.