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Terrestrial mud volcanoes are unique structures driven by tectonic pressure and fluids from the deep subsurface. These structures are mainly found in active tectonic zones, such as the area near the Los Bajos Fault in Trinidad. Here we report a chemical and microbiological characterization of three mud volcanoes, which included analyses of multiple liquid and solid samples from the mud volcanoes. Our study confirms previous suggestions that at least some of the mud volcano fluids are a mixture of deeper salt-rich water and surficial/precipitation water. No apparent water quality differences were found between sampling sites north and south of a major geological fault line. Microbiological analyses revealed diverse communities, both aerobic and anaerobic, including sulfate reducers, methanogens, carbon dioxide fixing and denitrifying bacteria. Several identified species were halophilic and likely derived from the deeper salt-rich subsurface water, while we also cultivated pathogenic species from the Vibrionaceae, Enterobacteriaceae, Shewanellaceae, and Clostridiaceae. These microorganisms were likely introduced into the mud volcano fluids both from surface water or shallow ground-water, and perhaps to a more minor degree by rain water. The identified pathogens are a major health concern that needs to be addressed.
Microcolonial black fungi are a group of ascomycetes that exhibit high stress tolerance, yeast-like growth and constitutive melanin formation. They dominate a range of hostile natural and man-made environments, from desert rocks and salterns to dishwashers, roofs and solar panels. Due to their slow growth and a lack of genetic tools, the underlying mechanisms of black fungi’s phenotypic traits have remained largely unexplored. We chose to address this gap by genetically engineering the rock-inhabiting fungus Knufia petricola (Eurotiomycetes, Chaetothyriales), a species that exhibits all characteristics of black fungi. A cell biological approach was taken by generating K. petricola strains expressing green or red fluorescent protein variants. By applying: (1) traditional gene replacement; (2) gene editing and replacement via plasmid-based or ribonucleoprotein (RNP)-based CRISPR/Cas9, and (3) silencing by RNA interference (RNAi), we constructed mutants in the pathways leading to melanin, carotenoids, uracil and adenine. Stable single and double mutants were generated with homologous recombination (HR) rates up to 100%. Efficient, partially cloning-free strategies to mutate multiple
genes with or without resistance cassettes were developed. This state-of-the-art genetic toolkit, together with the annotated genome sequence of strain A95, firmly established K. petricola as a model for exploring microcolonial black fungi.
Pseudomonas aeruginosa MPAO1 is the parental strain of the widely utilized transposon mutant collection for this important clinical pathogen. Here, we validate a model system to identify genes involved in biofilm growth and biofilm-associated antibiotic resistance. Our model employs a genomics-driven workflow to assemble the complete MPAO1 genome, identify unique and conserved genes by comparative genomics with the PAO1 reference strain and genes missed within existing assemblies by proteogenomics. Among over 200 unique MPAO1 genes, we identified six general essential genes that were overlooked when mapping public Tn-seq data sets against PAO1, including an antitoxin. Genomic data were integrated with phenotypic data from an experimental workflow using a user-friendly, soft lithography-based microfluidic flow chamber for biofilm growth and a screen with the Tn-mutant library in microtiter plates. The screen identified hitherto unknown genes involved in biofilm growth and antibiotic resistance. Experiments conducted with the flow chamber across three laboratories delivered reproducible data on P. aeruginosa biofilms and validated the function of both known genes and genes identified in the Tn-mutant screens. Differential Protein abundance data from planktonic cells versus biofilm confirmed the upregulation of candidates known to affect biofilm formation, of structural and secreted proteins of type VI secretion systems, and provided proteogenomic evidence for some missed MPAO1 genes. This integrated, broadly applicable model promises to improve the mechanistic understanding of biofilm formation, antimicrobial tolerance, and resistance evolution in biofilms.
Popioły pochodzące z instalacji do spalania komunalnych osadów ściekowych, znajdującej się na terytorium Polski, poddano procesom termochemicznego przekształcania, dzięki czemu otrzymano produkty nawozowe spełniające kryteria dopuszczające je do obrotu jako nawozy mineralne. Ponadto dokonano analizy możliwości wykorzystania popiołów na cele nawozowe zgodnie z obecnie przyjętymi dokumentami strategicznymi i planistycznymi dotyczącymi gospodarki odpadami na poziomie krajowym i europejskim.
Biofilms cause complications and high costs in both industry and medicine. Of particular interest are bacterial infections of prosthetic materials, which usually cannot be eliminated due to the high antibiotic resistance known for bacteria forming biofilms. The search for new materials and coatings with lower colonization potential and antibacterial activity is of great importance to reduce biofilm formation. However, there is no standardized procedure to examine the colonization characteristics of bacteria in the Biofilm state in situ. Here, we describe an automated epifluorescence microscopy system for the semi-quantitative analysis of three-dimensional (3D) biofilms on various surfaces. To analyze adherent bacteria, three materials (glass, steel and titanium) were incubated with bacteria in a flow chamber system. After fluorescence staining of the bacteria, automated image capturing, quantification of the bacteria, measurement of the colonized area and determination of the 3D biofilm height were carried out by using novel software.
Furthermore, the materials were examined for their surface topography using white light scanning interferometry. Titanium compared to glass showed a significantly higher number of adherent bacteria.
We argue that this was due to the higher microroughness of titanium. The colonized area was in accordance with the number of adherent bacteria and was also significantly larger on titanium coupons compared to glass. Maximum 3D biofilm height on glass coupons was significantly lower compared to the ones on steel and titanium. This novel method enables the standardized, automated investigation of the colonization with bacteria on different materials. This approach can considerably support the characterization of new material surfaces and their innovative coatings by analyzing the amount of attached Bacteria and thickness of biofilms in situ and eliminates the need of conventional cultivation.
With the advent of widely accessible and cost-effective next-generation sequencing technologies, it has become increasingly feasible to study insect immunity on a deep genomic or transcriptomic level. Here we introduce a protocol that is aimed at exploiting transcriptomic data to study immunity in non-model insect organisms. We provide instructions for an entire workflow, starting with successfiil extraction of insect RNA through to bioinformatic guidelines for the effective analysis of mRNA sequencing data. The RNA extraction procedure is based on TRIzol Reagent and a spin-column clean-up Step. The bioinformatic pipeline is intended to help users identify immune genes from de novo transcriptome data and includes guidelines for conducting differential gene expression analyses on transcriptomic data. The immune gene prediction method is based on inferring protein homologs with HMMERand Blastp and talces Advantage ofthe ImmunoDB database, which is a valuable resource for research on insect immune-related genes and gene families. The differential gene expression analysis procedure utilizes the DESeq2 package as implemented in R. We hope this protocol will serve as a usefi.il resource for researchers aiming to study immunity in non-model insect species.
Salt tolerance in the γ-proteobacterium Halomonas elongata is linked to its ability to produce the compatible solute ectoine. The metabolism of ectoine production is of great interest since it can shed light on the biochemical basis of halotolerance as well as pave the way for the improvement of the biotechnological production of such compatible solute. Ectoine belongs to the biosynthetic family of aspartate-derived amino-acids. Aspartate is formed from oxaloacetate, thereby connecting ectoine production to the anaplerotic reactions that refill carbon into the tricarboxylic acid cycle (TCA cycle). This places a high demand on these reactions and creates the need to regulate them not only in response to growth but also in response to extracellular salt concentration. In this work, we combine modeling and experiments to analyze how these different needs shape the anaplerotic reactions in H. elongata. First, the stoichiometric and thermodynamic factors that condition the flux distributions are analyzed, then the optimal patterns of operation for oxaloacetate production are calculated. Finally, the phenotype of two deletion mutants lacking potentially relevant anaplerotic enzymes: phosphoenolpyruvate carboxylase (Ppc) and oxaloacetate decarboxylase (Oad) are experimentally characterized. The results show that the anaplerotic reactions in H. elongata are indeed subject to evolutionary pressures that differ from those faced by other gram-negative bacteria. Ectoine producing halophiles must meet a higher metabolic demand for oxaloacetate and the reliance of many marine bacteria on the Entner-Doudoroff pathway compromises the anaplerotic efficiency of Ppc, which is usually one of the main enzymes fulfilling this role. The anaplerotic flux in H. elongata is contributed not only by Ppc but also by Oad, an enzyme that has not yet been shown to play this role in vivo. Ppc is necessary for H. elongata to grow normally at low salt concentrations but it is not required to achieve near maximal growth rates as long as there is a steep sodium gradient. On the other hand, the lack of Oad presents serious difficulties to grow at high salt concentrations. This points to a shared role of these two enzymes in guaranteeing the supply of oxaloacetate for biosynthetic reactions.
New antibacterial agents are urgently required to fight the emergence of antibiotic-resistant bacteria. We recently synthesized the first thioimidazolium ionene, which has antibacterial properties and can degrade in various media. This dual functionality is crucial in order to limit the environmental impact of these biocides. We have found that our polymer is stronger than benzalkonium chloride (BAC) against Pseudomonas aeruginosa and also readily degrades in the presence of base, while remaining stable in acidic environments. These results highlight a new emerging class of antibacterial degradable polymers.
The application of fracture mechanics to the determination of the fatigue behavior of weldments is discussed with the focus on classic fatigue, i.e., the overall fatigue life and the fatigue strength in terms of an S-N curve and the endurance limit. The following issues are addressed: specific features of short fatigue crack propagation, an adequate initial crack size, multiple crack propagation and its statistical treatment as well as welding residual stresses. As an example, an approach of the authors is applied to the determination of FAT classes for a butt weld with varying weld toe geometry.
In 2018, the production of Municipal SolidWaste (MSW) in EU-28 reached 250.6 Mt, with the adoption of different management strategies, involving recycling (48 wt %), incineration and thermal valorization (29 wt %) and landfilling (23 wt %). This work was based on the analysis of the baseline situation of MSW management in EU-28 in 2018, considering its progress in 2008–2018, and discussed the possible improvement perspectives based on a framework involving incineration and recycling as the only possible alternatives, specifically evaluating the capability of already-existing incineration plants to fulfill the EU needs in the proposed framework. The results of the assessment showed two main crucial issues that could play a pivotal role in the achievement of Circular Economy action plan targets: the need to increase the recycling quotas for specific MSW fractions through the separate collection, and therefore the improvement of definite treatment process chains; the optimization of the recovery of secondary raw materials from incineration bottom ash, involving the Recycling of ferrous and nonferrous metals and the mineral fraction. Both issues need to find an extensive application across all member states to decrease the actual differences in the adoption of sustainable MSW management options.
The growth rate of single bacterial cells is continuously disturbed by random fluctuations in biosynthesis rates and by deterministic cell-cycle events, such as division, genome duplication, and septum formation.
It is not understood whether, and how, bacteria reject these growth-rate disturbances. Here, we quantified growth and constitutive protein expression dynamics of single Bacillus subtilis cells as a function of cell-cycle progression. We found that, even though growth at the population level is exponential, close inspection of the cell cycle of thousands of single Bacillus subtilis cells reveals systematic deviations from exponential growth.
Newborn cells display varying growth rates that depend on their size. When they divide, growth-rate Variation has decreased, and growth rates have become birth size independent. Thus, cells indeed compensate for growth-rate disturbances and achieve growth-rate homeostasis. Protein synthesis and growth of single cells displayed correlated, biphasic dynamics from cell birth to division. During a first phase of variable duration, the absolute rates were approximately constant and cells behaved as sizers. In the second phase, rates increased, and growth behavior exhibited characteristics of a timer strategy. These findings demonstrate that, just like size homeostasis, growth-rate homeostasis is an inherent property of single cells that is achieved by cell-cycle-dependent rate adjustments of biosynthesis and growth.
We present here our methodological approach applied to the study of Egyptian inks in Late Antiquity. It is based on an interdisciplinary strategy, bringing together a variety of disciplines from humanities and natural sciences, and it aims at systematically collecting a statistically relevant amount of data regarding the composition of the inks. The application of a wellestablished, non-invasive protocol that includes near-infrared imaging and X-ray fluorescence spectroscopy for in situ measurements enables the identification and characterisation of inks dating from the end of Late Antiquity onwards. However, sometimes this method limits our understanding when characterising more ancient inks. Trying to overcome these limitations, the potential of a new device for the characterisation of organic compounds is here explored by conducting preliminary tests on mock samples.
In this work, we present the results from 77 codicological units that include some of the earliest manuscripts of our corpus that presently lists 159 units.
Phosphorus (P) fertilizers from secondary resources became increasingly important in the last years. However, these novel P-fertilizers can also contain toxic pollutants such as chromium in its hexavalent state (Cr(VI)). This hazardous form of chromium is therefore regulated with low limit values for agricultural products even though the correct determination of Cr(VI) in these fertilizers may be hampered by redox processes, leading to false results. Thus, we applied the novel diffusive gradients in thin-films (DGT) technique for Cr(VI) in fertilizers and compared the results with the standard wet chemical extraction method (German norm DIN EN 15192) and Cr K-edge X-ray Absorption near-edge structure (XANES) spectroscopy. We determined an overall good correlation between the wet chemical extraction and the DGT method. DGT was very sensitive and for most tested materials selective for the analysis of Cr(VI) in P-fertilizers. However, hardly soluble Cr(VI) compounds cannot be detected with the DGT method since only mobile Cr(VI) is analyzed. Furthermore, Cr K-edge XANES spectroscopy showed that the DGT binding layer also adsorbs small amounts of mobile Cr(III) so that Cr(VI) values are overestimated. Since certain types of the P fertilizers contain mobile Cr(III) or partly immobile Cr(VI), it is necessary to optimize the DGT binding layers to avoid aforementioned over- or underestimation. Furthermore, our investigations showed that the Cr K-edge XANES spectroscopy technique is unsuitable to determine small amounts of Cr(VI) in fertilizers (below approx. 1% of Cr(VI) in relation to total Cr).
There are viral, fungal, bacterial and trypanosomal pathogens that negatively impact the individual and superorganismal health of the western honey bee. One fungal pathogen, Ascosphaera apis , affects larvae and causes the disease chalkbrood. A previous genome analysis of As. apis revealed that its genome encodes for RNA interference genes, similar to other fungi and eukaryotes. Here, we examined whether As. apis -targeting double-stranded RNA species could disrupt the germination of As. apis.
We observed that when spores were co-incubated with As. apis -targeting dsRNA, fewer spores were activated for germination, suggesting an uptake of exogenous genetic material at the very onset of germination and consequent damage to essential transcripts needed for germination. Overall, these results indicate that the causative agent of chalkbrood disease, As. apis , can be successfully targeted using an RNAi-based strategy.
For osmoadaptation the halophilic bacterium Halomonas elongata synthesizes as its main compatible solute the aspartate derivative ectoine. H. elongata does not rely entirely on synthesis but can accumulate ectoine by uptake from the surrounding environment with the help of the osmoregulated transporter TeaABC. Disruption of the TeaABC-mediated ectoine uptake creates a strain that is constantly losing ectoine to the medium. However, the efflux mechanism of ectoine in H. elongata is not yet understood. H. elongata possesses four genes encoding mechanosensitive channels all of which belong to the small conductance type (MscS). Analysis by qRT-PCR revealed a reduction in transcription of the mscS genes with increasing salinity. The response of H. elongata to hypo- and hyperosmotic shock never resulted in up-regulation but rather in downregulation of mscS transcription. Deletion of all four mscS genes created a mutant that was unable to cope with hypoosmotic shock. However, the knockout mutant grew significantly faster than the wildtype at high salinity of 2 M NaCl, and most importantly, still exported 80% of the ectoine compared to the wildtype. We thus conclude that a yet unknown system, which is independent of mechanosensitive channels, is the major export route for ectoine in H. elongata.
In this study, we compare combustion ion chromatography (CIC) and high resolution-continuum source-graphite furnace molecular absorption spectrometry (HR-CS-GFMAS) with respect to their applicability for determining organically Bound fluorine sum parameters. Extractable (EOF) and adsorbable (AOF) organically bound fluorine as well as total fluorine (TF) were measured in samples fromriver Spree in Berlin, Germany, to reveal the advantages and disadvantages of the two techniques used as well as the two established fluorine sum Parameters AOF and EOF. TF concentrations determined via HR-CS-GFMAS and CIC were comparable between 148 and 270 μg/L. On average, AOF concentrations were higher than EOF concentrations, with AOF making up 0.14–0.81% of TF (determined using CIC) and EOF 0.04–0.28% of TF (determined using HR-CSGFMAS).
The results obtained by the two independent methods were in good agreement. It turned out that HR-CS-GFMAS is a more sensitive and precise method for fluorine analysis compared to CIC. EOF and AOF are comparable tools in Risk evaluation for the emerging pollutants per- and polyfluorinated alkyl substances; however, EOF is much faster to conduct.
The reliable measurement of very volatile organic compounds (VVOC) in indoor air by use of thermal desorption gas chromatography (TD-GC) in order to include them into evaluation schemes for building products even nowadays is a great challenge. For capturing these small molecules with carbon numbers ranging from C 1 –C 6 , strong adsorbents are needed. In the present study, recovery rates of nine suitable adsorbents of the groups of porous polymers, graphitised carbon blacks (GCB) and carbon molecular sieves (CMS) are tested against a complex test gas standard containing 29 VVOC. By consideration of the recovery and the relative humidity (50% RH), combinations of the GCB Carbograph 5TD, the two CMS Carboxen 1003 and Carbosieve SII as well as the porous polymer Tenax® GR were identified to be potentially suitable for sampling the majority of the VVOC out of the gas mix. The results reveal a better performance of the adsorbents in combination than being used alone, particularly under humid sampling conditions. The recovery rates of the chosen compounds on each adsorbent should be in the range of 80–120%.
This work presents a spectroscopic study of metallic pigments and metal foils used in reverse glass paintings that were created between 1912 and 1954. Metallic pigments induce a notable sparkling effect by means of the lateral incidence of light, whereas metal foils enhance the gloss and create a glittering effect when the painting is viewed in reflected light. Both effects were desired features especially in modern reverse paintings on glass and applied by artists in manifold creative manner. The paper gives an overview on the composition of the metalfoils and metal pigments in 14 works as determined in situ by X-ray spectrometry. Metal foils made of tin, brass, aluminium and silver were found in nine paintings. Gold imitating Cu-Zn pigments in different hues and with various Cu/Zn net intensity ratios were recorded for six paintings. Silvery pigments were identified mainly as Al pigments, but also as Cu-Zn-Ni alloy in one case. Other uncommon metallic pigments were detected in Arlequin vindicatif (c. 1925) by Floris Jespers, who used not only Cu-Zn and Al pigments, but also brownish Cu and Cu-Zn-Sn pigments. Non-invasive diffuse reflectance infrared Fourier-transform spectroscopy (DRIFTS) was used to classify the binding media of the metallic pigments. Drying oil and natural resin probably mixed with oil were the most common binding media. A polysaccharide-based binder was found in the silvery Cu-Zn-Ni pigment of Ohne Titel (1954) by Marianne Uhlenhuth.
The feasibility of microbial hydrogen consumption to mitigate the hydrogen embrittlement (HE) under different cathodic potentials was evaluated using the Devanathan-Stachurski electrochemical test and the hydrogen permeation efficiency h. The hydrogen permeation efficiency h in the presence of strain GA-1 was lower than that in sterile medium. The cathodic potential inhibited the adherence of strain GA-1 to AISI 4135 steel surface, thereby reducing the hydrogen consumption of strain GA-1. The adherent GA-1 cells were capable of consuming ‘cathodic hydrogen’ and reducing the proportions of absorbed hydrogen, indicating that it is theoretically possible to control HE by hydrogen-consuming microbes.