4 Material und Umwelt
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- 4 Material und Umwelt (162)
- 4.5 Kunst- und Kulturgutanalyse (45)
- 4.1 Biologische Materialschädigung und Referenzorganismen (43)
- 4.0 Abteilungsleitung und andere (27)
- 4.2 Material-Mikrobiom Wechselwirkungen (27)
- 4.4 Thermochemische Reststoffbehandlung und Wertstoffrückgewinnung (24)
- 4.3 Schadstofftransfer und Umwelttechnologien (10)
- 6 Materialchemie (7)
- 6.2 Material- und Oberflächentechnologien (5)
- 1 Analytische Chemie; Referenzmaterialien (4)
Eingeladener Vortrag
- nein (73)
An approach to achieve 'zero leakage' is discussed with respect to experience in Germany, where strict regulations for landfill lining and capping Systems have been developed and issued because of large environmental Problems related to landfills that accumulated in the 1970s and 1980s. Using a thick, high-quality high-density Polyethylene (HDPE) geomembrane (GM) that is installed free of residual waves and wrinkles in intimate contact with a compacted clay liner or geosynthetic clay liner of very low permeability, by a qualified, experienced, well-equipped and properly third-party-controlled installer, and which is protected by heavy protection layers designed with respect to the long-term performance of the GM may result in a liner or capping system of practically no leakage. This is demonstrated by analysing results of measurements obtained from permanently installed leak-detection Systems in combination with HDPE GMs. The survey was based on 32 German landfills with 1.276.500 m² of installed GMs.
In the transition to the Circular Economy (CE) model, where the added value of products is kept as long as possible and waste is eliminated, the sustainable management of raw materials plays a key role. In above CE model, especial attention is paid to CRMs which are economically and strategically important for the European economy, but have a high-risk associated with their supply.
One of the most important element which can not be replaced and is an essential element for human nutrition, yet limited resource is phosphorus (P). An importance of issues related to sustainable P management results from EU legislation, which indicated P as a Critical Raw Material (CRM).
The sustainable management of P-resources is especially important for the Baltic region. A consequence of waterborne loads passing into the sea, mainly as wastewater with a high P content is the eutrophication of the Baltic Sea environment. Due to the largest inputs of P (37%) into the Baltic Sea originate from Poland, the development of sustainable solutions aimed at more rational P management for this country is externally important.
Hundreds of papyrus rolls, carbonized during the 79CE eruption of Mount Vesuvius, were discovered in 1754 at Herculaneum. Sophisticated mechanical methods for unrolling the best-preserved scrolls have been applied, with varying success. However, such processes have been abandoned, to prevent risk from irremediable damage or loss and to preserve the integrity of the extremely fragile rolls. Following the development of X-ray based non-invasive techniques, attempts to virtually unroll the scrolls were made. The most common ink in Antiquity was carbon-based, and the main element of carbonized papyrus is carbon, making these investigations difficult. However, some attempts with synchrotron X-ray phase-contrast tomography (XPCT) were successful. Recently, the identification of antique inks containing metals raised hope that if some of the inks contain metal the rolls can be virtually unrolled using conventional CT- technique. We are presenting here the first results of a preliminary analysis, which aimed at identifying scrolls whose ink contains metals.
Due to the vital importance of phosphorus (P) and its increasing scarcity as a natural resource, phosphorus recovery has recently gained significant scientific and technical interest. An interesting sources of phosphorus are sewage sludge (SS) and sewage sludge ash (SSA) due to the major part of the phosphate from P rich wastewater is transferred to the sludge (approx. 90%). Despite the fact that the raw materials base is large (PURE report indicates that in 2020 the amount of sewage sludge generated in Poland will reach 180% of the dry matter of sewage sludge produced in 2010), at present recycling of phosphorus is not a commonly used practice in Poland.
Blast furnace (BF) sludge and electric arc furnace (EAF) dust are typical wastes that incur from iron and steel production. In addition to iron, calcium, carbon, and silicon they usually contain high concentrations of heavy metals such as zinc, lead, and cadmium that are potentially hazardous to the environment, rendering disposal in landfills ecologically problematic and costly. Consequently, pyrometallurgical, hydrometallurgical, and hybrid methods for selective elimination of non-ferrous heavy metals from BF sludge and EAF dust have been conceived, of which only the carbothermic reduction route taken in the so-called Waelz rotary kiln process has been proven to be economically successful. However, this process has several drawbacks regarding efficiency of heavy-metal removal and recovery of iron, and it does not allow processing of BF sludge. In this study, we investigated the efficiency and feasibility of selective chlorination and evaporation of non-ferrous heavy metals, particularly zinc and lead, in both BF sludge and EAF dust as an alternative, thermochemical processing route. To this end, hydrochloric acid and iron(II) chloride solution have been used as chlorinating agents, and the process of heavy-metal chlorination and evaporation has been investigated under inert operating conditions, at variable chlorine concentrations, and at temperatures between 500 and 1200 °C.
High zinc and lead removal efficiencies of > 99.5 % were achieved with both chlorinating agents, but iron(II) chloride turned out to be overall more efficient for removal of zinc and lead from BF sludge and EAF dust. Interestingly, and in contrast to previous studies, the iron was completely retained in the processed solid residue, therefore rendering the processed residues virtually zinc- and lead-free raw materials that may either be used internally (e.g., feeding processed BF sludge and EAF dust back into the respective furnaces) or externally (e.g., for cement production).
An existing pyrometallurgical process for tantalum and niobium recovery, mainly from low grade pyrometallurgical residues, was investigated. Series of melting experiments were carried out in a pilot-scale electric arc furnace to study how the amount, the grain size and the way of feeding affect the activity of carbon as a reducing agent. During the pyrometallurgical treatment refractory metals such as tantalum and niobium are reduced to their carbide form and enriched in the molten iron-based metal phase. The cooled down slag and metal phase were analysed to investigate thermodynamic and kinetic conditions of the carbide formation. FACT Sage simulations were also used to investigate the material system in state of thermodynamic equilibrium. Results show that mass transfer and kinetics may play an important role if compared to equilibrium analyses using FACT Sage.
This work highlights the rediscovery of the technique of reverse glass painting by the artists of the “Blaue Reiter” collective in the early 20th-century and focusses particularly on the role of Wassily Kandinsky (1866–1944). Kandinsky created more than 70 reverse paintings on glass and showed several of them in exhibitions together with paintings on canvas and cardboard, implying a coequal importance of these techniques. Four of his early (1911–1914) reverse glass paintings (Auferstehung, Allerheiligen II, Rudern, Apokalyptischer Reiter II) were selected for investigation and their iconography, painting techniques and painting materials were examined. Two paintings were executed on so-called cathedral glass, revealing a “hammered surface”, whereas Kandinsky used a corrugated glass panel for Rudern. A multi-analytical, non-invasive approach [X-ray fluorescence (XRF), diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS), VIS spectroscopy (VIS), Raman spectroscopy] was taken to identify the pigments and classify the binding media. The results reveal a broad palette of materials. Several pigments like lead white, zinc white, Strontium yellow, Prussian blue, viridian, cadmium yellow, ultramarine blue, cinnabar and carbon black were found in most of the four paintings. The use of the rare synthetic organic pigments PR60 and PB52 is discussed. In two works of art, cadmium carbonate is associated with cadmium yellow. The identification of aluminium foil along with tin foils in Rudern indicates an early use of this material for reverse glass paintings.
This work addresses the identification of synthetic organic pigments (SOP) in eight modern reverse paintings on glass (1913–1946) by means of an in-situ multi-analytical approach. We combined the complementary properties of mobile Raman spectroscopy and diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS) to overcome the main disadvantages of each method: extensive band overlapping in DRIFT spectra and fluorescence problems in Raman spectra. A collection of DRIFTS reference spectra enables a precise pigment identification by DRIFTS and establishes this method as a serious non-destructive alternative for the identification of SOP. The group of β-naphthol pigments yielded valuable results for both methods, whereas synthetic alizarin (PR83) was preferentially detected by DRIFTS. Finally, uncommon triaryl carbonium pigments and two azo group-based yellows were identified in the paintings by means of Raman spectroscopy.
This work addresses the identification of synthetic organic pigments (SOP) in ten modern reverse paintings on glass (1912-1946) by means of an in-situ multi-analytical approach. The combination of the complimentary properties of mobile Raman spectroscopy and diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS) enabled the detection of sixteen SOP even in complex mixtures with inorganic compounds and binders. For the β-naphthol pigments, both Raman and DRIFTS yield appropriate results. DRIFTS was the preferred method for the detection of synthetic alizarin (PR83). Its diagnostic band pattern even allows its detection in complex mixtures with mineral pigments, binders and fillers. Raman spectroscopy yielded distinctive spectra for the triaryl carbonium pigments (PG1, PV2, PR81) and the two-yellow azo SOP (PY3, PY12), whereas DRIFT spectra were affected by extensive band overlapping. This may also occur in Raman spectra, but in less problematic amounts. Fluorescence is the major problem with Raman and it significantly hampers the SOP spectra even with the 785 nm laser. On the one hand the big spot size of DRIFTS (10 mm) limits the technique to rather large sampling areas, whereas the use of a 50× objective for in-situ Raman measurements permits a focus on small spots and aggregated SOP flakes. Moreover, “environmental” factors like temperature changes, artificial light, limited space and vibrations when people pass by need to be considered for in-situ measurements in museums.
Finally, the results show the experimental use of SOP in modern reverse glass paintings. Among several rare SOP (e.g. PB52, PR81), two of them (PG1, PV2) have never been reported before in any artwork.
First insights into Chinese reverse glass paintings gained by non invasive spectroscopic analysis
(2019)
A non-invasive methodological approach (X-ray fluorescence (XRF), diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS), Raman spectroscopy) has been carried out to identify the pigments and classify the binding media in two Chinese reverse glass paintings (The Archer, Yingying and Hongniang) from the late 19th and early 20th centuries. The results reveal a combined use of traditional Chinese and imported European materials. Several pigments like cinnabar, lead white, orpiment, carbon black and copper-arsenic green (probably emerald green) were found in both paintings; red lead, artificial ultramarine blue, Prussian blue and ochre appear in at least one of the paintings.
The presence of portlandite (Ca(OH)2) along calcite (CaCO3) in the fine-grained, white backing layer of Yingying and Hongniang indicates the presence of limewash. In Chinese tradition, limewash was produced from clamshells, and was then sold as clamshell white. In contrast to the Japanese pigment, Chinese clamshell white was made of finely grounded shells, which were heated over a low fire. The residue (CaO) forms portlandite (Ca(OH)2) when water is continuously added. This water-rich mixture is applied on the painting. Portlandite reacts with atmospheric CO2 during drying and forms fine-grained calcite (CaCO3) [1,2].
The identification of emerald green (The Archer) suggests an earliest manufacturing date in the 1830s [3] and promotes the sinological dating of the painting. Drying oil was classified as a binding media in most areas of both paintings. However, the orange background of The Archer yielded prominent bands of both proteinaceous and fatty binder.
The church of Koszewko (Poland) is a brick building edified in the 15th century built on cobblestone foundations. There are five windows in the sanctuary. Three of them enclose heraldic panels from the Küssow’s family from the 15th century which are surrounded with Goethe glass from the 18th century to complete the windows. The colored heraldic panels are strongly damaged and corroded with massive paint layer losses, glass- and leadbreakages. Those medieval glass fragments have been shortly discovered and are of particular interest for Poland since only few medieval glazing have been conserved.
The damages as well as the glass compositions have been investigated with ESEM/EDX. Two categories of medieval glass compositions have been identified. The blue glass is particularly sensible to corrosion because of his high content in K2O. The colorless and the red glass samples belong to a stable glass type. Due to the thickness of the gel layer, it is easy to see that the degradation is strongly proceeded. The protection of those medieval stained-glass panels is absolute necessary.
The medieval panels have been restored and surrounded from a copper frame. Then they have been fixed on the wood frame in the church. The exterior glazing has been closed with a panel of Goethe glass. The gap between the Goethe- and the medieval glass is about 3 cm. The Goethe glass panel has been stabilized with a film based on polyester to protect the medieval glasses against any damages. In this way, a low cost protective glazing has been installed for a long-term conservation of each medieval stained-glass panels. The climate measurements over the period of one year on the restored windows are in process. The temperature and the relative humidity are recorded in the church interior, in the gap between the original and the Goethe glass and outdoors.
Z02 is one of the three technically supporting projects at the Centre for the Study of Manuscript Cultures (CSMC). In collaboration with the other two service projects, Z01 and Z03, it aims at bridging the gap between humanities and natural sciences and technology. To that purpose, we set up a laboratory with a range of high-end instruments, most of them mobile, allowing thorough non-destructive analysis of manuscripts. In addition to working on constantly improving the laboratory and the methods of analysis, a substantial part of our activities is dedicated to service, by supporting different research projects conducted at the centre. In this talk, we will present our equipment and the possibilities offered by the different techniques available regarding the different kinds of missions: typology and classification of inks, provenance studies, recovery of faded inscriptions and palimpsests, reconstruction of the history of manuscripts, authentication and dating. We will give a brief overview of our past and ongoing activities in the frame of the second phase of the CSMC. Finally, a selection of three projects will be presented in greater detail to highlight the possibilities of our laboratory and the diversity of missions which can be carried out.
Microbiologically influenced corrosion (MIC) of iron is usually attributed to sulfate-reducing microorganisms (SRM) in oil and gas facilities. SRM act upon the metal by the re-activeness of hydrogen sulfide (HS-), and by withdrawal of the available electrons in electrical contact with the metal (EMIC). Methanogenic archaea (MA) can also cause MIC (Mi-MIC). Several MAs were identified to be corrosive by using elemental iron as the sole electron donor for methanogenesis, including Methanobacterium-affliated IM1 and Methanococcus maripaludis Mic1c10. Currently, low corrosion rates were reported for MA, possibly due to the formation of siderite (4Fe + 5HCO3- + 5H+ ® 4FeCO3 + CH4 + 3H2O). Since MA do not produce HS-, withdrawal of electrons may be their main corrosive mechanism; however, mechanistic details and kinetics of the overall process are poorly understood.
To investigate the corrosion potential of MA, we studied the EMIC methanogenic strains (IM1 and Mic1c10) individually or part of a syntrophic co-culture with SRM. Corrosion studies were conducted using an in-house developed flow-through system to simulate fluctuating environmental conditions. Results indicate that the rates of iron corrosion by MA (up to 0.4 mm/yr) are higher to that caused by the marine SRM Desulfovibrio alaskensis (0.15 mm/yr) and the co-culture (0.1 mm/yr). Scanning electron microscopy (SEM) images of the metal incubated with MA showed severe pitting corrosion. Genomic analysis of the EMIC MA was conducted to provide an insight on the possible cellular mechanisms that could be involved. Furthermore, low concentrations of MA-targeting biocides will be applied to EMIC MA in static and flow conditions to gain insights for possible mitigation strategies. Such knowledge and deeper understanding also from an electrokinetic point of view may not only provide further models in microbial electrophysiology, but also contribute to mitigation strategies in MIC.
An existing pyrometallurgical process for tantalum and niobium recovery, mainly from low grade pyrometallurgical residues, was investigated. Series of melting experiments were carried out in a pilot-scale electric arc furnace to study how the amount, the grain size and the way of feeding affect the activity of carbon as a reducing agent. During the pyrometallurgical treatment refractory metals such as tantalum and niobium are reduced to their carbide form and enriched in the molten iron-based metal phase. The cooled down slag and metal phase were analysed to investigate thermodynamic and kinetic conditions of the carbide formation. FACT Sage simulations were also used to investigate the material system in state of thermodynamic equilibrium. Results show that mass transfer and kinetics may play an important role if compared to equilibrium analyses using FACT Sage.
The collection Manuscripta Americana in Berlin consists of fragmented codices acquired by Alexander von Humboldt in Mexico. Some of these Humboldt Codices are published as hieroglyphic writings of the Aztecs in “Vues des Cordilleres et Monuments des Peuples indigènes de l’Amerique”. Starting from a special compilation of seven fragments on plate 36 in the “Atlas”, we investigated the corresponding original fragments using material analysis in order to clarify their historical relations. The analyses were carried out with X-ray fluorescence analysis, VIS spectroscopy, and infrared spectroscopy in diffuse reflection and revealed typical indigenous colors like cochineal, indigo, and organic yellows (e.g. mangrove and zacatlaxcalli). Four fragments under investigation show exactly the same material and thus must once have belonged together in one compendium. An additional manuscript from Mizquiahuala also shown on plate 36 can be matched to another one that is not published in the “Atlas”.
The miraculously preserved 2000-year-old Dead Sea Scrolls, ancient texts of invaluable historical significance, were discovered in the mid-20th century in the caves of the Judean desert. The texts were mainly written on parchment and exhibit vast diversity in their states of preservation. One particular scroll, the 8-m-long Temple Scroll is especially notable because of its exceptional thinness and bright ivory color. The parchment has a layered structure, consisting of a collagenous base material and an atypical inorganic overlayer. We analyzed the chemistry of the inorganic layer using x-ray and Raman spectroscopies and discovered a variety of evaporitic sulfate salts. This points toward a unique ancient production technology in which the parchment was modified through the addition of the inorganic layer as a writing surface. Furthermore, understanding the properties of these minerals is particularly critical for the development of suitable conservation methods for the preservation of these invaluable historical documents.
Our experimental knowledge about the inks of antiquity and late antiquity rarely goes beyond their visual description. In rare cases, inks typology has been determined by means of microscopy and reflectography, i.e. using their physical and optical properties, respectively. Since carbon, plant and iron gall inks belong to different classes of compounds they could be easily distinguished had only pure inks been used. Even these crude observations suggest that the inks used differed greatly in their composition.
Reconstruction of the ink recipes with the help of advanced non-destructive analytical techniques could serve as a powerful accessory for in the studies of ancient papyri.
The proposed paper will present a short survey of the methods of material analysis and the challenges offered by ancient inks. The examples of the ink studies from the collections of the Israel Museum in Jerusalem and Egyptian Museum in Berlin will conclude the paper.
Larvae and adults of Korynetes caeruleus (de Geer 1775) (Coleoptera: Cleridae) were collected from old churches and reared in the laboratory on Anobium punctatum (de Geer 1774) (Coleoptera: Ptinidae). Breeding success of K. caeruleus was low, but basic parameters of this species’ developmental cycle were identifi ed. At 21°C and 75% relative humidity and a fourmonth cold period at 4°C, the development of K. caeruleus from egg to adult appearance lasted 2 years. The pupal stage may be reached and completed after one and a half years. Feeding on larvae of A. punctatum by larvae of K. caeruleus was observed and consisted of a combination of sucking haemolymph and consuming body parts. The sickle-like mandibles of larvae of K. caeruleus penetrate the cuticle of prey larvae; this is followed by pumping and sucking body movements. Adult beetles of A. punctatum were not attacked by K. caeruleus larvae. Feeding behaviour of adult K. caeruleus was not investigated.
Fungi that share light-flooded habitats with phototrophs may profit from excess photosynthetic products. But to cope with sunlight-associated stresses it is important for fungi to accurately sense and respond to changes in light. To test the hypothesis that light is an environmental cue that Ascomycota use to coordinate growth, stress responses as well as to establish pathogenic or symbiotic relationships, the photoreceptor (PR) distribution in species from different ecological niches was analysed. The genomes of black fungi from phyllosphere and exposed solid surfaces contain multiple photoreceptors (PRs). The filamentous foliar plant pathogen Botrytis cinerea (Leotiomycetes) has a highly sophisticated photosensory and signalling system that helps to avoid light and to locate susceptible hosts. Rock-inhabiting Dothideo- and Eurotiomycetes including Knufia petricola possess equal numbers of PRs along with the same set of protective pigments. This similarity between black fungi from plant and rock surfaces suggests that photoperception and photoregulation are important for fungi that avoid loss of energy and nutrients through cooperation with phototrophs.
Social insects nesting in soil environments are in constant contact with entomopathogens and have evolved disease resistance mechanisms within a colony to prevent the occurrence and spread of infectious diseases. Among these mechanisms: mutual grooming reduces the cuticular load of pathogens, and burial of cadavers and cannibalism can prevent pathogens from replicating within the group. We explored how the rate and type of collective behavioural response is determined by stepwise infection dynamics operating at the level of the individual. Specifically, we infected the eastern subterranean termite Reticulitermes flavipes with different types of infectious particle and infection route of the entomopathogenic fungus Metarhizium anisopliae and recorded behavioural responses of nestmates to individuals at different times during the progression of infections. As expected, termites groomed conidia-exposed individuals significantly more than controls. Interestingly, grooming was significantly elevated after fungal germination than before, suggesting that pathogen growth cues act as strong stimulators of allogrooming. Conidia-exposed termites were cannibalized, but only after they became visibly ill. By contrast, termites did not groom blastospore-injected individuals more than controls at any time-point following infection. Instead, we found that blastospore-injected individuals were continually cannibalized at a low-level following injection with either viable or heat-killed blastospores, with a marked increase in cannibalism after termites injected with viable blastospores became visibly ill and were close to death. Together, these findings point to the importance of host condition as a cue for social hygienic behavior, and that the host itself appears to emit essential sickness cues that act as targets for its own sacrifice. This demonstrates that termites have independently evolved to both identify and destructively respond to sickness.
The termites are a derived eusocial lineage of otherwise non-social cockroaches. Understanding the proximal and ultimate drivers of this major evolutionary transition represents an important goal in biology. One outstanding question concerns the evolution of termite immunity, which is thought to have undergone broad-sweeping adaptations in order to enhance group-level immune protection. To understand the evolutionary origins of termite immunity, we conducted qualitative and quantitative transcriptome analyses along a gradient of sociality. Firstly, we aimed to identify large-scale genetic shifts in immune traits linked to eusociality by comparing immune gene repertoires in solitary and subsocial cockroaches and across a range of eusocial termite lineages. Secondly, we compared the responses of a solitary cockroach, a subsocial wood-roach and different castes of a lower termite species to a non-specific immune challenge, in order to understand how sociality may have influenced the evolution of immune gene regulation. Firstly, we found that termites have a broadly representative repertoire of canonical immune genes as compared to gregarious cockroaches and subsocial wood-roaches. Secondly, with respect to immune challenge, the solitary cockroach and the subsocial wood-roach displayed a similarly comprehensive induced response, while the termite response was considerably dampened by comparison and strongly influenced by caste; with reproductives displaying a generally higher constitutive level of immune-gene expression compared to sterile castes. In summary we did not find termite eusociality to be associated with significant changes in immune gene diversity, but rather to be linked with significant modifications to the regulation of immunity following the origin of division of labour.
Results suggest a reduction in immune gene repertoires in termites and possible complementary expression between termite castes. With comparative genomics we will investigate the evolution of gene families related to immunity, try to understand where reductions and expansions take place and relate these changes to shifts in sociality and ecology. The role of TEs in expansions and contractions of immune gene families will be investigated. For these analyses, we propose to generate high quality, highly contiguous genomes of species from different levels of sociality, covering all major termite families. With comparative transcriptomics we will investigate the expression of immune genes in different castes. Via network analyses we will identify pathways indicated in differential immunity between castes and between species of different sociality levels. We will investigate how these pathways have been rewired along the transitions to higher levels of sociality and how, intra-specifically, they change between castes.
A broad suite of immune adaptations have evolved in social insects which hold close parallels with the immune systems of multicellular individuals. However, comparatively little is known about the evolutionary origins of immunity in social insects. We tackle this by identifying immune genes from 18 cockroach and termite species, spanning a gradient of social lifestyles. Termites have undergone contractions of major immune gene families during the early origin of the group, particularly in antimicrobial effector and receptor proteins, followed by later re-expansions in some lineages. In a comparative gene expression analysis, we find that reproductive individuals of a termite invest more in innate immune regulation than other castes. When colonies encounter immune-challenged nestmates, gene expression responses are weak in reproductives but this pattern is reversed when colony members are immune-challenged individually, with reproductives eliciting a greater response to treatment than other castes. Finally, responses to immune challenge were more comprehensive in both subsocial and solitary cockroaches compared to termites, indicating a reduced overall ability to respond to infection in termites. Our study indicates that the emergence of termite sociality was associated with the evolution of a tapered yet caste-adapted immune system.
Termitidae comprises 80% of all termite species that play dominant decomposer roles in Tropical cosystems. Two major events during Termite evolution were the loss of cellulolytic gut protozoans in the ancestor of Termitidae and the subsequent gain in the termitid subfamily Macrotermitinae of fungal symbionts cultivated externally in ‘‘combs’’ constructed within the nest. How these symbiotic transitions occurred remains unresolved. Phylogenetic analyses of mitochondrial data previously suggested that Macrotermitinae is the earliest branching termitid lineage, followed soon after by Sphaerotermitinae, which cultivates bacterial symbionts on combs inside its nests. This has led to the hypothesis that comb building was an important evolutionary step in the loss of gut protozoa in ancestral termitids. We sequenced genomes and transcriptomes of 55 termite species and reconstructed phylogenetic trees from up to 4,065 orthologous genes of 68 species. We found strong support for a novel sister-group relationship between the bacterial comb-building Sphaerotermitinae and fungus comb-building Macrotermitinae.
This key finding indicates that comb building is a derived trait within Termitidae and that the creation of a comb-like ‘‘external rumen’’ involving bacteria or fungi may not have driven the loss of protozoa from ancestral termitids, as previously hypothesized.
Instead, associations with gut prokaryotic symbionts, combined with dietary shifts from wood to other plant-based substrates, may have played a more important role in this symbiotic transition. Our phylogenetic tree provides a platform for future studies of comparative termite evolution and the evolution of symbiosis in this taxon.
A Genetic Toolbox for Exploring the Life Style of the Rock-inhabiting Black Fungus Knufia petricola
(2019)
After their discovery on rock surfaces in cold and hot deserts, a polyphyletic group of ascomycetous black fungi was found to dominate a range of hostile environments – natural and man-made, from salterns to dishwashers, roofs and solar panels. Together with bacteria and algae they may establish sub-aerial biofilms and cause weathering of the surfaces they grow on. Their impressive survival abilities as well as their constitutive protective pigmentation and cluster-like microcolony organisation are similar in environmental isolates as well as in heat-tolerant opportunistic pathogens of animals and humans. The exact genetic properties that ensure their survival in extreme environments can be studied if some black fungi were amenable to genetic manipulations. We selected the rock-inhabiting fungus Knufia petricola (class Eurotiomycetes, order Chaetothyriales) that grows moderately in axenic culture and exhibits all the characteristics of black yeasts such as yeast-like cell growth, absence of reproductive structures and constitutive dihydroxynaphthalene (DHN) melanogenesis. For this environmental strain we developed protocols to efficiently generate and transform protoplasts resulting in stable homokaryotic transformants by targeting genes involved in pigment synthesis and expressing fluorescent reporter genes. Hence, endogenous and foreign genes can be expressed from episomal AMA1-containing plasmids and genome-integrated DNA constructs. Moderate rates of homologous recombination allow for both ectopic and targeted integrations. CRISPR-Cas9 was further validated as a strategy for obtaining selection marker-free mutants and silencing via RNA interference as an approach to study essential genes. Availability of this genetic toolbox and an annotated genome sequence is paving the way for studying interactions of K. petricola and other black yeasts with environmental stressors, material surfaces, soil matrices and phototrophic symbionts.
Black microcolonial fungi (MCF) are persistent inhabitants of rock surfaces in hostile desert environments. In these niches, MCF have evolved mineral-weathering and symbiotic capabilities as well as mechanisms to cope with multiple stresses such as solar irradiation, temperature extremes and low water activity. Due to their stress tolerance these ascomycetes are prominent in modern terrestrial ecosystems – like man-made material surfaces from roof to solar panels. MCF interactive capabilities support their facultative symbiotic relationships with cyanobacteria and ensure their rock-weathering geochemical activity.
Using the rock-inhabiting fungus K. petricola A95 (Chaetothyriales), we developed transformation protocols and deleted genes responsible for production of the protective pigments melanins and carotenoids. To confirm that the mutant phenotypes were not due to hidden mutations, melanin synthesis was restored by complementing the mutants with the respective wild type genes. Strains of K. petricola carrying gene variants for fluorescent proteins EGFP and DsRed are available. We successfully labelled the cytoplasm, nuclei, peroxisomes and mitochondria. Targeted and ectopic integrations result in stable transformants suitable for further phenotypical characterization. As K. petricola is a non-pathogenic fungus with all characteristic features of MCF, including meristematic growth, melanized cell-walls, extracellular polymeric substances and extensive pigment production, our results will shed light on protective role of pigments during cell wall maturation and oxidative stress defence in rock-inhabiting MCF. Genes involved in environmental sensing or substrate and phototroph interactions are currently targeted. With the help of a mutant collection and fluorescently labelled K. petricola we will be able to investigate interactions of MCF with environmental stressors, mineral substrates, soil matrices and phototrophic symbionts.
Black yeasts form a polyphyletic group of Ascomycota that colonize bare surfaces like rocks, facades of buildings, and solar panels. Their protective adaptations enable an adequate response to fluctuating and diverse temperature, water and UV radiation stresses. Together with bacteria and algae they form sub-aerial biofilms (SAB) this way discoloring and weathering the surfaces they grow on. Strain A95 of Knufia petricola (Eurotiomycetes, Chaetothyriales) displays both typical yeast-like cell growth and constitutive dihydroxynaphthalene (DHN) melanogenesis. Along with the cyanobacterium Nostoc punctiforme as photobiont, it is already used in a model system for studying SAB formation and bio-weathering. Applying the recently developed tools for the generation of deletion mutants will allow to define gene functions and to identify genes critical for abiotic and biotic interactions. We present a chromosome-level genome assembly and annotation for K. petricola A95. The genome was assembled with MaSuRCA using a hybrid assembly approach of Illumina MiSeq and PacBio SMRT sequencing data. The resulting assembly consists of 17 contigs including the complete mitochondrial genome and five complete chromosomes. It shows indication of repeat-induced point mutations (RIP). Supported by RNA sequencing data from eight different growth conditions, 10,994 genes were predicted with the BRAKER2 pipeline. Functional annotation of genes was obtained from general functional annotation databases and the fungal specific database FungiPath. Comparative analyses are in progress to identify genes specific to black yeasts, that may facilitate the survival on exposed surfaces. In sum, the genome sequence of K. petricola is a valuable resource to gain insight into the protein inventory and functional pathways of extremotolerant and symbiosis-capable fungi.
Fungi that share light-flooded habitats with phototrophs may profit from excess photosynthetic products. But to cope with sunlight-associated stresses it is important for fungi to accurately sense and respond to changes in light. To test the hypothesis that light is an environmental cue that Ascomycota use to coordinate growth, stress responses as well as to establish pathogenic or symbiotic relationships, the photoreceptor (PR) distribution in species from different ecological niches was analysed. The genomes of black fungi from phyllosphere and exposed solid surfaces contain multiple photoreceptors (PRs). The filamentous foliar plant pathogen Botrytis cinerea (Leotiomycetes) has a highly sophisticated photosensory and signalling system that helps to avoid light and to locate susceptible hosts. Rock-inhabiting Dothideo- and Eurotiomycetes including Knufia petricola possess equal numbers of PRs along with the same set of protective pigments. This similarity between black fungi from plant and rock surfaces suggests that photoperception and photoregulation are important for fungi that avoid loss of energy and nutrients through cooperation with phototrophs.
Wild bees are important pollinators for agricultural crops and solitary species such as Osmia bicornis are particularly suitable for pollination management. Wild bees share floral resources with managed honey bees and may be exposed to emerging infectious diseases. Although studies have explored the prevalence of pathogens in solitary wild bee species, data regarding the impact of pathogens on solitary bee health are lacking.
We carried out experiments examining whether the solitary bee species O. bicornis is susceptible to infection with the emerging pathogen The results obtained indicate that N. ceranae may be able to infect O. bicornis but its impact on host fitness is negligible: survival rates did not differ between Control and inoculated bees, although male survival was marginally lower after infection. To explore the possible field-relevance of our findings, we collected wild bees near an infected and a non-infected hive and showed that N. ceranae was shared between managed and wild bees, although only the in presence of infected honey bees.
The findings of the present study show that O. bicornis is susceptible to pathogen spillover and could act as a potential reservoir host for N. ceranae in pollinator networks. Additional studies on this species incorporating sublethal effects, multiple infections and other interacting stressors are warranted.
Wild bees are important pollinators of wild plants and agricultural crops and they are threatened by several environmental stressors including emerging pathogens. Honey bees have been suggested as a potential source of pathogen spillover. One prevalent pathogen that has recently emerged as a honey bee disease is the microsporidian Nosema ceranae. While the impacts of N. ceranae in honey bees are well documented, virtually nothing is known about its effects in solitary wild bees.
The solitary mason bee Osmia bicornis is a common pollinator in orchards and amenable to Commercial management. Here, we experimentally exposed larvae of O. bicornis to food contaminated with N. ceranae and document spore presence during larval development. We measured mortality, growth parameters, and timing of pupation in a semi-field experiment. Hatched individuals were assessed for physiological state including fat body mass, wing muscle mass, and body size. We recorded higher mortality in the viable-spore-exposed group but could only detect a low number of Spores among the individuals of this treatment. Viable-spore-treated individuals with higher head capsule width had a delayed pupation start. No impact on the physiological status could be detected in hatched imagines. Although we did not find overt evidence of O. bicornis infection, our findings indicate that exposure of larvae to viable N. ceranae spores could affect bee development.
Biocide mitigation strategies of microbiologically influenced corrosion (MIC) in the oil and gas industry have been primarily used to eliminate the growths of sulfate-reducing microorganisms (SRM). However, methanogenic Archaea (MA) can also be highly corrosive by using iron as an electron source for methanogenesis. Because of the fundamental physiological differences between archaea and bacteria, responses of MA towards SRM-specific biocides cannot be deduced using SRM. Due to the lack of information available on the effect of biocides on corrosive MA, we selected THPS, glutaraldehyde, nitrate and perchlorate to compare against corrosive SRM.
Preliminary results showed that at low concentrations of THPS (0-10 ppm), growth of MA was not affected, methane production and corrosion rates (0.1 mm/yr) were comparable between the different THPS concentrations. On the contrary, the SRM strain showed decreased corrosion rates (0.18 mm/yr to 0.03 mm/yr) with increasing THPS concentrations. Further corrosion tests including electrochemical measurements of different biocides on the growth of MA and SRM will be conducted. Such knowledge not only provide important insights on the physiological response of MA to biocides but also contribute to more effective mitigation strategies that can be both economic and environmentally beneficial.
A similar secretome disturbance as a hallmark of non-pathogenic Botrytis cinerea ATMT-mutants?
(2019)
The gray mold fungus Botrytis cinerea is a necrotrophic pathogen able to infect
hundreds of host plants, including high-value crops such as grapevine, strawberry and tomato. In order to decipher its infectious strategy, a library of 2,144 mutants was generated by random insertional mutagenesis using Agrobacterium tumefaciensmediated transformation (ATMT). Twelve mutants exhibiting total loss of virulence toward different host plants were chosen for detailed analyses. Their molecular characterization revealed a single T-DNA insertion in different loci. Using a proteomics approach, the secretome of four of these strains was compared to that of the parental strain and a common profile of reduced lytic enzymes was recorded. Significant variations in this profile, notably deficiencies in the secretion of proteases and hemicellulases, were observed and validated by biochemical tests. They were also a hallmark of the remaining eight non-pathogenic strains, suggesting the importance of these secreted Proteins in the infection process. In the twelve non-pathogenic mutants, the Differentiation of infection cushions was also impaired, suggesting a link between the Penetration structures and the secretion of proteins involved in the virulence of the pathogen.
The Weimar research project has set the goal of placing style-critical research on Old Master drawings on a new, methodologically proven foundation. The starting point is the stock of Dutch drawings possessed by the Klassik Stiftung Weimar, whose extent (ca. 1,400 items) and character make it one of the most significant outside the Netherlands. The focus here is on drawings that were executed with red chalk. This drawing material has different functions - one of which is the use as a transmission material. The methodological approach is double: first, the scientific indexing of this stock. The foundations for this, along with classic analysis of style, are innovative material-scientific methods of investigation.
Treatment and reusing of hazardous wastes have become significant issues of modern societies. Blast furnace sludge (BFS) and electric arc furnace dust (EAFD) are such typical wastes, because they usually contain high amounts of undesirable elements such as zinc (Zn) and lead (Pb).
These elements can cause operational problems in reusing of BFS and EAFD. In this study, the Ferro Duo GmbH and the Federal Institute for Materials Research and Testing have investigated a novel approach for eliminating and/or recovering Zn and Pb from both wastes. Applied was a selective chlorination and volatilization of Zn and Pb as chlorides at temperatures between 500 °C and 1100 °C. Both product obtained, Zn and Pb in the form of ZnCl2 and PbCl2 and the purified solid mineral compound are attractive materials for further use. Hydrochloric acid and iron(II) chloride were used as chlorination agents. Exceptionally high Zn and Pb removal efficiencies of >99.5 % could be achieved with both chlorination agents, whereby iron(II) chloride exhibited better performance.
This work presents a technical investigation of two Chinese reverse glass paintings from the late 19th and early 20th centuries. A multi-analytical, non-invasive approach (X-ray fluorescence (XRF), diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS), Raman spectroscopy) was used to identify the pigments and classify the binding media. The results reveal a combined use of traditional Chinese and imported European materials. Several pigments like cinnabar, lead white, orpiment, carbon black and copper-arsenic green (probably emerald green) were found in both paintings; red lead, artificial ultramarine blue, Prussian blue and ochre appear in at least one of the paintings. The proof of limewash (calcite and small amounts of portlandite) as a backing layer in Yingying and Hongniang indicates that clamshell white was also used for reverse glass paintings. Drying oil was classified as a binding media in most areas of both paintings. However, the orange background of The Archer yielded prominent bands of both proteinaceous and fatty binder.
A non-invasive methodological approach has been carried out to identify the pigments and classify the binding media of the reverse painting on glass Kreuzabnahme (1914/15) by Carlo Mense.
Mense was a member of the group Rhenish Expressionists around August Macke and got interested in the technique of painting on the reverse side of a glass panel. The studied painting indicates a remarkably painted reverse and front side, which creates a unique visual appearance of the glass. The applied noninvasive, spectroscopic approach includes X-ray fluorescence, VIS spectroscopy, Raman spectroscopy and diffuse reflectance infrared Fourier transform spectroscopy. The measurements revealed inorganic Pigments mixed with drying oil as binder. Together with common pigments, such as lead white, barium sulphate, bone black, cinnabar, chrome yellow, ochre, and viridian, an unusual additional material was found, Strontium white.
Column leaching tests are frequently used and accepted for investigation of release of hazardous substances from solid materials. Independent of differences due to the field of application or national regulations, column tests assume that local equilibrium is established in the experiment which facilitates transfer of results to field conditions. In the process of harmonization and standardization within Europe the question on the influence of flow rate and grain size distribution on the local equilibrium was raised. Thus, a set of experiments using two different masonry materials with varying grain size distribution and flow rate were conducted including stop/flow experiments. Results are compared to a numerical model which takes intraparticle pore diffusion-controlled release of Mo and V into the percolating water into account. Due to the relatively high intraparticle porosity of the materials (24–29%) data and model indicate that initially equilibrium-state conditions prevail followed by rapidly decreasing concentrations. The model fits data for Mo and V reasonably well; however, after the initial decline of concentrations (at L/S > 2) extended tailing is observed especially of elements occurring as oxides, which is not captured by the model.
Biocides are commonly applied to construction materials such as facade renders and paints in order to protect them from microbial spoilage. These renders and paints are exposed to weathering conditions, e.g., sunlight and rain. Pigments are interacting intensively with the spectrum of the incoming light; thus, an effect of paint pigments on phototransformation rates and reaction pathways of the biocides is hypothesized.
In this study, the phototransformation of four commonly used biocides (carbendazim, diuron, octylisothiazolinone (OIT) and terbutryn) in four different paint formulations differing solely in pigments (red and black iron oxides, white titanium dioxide, and one pigment-free formulation) were investigated. Paints surfaces were irradiated under controlled conditions. The results show that biocides degrade most rapidly in the pigment-free formulation. The degradation in the pigment-free formulation followed a first-order kinetic model with the respective photolysis rate constants: kp,Diuron=0.0090 h−1, kp,OIT=0.1205 h−1, kp,Terbutryn=0.0079 h−1.
Carbendazim concentrations did not change significantly. The degradation was considerably lower in the pigment-containing paints. The determination of several phototransformation products of terbutryn and octylisothiazolinone showed different transformation product ratios dependent on the pigment. Consequently, pigments not only reflect the incoming light, but also interact with the biocide photodegradation.
OECD test guideline on particle size and size distribution: Comparability of measurement results
(2019)
OECD-Prüfrichtlinien werden weltweit für regulatorische Zwecke angewandt, da sie eine harmonisierte und über internationale Ringversuche validierte Vorschrift zur Analyse vorgeben. Die bestehende Prüfrichtlinie TG 110 zur Größencharakterisierung von Partikeln und Fasern ist für viele Methoden und Materialklassen nicht geeignet, weswegen es Bedarf an einer Neuentwicklung gibt. Im Fokus der neuen TG steht die reproduzierbare und vergleichbare Anwendung von Methoden zur Bestimmung von Partikelgröße sowie Größenverteilung annährend sphärischer und faserförmiger Nanomaterialien im Bereich 1-1000 nm. Berücksichtigt werden die häufig angewandten Methoden zur Größencharakterisierung mit ihren unterschiedlichen physikalisch-technischen Limitierungen und die Bestimmung unterschiedlicher Durchmesser (hydrodynamischer, Feret…). Weiterhin sind materialspezifische Einflüsse durch die Partikel sowie das Messmedium zu berücksichtigen. Jede der Methoden soll unter Berücksichtigung von ISO-Standards und den materialspezifischen Limitierungen in der neuen Prüfrichtlinie beschrieben und über einen Ringversuch validiert werden.
The environmental compatibility of reactive fire-retardant coatings (intumescent paints) was investigated by a combination of leaching and ecotoxicological tests. Three representative fire-retardant coating systems were tested using two leaching procedures: “Horizontal Dynamic Surface Leaching Test” (DSLT) and the “Intermittent Immersion Test” (IIT). All eluate fractions (8 for DSLT and 9 for IIT) were analyzed for pH, conductivity, concentration of total organic carbon and selected anions und cations. Additionally, a GC-MS screening of selected fractions was conducted for identification of organic compounds. Eluate fractions 1 + 2 and fraction 7 of the DSLT were analyzed in four ecotoxicological tests (algae, daphnia, fish egg, luminescent bacteria) and in one genotoxicity test (umu). Concentration of most analytes was rather low or below limit of detection for many eluates. Analytes detected in eluates of all three products are Zn, Ba, SO4(2−) and PO4(3−). Release patterns do not indicate a general trend: some compounds show maximum release in the first fractions while for others the maximum was observed in later test stages. Ecotoxic effects in eluates were found, which were higher in the eluate fraction 7 (maximum lowest ineffective dilution for luminescent bacteria (LIDL) 256) than in the eluate fraction 1 + 2 (maximum LIDL = 24). The sensitivity of the test systems was very different with highest effects for luminescent bacteria, followed by algae and daphnia and without effects in the fish egg test and umu test. A biotest battery for the comprehensive assessment is therefore advisable.
Flow-back and produced waters from shale gas and shale oil fields contain high ammonium, which can be formed by methanogenic degradation of methylamines into methane and ammonium. Methylamines are added to fracturing fluid to prevent clay swelling or can originate from metabolism of the osmolyte triglycinebetaine (GB).
We analyzed field samples from a shale gas reservoir in the Duvernay Formation and from a shale oil reservoir in the Bakken formation in Canada to determine the origin of high ammonium. Fresh waters used to make fracturing fluid, early flow-back waters, and late flow back waters from the shale gas reservoir had increasing salinity of 0.01, 0.58, and 2.66 Meq of NaCl, respectively. Microbial community analyses reflected this fresh water to saline transition with halophilic taxa including Halomonas, Halanaerobium, and Methanohalophilus being increasingly present. Early and late flow-back waters had high ammonium concentrations of 32 and 15 mM, respectively.
Such high concentrations had also been found in the Bakken produced waters.
Enrichment cultures of Bakken produced waters in medium containing mono, di-, or trimethylamine, or triglycinebetaine (GB) converted these substrates into ammonium (up to 20 mM) and methane. The methylotrophic methanogen Methanohalophilus, which uses methylamines for its energy metabolism and uses GB as an osmolyte, was a dominant community member in these enrichments. Halanaerobium was also a dominant community member that metabolizes GB into trimethylamine, which is then metabolized further by Methanohalophilus. However, the micromolar concentrations of GB measured in shale reservoirs make them an unlikely source for the 1,000-fold higher ammonium concentrations in flow-back waters. This ammonium either originates directly from the reservoir or is formed from methylamines, which originate from the reservoir, or are added during the hydraulic fracturing process. These methylamines are then converted into ammonium and methane by halophilic methylotrophic methanogens, such as Methanohalophilus, present in flow-back waters.
The presented Humboldt Codices are from the early colonial period of Mexico and originate from different localities
1. typical indigen colors and mixtures:
cochineal (red), indigo (blue and green), carbon (black),
mangle, zacatlaxcalli, organic (brown, yellow)- critical!
2. colonial influence:
iron gall ink for writing
3. single fragments belong together:
are cut (fragments IX-XII),
from Mizquahuala distributed (VII, XIII, XIV, [XV]) (VII, XIII in Mexico, XIV in Paris)
Since the early 19th century microorganisms were studied on their capabilities of causing microbiologically influenced corrosion (MIC) of metals. The most studied ones are sulfate-reducing bacteria (SRB), but others can corrode metals as well, e.g. acid-producing bacteria or methanogenic archaea (MA). However, these studies were mostly focused on metals related to the petroleum industry but metals for other industries, e.g. dentistry, are also susceptible to corrosion. The inert Titanium (Ti) is often used as an implant material, but it is a base metal. The formation of a passivating oxide layer allows Ti to be corrosion resistant at normal conditions.
Nonetheless, scanning electron microscope images on dental implants from patients with acute peri-implantitis showed clear signs of corrosion. Currently, the corrosion mechanism of dental implants is unknown, but many indications suggest that oral microorganisms, including MA (Methanobrevibacter oralis) and SRB (Desulfomicrobium orale), could be involved.
To determine if MA or SRB can corrode Ti (pure Ti or Ti-6Al-4V alloy), corrosion rate, methane and sulfide concentrations were analyzed. Electrical potential measurements using in-house developed electrochemical cells indicated a potential change on Ti in the presence of a corrosive MA strain compared to an abiotic control.
Microbial composition comparison will be analyzed using samples from dental pockets of 150 infected patients by considering the quality of the implant and 50 healthy people by means of amplicon sequencing. Enrichments and isolation of pure cultures from the dentals samples are also examined for their corrosion behavior. Overall, this is the first study investigating the susceptibility of dental implant material to corrosion using human related MA.
The European building sector is moving towards more complex and high-tech building approaches. While focusing on energy efficiency, aspects e.g. occupant health, sustainability and life cycle costing are often neglected. This study highlights the potential of earthen plasters in combination with natural ventilation for low-tech solutions.
The EU funded project [H]house established the outstanding performance of earthen materials in light of hygrothermal and air purifying properties, which were further supported by experimental data from monitoring of naturally ventilated pilot buildings in Berlin. Additionally, [H]house demonstrated through LCC an increased cost efficiency of earth based low-tech solutions in comparison to conventional constructions relying on mechanical ventilation.
Microbiologically influenced corrosion (MIC) of iron is usually attributed to sulfate-reducing microorganisms (SRM) in offshore industries, such as the oil and gas pipelines, due to the high concentrations of sulfate in the seawater. SRM act upon the metal by the reactiveness of hydrogen sulfide (HS-), and by withdrawal of the available electrons (Fe --> Fe2+ + 2e-; E° = -0.47 V) in electrical contact with the metal (EMIC). However, methanogenic archaea can also cause MIC. Because they do not produce HS-, withdrawal of electrons may be their main corrosive mechanism; however, mechanistic details and kinetics of the overall process are poorly understood.
To investigate the corrosion potential of methanogens, we studied the EMIC methanogenic strains isolated from marine sediments (Methanobacterium-affiliated strain IM1) and crude oil tanks (Methanococcus maripaludis Mic1c10), in an in-house developed flow-through cell to simulate a fluctuating environment. A co-culture of M. maripaludis and D. alaskensis was also established to study the effect of syntrophic growth on metal corrosion that may occur in industrial pipelines. Results indicate that the rates of iron corrosion due to coupled methanogenesis (up to 0.4 mm/yr) are higher to that caused by the marine SRM Desulfovibrio alaskensis (0.15 mm/yr). Surface analyses of the metal showed severe pitting with high methane production. Genomic analysis of the EMIC methanogen M. maripaludis Mic1c10 will provide an insight on the mechanisms of MIC. Such knowledge and deeper understanding also from an electrokinetic point of view may not only provide further models in microbial electrophysiology, but also contribute to mitigation strategies in MIC.
Leaching processes are responsible for the release of biocides from treated materials into the environment.
Adequate modeling of emission processes is required in order to predict emission values in the framework of the risk assessment of biocidal products intended for long-term service life. Regression approaches have been applied to data obtained from the long-term monitoring of biocide emissions in experiments involving semi-field conditions. Due to the complex interaction of different underlying mechanisms such as water and biocide diffusion and desorption, however, these attempts have proven to be of limited usefulness e at least, for the available biocide emission data. It seems that the behavior of the biocide emission curve depends to a considerable extent on whichever underlying mechanism is slowest at a given point in time, thus limiting the amount of biocide available for release. Building on results obtained in the past few years, the authors propose a criterion for determining which mechanism controls the leaching process at a given point in time based on the slope of the log-log emission curve. In addition, a first-order approximation of this slope value is presented which displays advantages both in terms of computability and interpretability. Finally, an algorithm for the determination of breakpoints in the slope of the log-log emission curve is presented for the demarcation of phases within which one mechanism acts as a limiting factor.
Microbiologically influenced corrosion (MIC) of iron is usually attributed to sulfate-reducing microorganisms (SRM) in offshore industries, such as the oil and gas pipelines, due to the high concentrations of sulfate in the seawater. SRM act upon the metal by the reactiveness of hydrogen sulfide (HS-), and by withdrawal of the available electrons (Fe --> Fe2+ + 2e-; E° = -0.47 V) in electrical contact with the metal (EMIC). However, methanogenic archaea can also cause MIC. Because they do not produce HS-, withdrawal of electrons may be their main corrosive mechanism; however, mechanistic details and kinetics of the overall process are poorly understood.
To investigate the corrosion potential of methanogens, we studied the EMIC methanogenic strains isolated from marine sediments (Methanobacterium-affiliated strain IM1) and crude oil tanks (Methanococcus maripaludis Mic1c10), in an in-house developed flow-through cell to simulate a fluctuating environment. A co-culture of M. maripaludis and D. alaskensis was also established to study the effect of syntrophic growth on metal corrosion that may occur in industrial pipelines. Results indicate that the rates of iron corrosion due to coupled methanogenesis (up to 0.4 mm/yr) are higher to that caused by the marine SRM Desulfovibrio alaskensis (0.15 mm/yr). Surface analyses of the metal showed severe pitting with high methane production. Genomic analysis of the EMIC methanogen M. maripaludis Mic1c10 will provide an insight on the mechanisms of MIC. Such knowledge and deeper understanding also from an electrokinetic point of view may not only provide further models in microbial electrophysiology, but also contribute to mitigation strategies in MIC.zeige weniger
The objective of this study is to find out, to what extent the geochemical characteristics of lapis lazuli can be utilized in respect to its provenance. A wide range of variables is taken into consideration depending on the quantity of samples analysed from a specific geological region and the methods applied. In order to provide evidence, a multi-technique analytical approach using µXRF, ESEM, PGAA and PIXE is applied to samples from the most famous deposits of lapis lazuli. Special elements determined as fingerprints are compared in relation to the forming conditions obvious in textural features. The results and statistical output allow a differentiation that enables an optimized local classification of the blue stone. An absolute requirement for all geo-tracing performed on blue colored cultural objects of unknown provenance is awareness of the limits of analysis. The possible sources of lapis lazuli are tested by analysing the blue pigment used as paint on murals and ink on manuscripts from the Silk Road.
Textile floor coverings are often used in offices and residential buildings. Large areas like meeting rooms, cinemas, theaters and hotels are often equipped with such coverings. They contribute to the comfort of the users as they provide high pedaling comfort and sound absorption. The weakness of these building materials is due to the odor emission that is released from the floor covering, which affects the comfort of the users. A bad air quality and the resulting dissatisfaction can lead to lower employee productivity. The research project of the Hochschule für Technik und Wirtschaft Berlin (HTW-Berlin) is promoted by the German Environment Agency (UBA). The project has the following title: Low-emission and low-odor building products for energy-efficient buildings - Development of requirements and concepts for the Blue Angel from a climate protection perspective; investigates the emission and odor behavior of textile floor coverings (Emissions- und geruchsarme Bauprodukte für energieeffiziente Gebäude - Entwicklung von Anforderungen und Konzepten für den Blauen Engel aus Klimaschutzsicht; untersucht das Emissions- und Geruchsverhalten textiler Bodenbeläge).
The chemical emissions from products are tested by means of emission test chambers under defined conditions (climate, loading, air change rate). The standard method for the determination of volatile organic compounds (VOC) is the sampling onto Tenax-tubes followed by thermal desorption (TDS) and gas chromatography-mass spectrometry (GC-MS) analysis. The EU-LCI list includes some very volatile organic compounds (VVOC) and some VOC for which there are limitations when using the standard method. For VVOC additional sampling is required using stronger absorbers like Carbotrap or multi-bed adsorption tubes. The analysis of VVOC also requires a different GC oven program and a different column for the separation. For the determination of formaldehyde and other low boiling aldehydes (e.g. acetaldehyde, acetone, propanal, propenal) DNPH-cartridges are used which are extracted with acetonitrile followed by liquid chromatography (HPLC-UV) analysis. The derivatisation of propenal and other unsaturated aldehydes (e.g. 2-butenal) with DNPH might lead to lower findings due to incomplete derivatization and forming of by-products. For a better quantification of acetic acid the use of ion chromatography (IC) is recommended because the analysis of acetic acid with the standard method (TDS) leads to lower findings due to break through during sampling. The use of ion chromatography for the analysis of organic acids requires a third sampling technique. The acids are adsorbed onto silica-gel and extracted with water.
EN 16516 sets the test method and requirements for the determination of emissions of Volatile Organic Compounds (VOCs) from building materials into indoor air. To address the quality control requirements for the class of semi-VOCs (SVOCs), VSL developed gaseous reference materials. A novel home-made dynamic gas mixture preparation system, operating according to ISO 6145-4 (continuous injection method), has recently been developed and validated. Thanks to the stable temperature control up to 100 oC, the system can prevent condensation of the SVOCs in air at indoor air concentration levels. The in-situ obtained SVOC gas standards can be sampled in sorbent tubes to obtain SVOC transfer standards. A study was performed to determine the optimal sorbent material and storage conditions. This study will be presented together with the results of the 2018 Round Robin test for emission test chamber measurements organised by BAM. Using the novel system, VSL prepared transfer standards with known amounts of VOCs and SVOCs for participants to evaluate their analytical performance.
A reliable analysis of aerosol particle is curial for enforcing EU air quality regulations to protect human health, and for research on climate change effects [1]. Although metrics such as PM10 and PM2.5 are currently in use, the level of uncertainty of aerosol metrics is too high and the traceability is insufficient. Within the AEROMET project [2] procedures are developed aiming at reducing the uncertainties of particle mass, size, and number concentration measurements including the characterization of regulated components in airborne particles. Here, we present an approach how to improve the uncertainties of the particle mass by mobile total reflection x-ray fluorescence (TXRF) analysis. The combination of TXRF and aerosols sampling techniques supported by reference-free synchrotron radiation-based XRF enables a quantitative real-time analysis of particle mass. During in-field campaigns, the procedure was tested, monitoring the size dependent mass concentrations of specific elements in ambient aerosols under dynamic conditions. This approach allows a direct time and size-resolved analysis without laborious digestion steps and a reduced risk of contamination.
Aerosol particles were sampled in a 13-stage DLPI impactor on acrylic discs. TXRF analysis was performed on-site with the transportable spectrometer S2 PICOFOX (Bruker Nano GmbH). The TXRF quantification was based on internal standardization. At moderate air pollution levels (PM10 20 µg/m³) sampling times of less than 2 hours were enough to detect elements in different particle size bins. The on-site approach and the high sensitivity of TXRF enables the observation of rather quick changes in the quantity and distribution of elements in an ambient aerosol on the day of sampling. The analysis of the morning and afternoon sampling shifts reveals the occurrence of the elements Fe, Ca and Si in different size bins as well as their temporal change in respective mass concentrations over the day while the distributions of several other elements remain unchanged.
Museums worldwide are equipped with different display cases. Exhibit display cases should protect cultural objects from dust as well as from mechanical and physical damage. To ensure a stable climate inside the display cases, a low air exchange rate is maintained. Typically air exchange rates are often smaller than 0.1 d 1, which can result in rising concentrations of potential harmful immissions inside of the display cases due to emissions from materials. Especially high concentrations of organic acids, which can emit from e.g. sealing materials, can produce damage of cultural objects. In 2012 BAM introduced a procedure witch is called: BEMMA-Scheme (Bewertung von Emissionen aus Materialien für Museumsausstattungen) which stands for: “Assessment of Emissions from Materials for Museum Equipment”. Micro chambers are used for VOC emission tests of display case construction materials, e.g. textiles, plastics, sealing material, coatings and others. Each sampling procedure is carried out in duplicate. Emissions like formic acid, acetic acid, formaldehyde and oximes are excluded and the sum of emissions of VVOCs, VOCs and SVOCs is limited. For a positive assessment all listed criteria must be fulfilled; otherwise the display construction material fails the BEMMA scheme. The BEMMA scheme is not a guarantee for an emission free display case, but a necessary requirement for the choice of suitable materials for emission and immission reduced display cases.
Environmental sustainability and –stability of materials concerning the migration of pollutants
(2019)
In addition to previously reported results on the accelerated weathering of polystyrene samples (PS) containing 1 wt. % hexabromocyclododecane (HBCD), we present the first results of our investigations of polypropylene samples (PP) containing 0.1 wt. % BDE-209. All studied polymer samples were exposed to a defined weathering schedule in a climate chamber in accordance to regulation EN ISO 4892-3:2006. For the determination of BDE-209 in the collected raining water the samples were prepared in accordance to a validated protocol. Before the analyses each sample was spiked with isotopically labeled BDE-209. Subsequently the samples were extracted with isooctane. The obtained extracts were concentrated, and the resulting solutions were analyzed by GC/MS. Additionally, the total bromine content was monitored for the weathered and untreated samples using laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) and X-ray fluorescence analysis (XRF) as a non-destructive and rapid method. In general, the resulting data from the accelerated weathering will be compared to those from the natural weathering experiments. Here, the surfaces of the test pieces were analyzed by LA-ICP-MS and XRF as well. Moreover, soil bed tests were conducted in a well characterized model soil. This soil (boulder-clay, sand with 12 % loam, particle size in total 0.2-4 mm) is filled in a free-draining basin inside of an air-conditioned cellar. In this manner, TOC, water capacity and humidity are recorded parameters. To induce a leaching process from the samples by the raining water, the target water content is calculated to 8%. The actual humidity is measured by a tensiometer, assuring the duration of the raining period. A defined humidity is a fundamental parameter for a biological activity in the soil. The test polymers were placed up to the half in the soil. Microbial activity of the soil is monitored by a reference polymer (polyurethane) and should induce the release of HBCD and BDE-209 out of the test materials. These released analytes will be captured by passive samplers (silicone tubes) placed in a distinct distance to the polymer samples in the soil. The soil bed experiments are complementary to the weathering experiments due to the biological activity in the soil.
Copper-based fungicides are routinely used for wood and plant protection, which can lead to an enrichment of copper-tolerant microbial communities in soil. To investigate the effect of such wood preservatives on the soil fungal and bacterial community compositions, five different vineyard and fruit-growing soil environments were evaluated using incubation studies over time. Pine sapwood specimens were impregnated with either water or different biocide treatment solutions containing a mixture of copper, triazoles, and quaternary ammonium compounds (CuTriQAC), a mixture of triazoles and quaternary ammonium compounds (TriQAC), or copper alone (Cu). Specimens were incubated in soil from each sample site for 8, 16, 24, and 32 weeks. The effects of preservative treatment on the modulus of elasticity (MOE) of the wood specimens and on the soil fungal as well as bacterial community composition at the soil-wood interface were assessed by quantitative PCR and amplicon sequencing of the fungal internal transcribed spacer (ITS) region and bacterial 16S rRNA gene. Specimens impregnated with CuTriQAC and Cu showed decreased MOE and reduced fungal and bacterial copy numbers over time compared to those impregnated with water and TriQAC. Fungal but not bacterial community composition was significantly affected by wood preservative treatment. The relative abundance of members of the family Trichocomaceae compared to other genera increased in the presence of the Cu and CuTriQAC treatments at three sites, suggesting these to be Cu-tolerant fungi. In conclusion, the copper-containing treatments resulted in marginally increased MOE, lowered microbial gene copy numbers compared to those in the TriQAC and water treatments, and thus enhanced wood protection against soil microbial wood degradation
In arid sun-exposed environments, rock-inhabiting fungi dominate microbial communities on rock surfaces. Fungi are known to enhance rock weathering, although the exact mechanisms are not completely understood. These natural processes can be simulated by reproducible experimental systems. Particularly attractive are genetically amenable rock-weathering fungi, where certain traits can be knocked-out to identify weathering-relevant functions. Here, we studied the effect of the rock-inhabiting fungus Knufia petricola A95 and its melanin-deficient mutant (ΔKppks) on the weathering of a Fe-bearing olivine. We examined the olivine dissolution kinetics and looked at the olivine-fungus interface. For the former we inoculated olivine powder with the fungus in batch and mixed flow reactors and analysed the medium solution by ICP-OES, for the latter biofilm-covered olivine sections from flow-through reactors were analysed by FIB-TEM.
In general olivine dissolution was lower when olivine was incubated without fungi: the abiotic olivine dissolution rates were lowest and the surface of the abiotically reacted olivine sections showed no etching. Various chemical analyses show Fe (oxyhydr)oxide precipitation on top of the abiotically reacted olivine, indicating its role in inhibiting olivine dissolution. Both the wild type (WT) and ΔKppks solubilised and bound significant amounts of Fe released by olivine dissolution. Fe (oxyhydr)oxide precipitation was thus reduced, explaining the enhanced olivine dissolution in their presence. Particularly efficient at preventing Fe precipitation were attached WT cells: the inhibition of olivine dissolution almost completely disappeared. The attachment capacity of the WT is most likely caused by the production of WT-specific extracellular polymeric substances. Our presented experimental systems allowed the precipitation of Fe (oxyhydr)oxides and included a rock-inhabiting fungus and thus simulated weathering processes relevant to natural ecosystems.
Laser Powder Bed Fusion (L-PBF) is a promising additive manufacturing (AM) technology for metal part production especially for complex and lightweight structures or functional designs. In L PBF processes several by-products including welding plume and its condensates, spatter and ejected powder are generated during laser exposure. Investigations of micro- and nano-sized by-products have received little attention in literature. This study focuses on the analysis of particle emissions in L PBF of 316L stainless steel using a scattered light aerosol spectrometer and a fast mobility particle sizer spectrometer during the process which allows for in-situ analysis of particle sizes in the range of 6 nm to 100 µm. A distinct correlation of emission signals to part position can be revealed. In addition, a significant influence of laser scanning vector directions on emission signals is presented. Furthermore, differing powder layer thicknesses can be recognised by deviations in emission signals.
Laser Powder Bed Fusion (L-PBF) is a promising additive manufacturing (AM) technology for metal part production especially for complex and lightweight structures or functional designs. In L PBF processes several by-products including welding plume and its condensates, spatter and ejected powder are generated during laser exposure. Investigations of micro- and nano-sized by-products have received little attention in literature. This study focuses on the analysis of particle emissions in L PBF of 316L stainless steel using a scattered light aerosol spectrometer and a fast mobility particle sizer spectrometer during the process which allows for in-situ analysis of particle sizes in the range of 6 nm to 100 µm. A distinct correlation of emission signals to part position can be revealed. In addition, a significant influence of laser scanning vector directions on emission signals is presented. Furthermore, differing powder layer thicknesses can be recognised by deviations in emission signals.
The project ProMoAM is presented. The goal of the project is to evaluate which NDT techniques or combination of techniques is suited for in-situ quality assurance in additive manufacturing of metals. To this end, also 3d-data fusion and visualization techniques are applied. Additional ex-situ NDT-techniques are used as references for defect detection and quantification. Feasability studies for NDT-techniques that are presently not applicable for in-situ use are performed as well.
The presentation gives a brief overview of the whole project and the different involved NDT-techniques.
Since it was realized that sites contaminated with metals and radionuclides needed treatment, various remediation methods have been and are being developed. Depending on the size of the contaminated site and urgency of intervention, conventional or recently introduced techniques have been used. Conventional techniques include excavation and removal for treatment of soil and contaminants, or the so-called “pump-and-treat” method, in which contaminated groundwater is removed from the ground by pumping and treated in a treatment plant on the surface. It has the advantage of using proven techniques and is easy to control, and the treated groundwater can be reinjected into the ground or discharged in rivers or lakes. Novel methods include permeable reactive barriers, biomineralization, and electrokinetic remediation.
Waste, Evaluation Methods
(2019)
It is difficult to decide which waste management system fulfills best predefined sustainability goals such as maximum materials and energy recovery, least environmental impact, and lowest societal cost. Such decisions are based on many parameters. However, several evaluation methods exist with different scope and output, and the application of one or more evaluation methods provides an objective comparison of alternatives.
Measurements of aerosol particles are vital for enforcing EU air quality regulations to protect human health, and for research on climate change effects. Although metrics such as PM10 and PM2.5 are currently in use, the level of uncertainty of aerosol metrics is too high and the traceability is insufficient. The project AEROMET, which has been started in June 2017 aims at implementing improvements in a) the uncertainty of particle mass, size and number concentration measurements and b) in the characterization of regulated components in airborne particles. Both are demanded by existing networks within the EU as well as by global atmospheric research.
On-site measurement campaigns
One of the objects is the application of mobile x-ray spectroscopy techniques combined with aerosol sampling techniques for quantifying particle compositions in the field for real time analysis. During two in-field measurement campaigns in Budapest, Hungary in May 2018 and Cassino, Italy in September 2018 the size dependent mass concentrations of specific elements in ambient aerosols were monitored under dynamic conditions.
Typically, airborne particles are sampled on filter substrates. During this project new sampling methods with specially designed substrate holders for an in-situ TXRF analysis were developed and applied for the first time. This approach allows a direct time and size resolved analysis without laborious digestion steps and a reduced risk of contamination.
Aerosol particles were sampled in a 13-stage DLPI impactor - size range from 0,03 µm to 10 µm - which was equipped with special adapters for acrylic discs of 30 mm diameter, serving as substrates. TXRF analysis was performed on site with the transportable spectrometer S2 PICOFOX (Bruker Nano GmbH) equipped with a Mo X-ray tube and a 30 mm² Silicon Drift Detector (SDD). Excitation conditions were 50 kV, 600 µA, measurement time 1000 s. Quantification was based on internal standardization using 50 ng of Y in solution, which was pipetted into the centre of the discs prior to sampling.
At moderate air pollution levels, i.e. PM10 ~ 20 µg/m³, sampling times of less than 2 hours were enough for the detection of elements in different particle size bins. The in-situ approach and the high sensitivity of TXRF enables the observation of rather quick changes in the quantity and distribution of elements in an ambient aerosol on the day of sampling, as the below example from the Cassino field campaign on 11 Sept. 2018 shows: The analysis of the morning and afternoon sampling shifts reveals the occurrence of the elements Fe, Ca and Si in different size bins as well as their significant temporal change in respective mass concentrations over the day while the distributions of several other elements in the aerosol remain unchanged.
The validation of these results by backup measurements is planned.
Traceable and reliable chemical element analysis of aerosols by X-ray spectrometry was investigated using aerosol samples from field campaigns which have been measured in the GIXRF-beamline at BESSY.
The reference-free XRF approach allows for a traceable analysis of the mass deposition.
Traceable quantification by means of XRF can be transfered to benchtop instrumentation used in the laboratory Chemical and dimensional analysis of deposited aerosol allows for a comprehensive analysis of aerosols, e.g. for toxicity assessment and determination of the source The folowing elements could be identified and quantified in the field samples: Al, Si, P, S, Cl, K, Ca, Ti, Cr, Mn, Fe, Ni, Cu, Zn, As, Se, Br, Rb, Sr, Y, W, Pb.
Simple particulate matter sensors are gaining popularity due to their low price, easy handling and good temporal resolution. In this presentation, we report on the performance of a Sharp optical PM sensor GP2Y1010AU0F, which costs less than 15 €.
The sensor is built around an infrared emitting diode (ILED) and a phototransistor detecting the light scattered from the aerosol particle. An electronic circuit shapes the detected light in a pulsed signal. The manufacturer advises sampling the output signal 280 microseconds after the ILED pulse. The measured output voltage is an indicator of dust concentration.
We have built two identical simple PM monitoring devices using Raspberry Pi 3 computer interfacing the PM sensor with Microchip’s MCP3002 ADC via SPI. The ADC is capable of more than 100 ksamples/s at 10-bit resolution. The Rpi3 was pulsing the sensor at 10Hz, digitizing and saving the data and sending the results wirelessly.
Sensor’s output pulse shape was sampled with 10 microsecond time steps and saved, thus making offline analysis possible. A time jitter of output pulses can be observed and suggests a peak fitting as a better approach to the signal readout compared to the single sampling at a fixed time after pulse triggering We compared both methods.
A mobile Bruker S2 Picofox TXRF spectrometer has been used in two field campaigns within the EMPIR env07 AEROMET project for the on-site analysis of cascade impactor aerosol samples.The results show that even at moderate air pollution levels – i.e.PM10 fairly below 20 μg/m³ - element mass concentrations in air in the range of 100 pg/m³could be measured in up to 13 size bins after sampling times of less than only 0.5 days.
In this study, femtosecond laser-induced sub-micrometer structures are generated to modify polyethylene (PE) surface topographies. These surfaces were subjected to bacterial colonization studies with Escherichia coli and Staphylococcus aureus as test strains. The results reveal that the nanostructures do not influence S. aureus coverage, while the adhesion of E. coli is reduced.
Question:
Biocides are in contact with soils through direct application and passive leaching from protected materials. Consequently, soil microorganisms are exposed to toxic biocides even though they are not their primary targets. Soil is a large reservoir of microbial diversity and has been hypothesized to be a crucial factor for the evolution and spread of antimicrobial resistance. Currently, there is little knowledge on how biocides used to protect materials affect the evolution and spread of resistance. Thus, our aim is to investigate the risk for the evolution of biocide resistance and cross-resistance to antibiotics. In addition, we aim to elucidate the affect of biocides on the spread of resistance via horizontal gene transfer (HGT).
Methods:
In laboratory evolution experiments we culture selected model soil microorganism with representative biocides followed by antibiotic cross-resistance determination and genome sequencing. Moreover, we investigate if the selected biocides affect the HGT frequency of plasmids that carry resistance genes among soil microorganism and the consequences for survival of the affected populations.
Results:
Our initial results show only small increases of biocide resistance during serial transfers in the presence of biocides. One reason for this might be the narrow selective window for biocide resistance due to steep dose-response relationships. Furthermore, we will present results from ongoing experiments on the effects of material preservatives on HGT frequencies facilitating microbial community adaptation to stress.
Conclusions:
The results will enable future risk assessment regarding resistance evolution for biocides used as material preservatives.
Question:
Biocides are in contact with soils through direct application and passive leaching from protected materials. Consequently, soil microorganisms are exposed to toxic biocides even though they are not their primary targets. Soil is a large reservoir of microbial diversity and has been hypothesized to be a crucial factor for the evolution and spread of antimicrobial resistance. Currently, there is little knowledge on how biocides used to protect materials affect the evolution and spread of resistance. Thus, our aim is to investigate the risk for the evolution of biocide resistance and cross-resistance to antibiotics. In addition, we aim to elucidate the affect of biocides on the spread of resistance via horizontal gene transfer (HGT).
Methods:
In laboratory evolution experiments we culture selected model soil microorganism with representative biocides followed by antibiotic cross-resistance determination and genome sequencing. Moreover, we investigate if the selected biocides affect the HGT frequency of plasmids that carry resistance genes among soil microorganism and the consequences for survival of the affected populations.
Results:
Our initial results show only small increases of biocide resistance during serial transfers in the presence of biocides. One reason for this might be the narrow selective window for biocide resistance due to steep dose-response relationships. Furthermore, we will present results from ongoing experiments on the effects of material preservatives on HGT frequencies facilitating microbial community adaptation to stress.
Conclusions:
The results will enable future risk assessment regarding resistance evolution for biocides used as material preservatives.
Introduction: Biofilms are regarded as a common cause of chronic infections on medical devices. Preventive and therapeutic strategies against biofilm infections commonly involve applications of multiple antimicrobial substances: antimicrobial coatings on the implanted biomaterials in combination with systemically administered antibiotics. While this practice of combination therapy harbours the risk of developing cross-resistance, it might also provide the possibility to implement specific antimicrobial-antibiotic combinations (AACs) that can slow down the selection of antibiotic resistant strains.
Hypothesis and aims: Specific AACs can exert combinatorial effects on the growth of susceptible and antibiotic-resistant Pseudomonas aeruginosa that either suppress or increase their individual effects.
Our aim is to identify AACs with antagonistic or synergistic effects on pseudomonal biofilms and to understand their impact on selection of resistant strains. Specifically, we want to identify AACs that select for and against antibiotic resistance during biofilm formation.
Methodology: We screened for AACs that cause antagonistic or synergistic effects on planktonic P. aeruginosa.
To study the effect of antimicrobial-antibiotic exposure on resistance selection in bacterial biofilms, we will grow resistant and sensitive strains on PDMS surfaces with and without antimicrobial coatings and expose them to antibiotics.
Results: Several combinations with synergistic or antagonistic interaction on the growth rate of P. aeruginosa were detected. We observed a strong antagonism when combining the antimicrobial substance chlorhexidine with the carbapenem drug meropenem. A meropenem-resistant mutant showed a selection advantage in low concentrations of chlorhexidine combined with a sub-inhibitory concentration of meropenem over the wild-type. No antagonistic effect was observed for the same combination when E. coli was exposed to chlorhexidine and meropenem, suggesting a non-chemical basis for the observed effect on P. aeruginosa.
Conclusion: Gaining a better understanding about resistance selection during biofilm formation on biomedical surfaces will enable us to mitigate against biofilm-associated antimicrobial resistance.
Question:
One cornerstone to prevent the spread of bacteria in clinical and industrial settings is the application of biocides including disinfectants and preservatives. However, bacteria can evolve resistance to biocides, which in turn can confer cross-resistance to antibiotics. Additionally, clonal bacterial populations can display phenotypic heterogeneity with respect to the tolerance of antibiotic stress leading to a prolonged survival of a sub-population; this phenomenon is termed persistence. Persistence to antibiotics is an evolvable trait and can serve as a stepping stone for the evolution of genetically encoded resistance. Until now, there is a lack of systematic studies that investigate if bacterial populations establish persister subpopulations that tolerate disinfectant stress longer than most of the population. Our objective is to investigate if persistence is a bacterial survival strategy against biocides. Furthermore, we investigate the mechanisms of biocide persistence and if persistence can evolve in the face of fluctuating exposure to biocides. Lastly, we test if the evolved mechanisms of biocide tolerance lead to biocide resistance and if they confer cross-tolerance and cross-resistance against antibiotics.
Methods:
We use time-kill assays in the presence of biocides and experimental evolution combined to whole-genome sequencing in the model organism E. coli.
Results:
We find persister sub-populations against chlorhexidine and quaternary ammonium compounds, but not to alcohols, aldehydes and oxidative compounds. We will present the relationship of mechanisms known to underlie antibiotic persister formation to the formation of persisters against biocides. In addition, we will present data from an ongoing evolution experiment for persistence against biocides.
Conclusion
There is a link between antibiotic and biocide persistence with possible implications for antibiotic resistance evolution and spread.
Microorganisms perform multiple metabolic functions that shape the global cycling of elements, health and disease of their host organisms, and biotechnological processes. The rates, at which different metabolic activities are performed by individual cells, can vary between genetically identical cells within clonal populations. While the molecular mechanisms that result in such metabolic heterogeneity have attracted considerable interest, the environmental conditions that shape heterogeneity and its consequences have received attention only in recent years. Here, we review the environmental drivers that lead to metabolic heterogeneity with a focus on nutrient limitation, temporal fluctuations and spatial structure, and the functional consequences of such heterogeneity. We highlight studies using single-cell methods that allow direct investigation of metabolic heterogeneity and discuss the relevance of metabolic heterogeneity in complex microbial communities.
Questions:
Biocides are in contact with soils through direct application and passive leaching from protected materials. Consequently, soil microorganisms are exposed to toxic biocides even though they are not their primary targets. Soil is a large reservoir of microbial diversity and has been hypothesized to be a crucial factor for the evolution and spread of antimicrobial resistance. Currently, there is little knowledge on how biocides used to protect materials affect the evolution and spread of resistance. Thus, our aim is to investigate the risk for the evolution of biocide resistance and cross-resistance to antibiotics. In addition, we aim to elucidate the affect of biocides on the spread of resistance via horizontal gene transfer (HGT).
Methods:
In laboratory evolution experiments we culture selected model soil microorganism with representative biocides followed by antibiotic cross-resistance determination and genome sequencing. Moreover, we investigate if the selected biocides affect the HGT frequency of plasmids that carry resistance genes among soil microorganism and the consequences for survival of the affected populations.
Results:
Our initial results show only small increases of biocide resistance during serial transfers in the presence of biocides. One reason for this might be the narrow selective window for biocide resistance due to steep dose-response relationships. Furthermore, we will present results from ongoing experiments on the effects of material preservatives on HGT frequencies facilitating microbial community adaptation to stress.
Conclusions:
The results will enable future risk assessment regarding resistance evolution for biocides used as material preservatives.
A fundamental question in ecology is how biodiversity affects ecosystem function. Biodiversity is commonly estimated based on genetic variation. We investigated a new approach that defines and measures biodiversity in complex microbial communities. We used the variation in multiple functionally-relevant, phenotypic traits measured in parallel in single cells as a metric for microbial phenotypic diversity. We studied phenotypic diversity and ecosystem functioning throughout different photosynthetic layers dominated by divergent microbial communities in the gradient of Lago di Cadagno. We determined genetic diversity by 16S and 18S amplicon sequencing and bulk ecosystem functioning (photosynthesis). In addition, we determined phenotypic diversity using single-cell technologies such as nanometer-scale secondary ion mass spectrometry (NanoSIMS) correlated with confocal laser scanning microscopy (CLSM) and scanning flow-cytometry. We measured functional trait variation between individuals in 13CO2 fixation, 15NH4+ uptake, and variation in physio-morphological cell traits, such as cell size, shape, and auto-fluorescence for various pigments related to photosynthesis. We used the distances between individuals in a multidimensional trait space to derive phenotypic trait-based diversity indices, such as trait richness, trait evenness, and trait divergence. We find that phenotypic trait divergence associates with ecosystem functioning, whereas genetic diversity does not. Including activity-based, single-cell phenotypic measurements with NanoSIMS provided an additional accuracy to the trait-based diversity assessment and allowed us to formulate hypotheses on the mechanisms that shape the correlation between phenotypic diversity and eco-system function. Together, our results show that phenotypic diversity is a meaningful concept to measure microbial biodiversity and associate it with ecosystem functioning.
Microbiologically induced corrosion due to bacterial biofilms causes several problems in industrial systems, technical applications and in medicine. Prior to the formation of a biofilm on a substrate, planktonic cells attach on the surface. Hence, the properties of the surface play a key role in biofilm formation and are of great importance for the development of strategies to prevent bacterial attachment and biofilm formation.
This project aims at clarifying to which extent surface micro-/nanostructuring and chemical functionalization affects bacterial attachment and whether a synergistic combination of the two can be used to control bacterial adhesion. To answer these questions, model surfaces with regular patterns of 5-10 micrometers in size have been prepared, which provide distinct zones differing in terms of their chemistry or nano-roughness. This was achieved by micro contact printing of self-assembled monolayers with different functional groups and deposition of patterned ZnO nanorod arrays for studying the effect of surface chemistry and morphology, respectively. Typical contrasts studied were combinations of positively/negatively charged, hydrophobic/hydrophilic or flat/rough.
The attachment behavior of bacteria on tailored surfaces were studied in a flow chamber as a function of time. The strain Pseudomonas fluorescens SBW25 was chosen as a model organism. DNA-intercalating dyes such as Syto9 have a high affinity to adsorb on ZnO nanorods. To overcome this limitation a genetic modification was performed by introducing a gene which expresses a green fluorescent protein in P. fluorescens SBW25 enabling the quantitative evaluation of the flow chamber studies by means of fluorescence microscopy. Further analysis of the attachment behavior was performed by means of scanning electron microscopy.
The presentation will summarize the results of our systematic study on the role of individual parameters on bacterial attachment and highlight synergistic combinations, showing an inhibition or enhancing effect. As the investigations with model substrates enable a precise control of the surface parameters, this approach can be applied to different microorganisms and material systems to achieve a correlative description of bacterial adhesion on solid surfaces.
A fundamental question in ecology is how biodiversity affects ecosystem function. Biodiversity is commonly estimated based on genetic variation. We investigated a new approach that defines and measures biodiversity in complex microbial communities. We used the variation in multiple functionally-relevant, phenotypic traits measured in parallel in single cells as a metric for microbial phenotypic diversity. We studied phenotypic diversity and ecosystem functioning throughout different photosynthetic layers dominated by divergent microbial communities in the gradient of Lago di Cadagno. We determined genetic diversity by 16S and 18S amplicon sequencing and bulk ecosystem functioning (photosynthesis). In addition, we determined phenotypic diversity using single-cell technologies such as nanometer-scale secondary ion mass spectrometry (NanoSIMS) correlated with confocal laser scanning microscopy (CLSM) and scanning flow-cytometry. We measured functional trait variation between individuals in 13CO2 fixation, 15NH4+ uptake, and variation in physio-morphological cell traits, such as cell size, shape, and auto-fluorescence for various pigments related to photosynthesis. We used the distances between individuals in a multidimensional trait space to derive phenotypic trait-based diversity indices, such as trait richness, trait evenness, and trait divergence. We find that phenotypic trait divergence associates with ecosystem functioning, whereas genetic diversity does not. Including activity-based, single-cell phenotypic measurements with NanoSIMS provided an additional accuracy to the trait-based diversity assessment and allowed us to formulate hypotheses on the mechanisms that shape the correlation between phenotypic diversity and eco-system function. Together, our results show that phenotypic diversity is a meaningful concept to measure microbial biodiversity and associate it with ecosystem functioning.
Background:
One cornerstone to prevent the spread of antibiotic resistant bacteria in clinical settings is the application of disinfectants. However, bacteria can evolve resistance to disinfectants, which in turn can confer cross-resistance to antibiotics. Additionally, clonal bacterial populations can display phenotypic heterogeneity with respect to the tolerance of antibiotic stress leading to a prolonged survival of a sub-population; this phenomenon is termed persistence. Persistence to antibiotics is an evolvable trait and can serve as a stepping stone for the evolution of genetically encoded resistance. Until now, there is a lack of systematic studies that investigate if bacterial populations establish persister subpopulations that tolerate disinfectant stress longer than most of the population.
Objectives:
Our objective is to investigate if persistence is a bacterial survival strategy against disinfectants. Furthermore, we investigate the mechanisms of disinfectant persistence and if persistence can evolve in the face of fluctuating exposure to disinfectants. Lastly, we test if the evolved mechanisms of disinfectant tolerance lead to disinfectant resistance and if they confer cross-tolerance and cross-resistance against antibiotics.
Methods:
We use time-kill assays in the presence of disinfectants and experimental evolution combined to whole-genome sequencing in the model organism E. coli.
Results:
We find persister sub-populations against chlorhexidine and quaternary ammonium compounds, but not to alcohols, aldehydes and oxidative compounds. We will present the relationship of mechanisms known to underlie antibiotic persister formation to the formation of persisters against disinfectants. In addition, we will present data from an ongoing evolution experiment for persistence against disinfectants.
The global rise of antibiotic resistance has made the proper use of disinfectants more important than ever. Their application in clinical l settings is an integral part of antibiotics stewardship by preventing the occurrence and spread of infections. However, improper use of disinfectants also harbours the risk for the evolution of tolerance and resistance to disinfectants, but also to antibiotics. It is therefore crucial to understand whether and how bacteria can survive chemical disinfection and which conditions facilitate the evolution of tolerance and resistance.
Here, we study the heterogeneity in the response of isogenic E. coli populations exposed to different levels of commonly used disinfectants. At concentrations below the minimal inhibitory concentration (MIC), we find that certain disinfectants induce prolonged lag times in individual cells, a phenotype that has been associated with persistence against antibiotics. At concentrations above the MIC, we find heterogeneous killing for a range of the tested substances. Interestingly, for the three cationic surfactants that were tested, we find kill kinetics revealing the presence of a tolerant subpopulation that can withstand disinfection longer than most of the population. We will present results from an ongoing evolution experiment in which we test the potential for evolution of population-wide tolerance and resistance through intermittent exposure to lethal doses of a cationic surfactant.
Specific co-fertilization of nutrients can enhance their plant-availability and thus the yield of plants. To investigate this effect, we performed a pot experiment with three different P-fertilizers and ammonium nitrate sulfate as a co-fertilizer, without and with a nitrification inhibitor (NI), and analyzed the form of nitrogen (N) in the soil via novel X-ray spectroscopic method. The application of NI with the N fertilizer led to a higher dry matter yield of maize. Novel N K-edge micro-X-ray absorption near-edge structure (micro-XANES) spectroscopy identified that the application of a NI promotes the temporary formation of a non-exchangeable N in detectable hot-spots in the soil. The subsequent slow release and prolonged availability of N during plant growth leads to higher yield. It can be concluded that NIs lead to a temporary fixation of ammonium-N in a pool that can be accessed by plant roots. Those types of available nutrient pools meet the idea of so-called “next generation fertilizers” as plants have access to nutrients according to their current demand.
Phosphorus (P) is an essential element for all life forms, and P-availability thus an important driver of a functioning agriculture. However, phosphate rock resources for P-fertilizer production are only available in a few countries. Therefore, P-recovery from waste materials has become of increasing interest during the last decade and has been investigated worldwide. In order to characterize potential novel P-fertilizers made from recycled materials, a large array of P-compound characterizations, chemical extractions and growth experiments were performed. This review bundles the work carried out in that field over the last years. Overall, P-fertilizers from recycled materials show a broad range of P-compounds with very different chemical structure and solubility. Growth experiments performed to assess their fertilizing effects display high variations for most of the products. While these experiments have demonstrated that some fertilizers made of recycled materials may reach P effects in the same order of magnitude as water-soluble phosphate rock-based fertilizers, an important limitation in their interpretation is the fact that they often vary considerably in their experimental design. The existing data show clearly that standardization of growth experiments is urgently needed to achieve comparable results. Standard chemical extractants used to assess the chemical solubility of P-fertilizers were found to be of limited reliability for predicting plant P uptake. Therefore, alternative methods such as sequential fractionation, or the extraction of incubated soil/fertilizer mixtures with standard soil extractants or with P sink methods should be tested more intensively in the future to provide alternative options to predict the P-availability of fertilizers from recycled materials.
Phosphorus (P) recycling from sewage sludge for agricultural needs has to meet requirements for agricultural implementation, such as short and long-term P-plant-availability under field conditions. Field experiments often bring no evaluable results, because agricultural soils got a high potential of P-supply even if they are classified as low in P-supply according to the CAL extraction method. The present study presents a possible way to investigate the P-plant-availability of P-recycling-fertilizers under field-like conditions. The plant experiments are firstly performed in small Mitscherlich pots in growth chambers and subsequently in containers with a high soil volume of 170 kg under greenhouse conditions, in which plants can grow until ripening. The tested P-recycling fertilizers were produced from sewage sludge in a large-scale thermal process. It was a two-step treatment process performed with a pyrolysis of sewage sludge at 550°C (SSC-550) and a subsequent thermochemical post-treatment at 950°C with Na2SO4 (SSA-Na) and HCl + Na2SO4 (SSA-HCl/Na) as additives. The results show, that the P-recycling-products from pyrolysis got an adequate long-term but a 65% lower short-term P-plant-availability compared to triple superphosphate. SSA-Na and SSA-HCl/Na show both a high short and longterm P-plant-availability comparable to triple-superphosphate. This can be explained by their highly plant-available P-compound CaNaPO4.
In a rapidly evolving world, the demand on raw materials is increasing steadily and many technologies are dependent on a secure supply of hi-tech metals. Recently, scandium (Sc) has attracted attention since its use in high strength Al-alloys and solid-oxide-fuel-cells strongly improves the performance of those materials.
The element Sc is not exceptionally rare but quite resistant to geochemical enrichment processes, it is scarcely found enriched to high concentrations and is recovered as a by-product. Since Sc enrichment in Greek bauxite residues was shown by Ochsenkühn-Petropoulou et. al (1994), intensive research on this material and development of efficient Sc-recovery methods is ongoing.
This study investigates Sc-bearing species in bauxite residues from alumina production. It aims to provide direct evidence about the Sc-speciation’s in those secondary resources and tries to find the link to speciation’s in primary resources, e.g. bauxites and laterites.
Therefore, Sc K edge XANES (X-ray absorption near edge structure) spectroscopy is performed using synchrotron radiation to determine the presence of certain Sc-components and distinguish between adsorbed and chemically bonded Sc as was shown for lateritic deposits in Australia by Chassé et al. 2016. For comparison, reference standards of Sc-bearing and Sc-adsorbed species are synthesized. Indirect inferences from leaching behavior of bauxites, in cases supported by analyses with LA-ICP-MS, suggest Sc to be associated with either iron- or aluminum phases (Vind et al. 2017); (Suss et al.). It remains unclear how different primary materials influence Sc-speciation in the bauxite residue. Therefore, a comparison between different European bauxite residues is made in this study. The investigations should help to understand Sc chemistry and
behavior in different primary and secondary materials and provide fundamentals for metallurgical processing. The research is incorporated in the SCALE project (GA No. 730105) funded by EU Horizon 2020 research and innovation program.
Phosphorus (P) resource availability is declining and the efficiency of applied nutrients in agricultural soils is becoming increasingly important. This is especially true for P-fertilizers from recycled materials which often have a lower plant-availability compared to commercial P-fertilizers but are expected to play an increasingly important role into the future (Kratz et al. 2019). One promising way to increase the plant-availability of the fertilizer P is a co-fertilization with specific nitrogen (N) forms which can enhance the P uptake and make P-fertilizers from recycled material more competitive to commercial phosphate rock-based P-fertilizers (Rahmatullah et al. 2006; Vogel et al. 2018). To investigate this effect, we performed a pot experiment with three different P-fertilizers (sewage sludge-based, phosphate rock and triple superphosphate) and ammonium nitrate sulfate as a co-fertilizer, without and with a nitrification inhibitor (NI), and analyzed the form of N and P in soil via a suite of chemical and novel X-ray spectroscopic methods. The application of NI with the P and N fertilizers led to a higher dry matter yield and a higher P uptake of maize. Novel N K-edge micro-X-ray absorption near-edge structure (micro-XANES) spectroscopy identified that the application of a NI promotes the temporary formation of a non-exchangeable N in detectable hot-spots in the soil. The subsequent slow release and prolonged availability of N during plant growth leads to higher yield and nutrient uptake. It can be concluded that NIs lead to a temporary fixation of ammonium-N in a pool that can be accessed by plant roots. Those types of available nutrient pools meet the idea of so-called “next generation fertilizers” as plants have access to nutrients according to their current demand.
Phosphorus (P) fertilizers from secondary resources became increasingly important in the last years. However, these novel P-fertilizer can also contain toxic pollutants e.g. chromium (Cr) in the hexavalent state (Cr(VI)), which is regulated with low limit values in agricultural products (German fertilizer ordinance limit: 2 mg/kg Cr(VI)). The determination of Cr(VI) in these novel fertilizer matrices can be hampered by redox processes that lead to false results with the standard wet chemical extraction method (German norm DIN EN 15192). Therefore, we analyzed Cr(VI) in various P-fertilizers with the DGT technique. DGT devices equipped with a APA (polyacrylamide) diffusion layer and Cr(VI) selective N-methyl-D-glucamine (NMDG) binding layer were used for the study. After a 24 h conditioning period of the fertilizer at 60% of the water holding capacity (WHC), the fertilizers were brought to 100% WHC, transferred onto the DGT devices and deployed for 24 h at 25°C. The extraction of Cr from the DGT binding layer was carried out with 1 M HNO3 for 24 h. The Cr-concentrations of the extract were determined by means of ICP-MS. We found a good correlation between the standard wet chemical extraction and the DGT method for the whole range of P-fertilizers. However, partly soluble Cr(VI) compounds cannot be detected in full extent by the DGT method that is best suited for mobile Cr(VI). Furthermore, Cr K-edge XANES spectroscopy showed that the Cr(VI)-selective DGT binding layer also adsorbs mobile Cr(III) compounds from acid treatment of phosphates which can therefore cause an overestimation of Cr(VI). The DGT method was very sensitive and in most cases selective for the analysis of Cr(VI) in P-fertilizers made from recycled materials. However, the results of certain types of P-fertilizers containing mobile Cr(III) or partly immobile Cr(VI) show that still some optimization of the method is required to avoid over- or underestimation of Cr(VI).
P recycling fertilizers are gaining increasing importance in our efforts to close nutrient cycles. An unsatisfactory performance of standard chemical extraction methods to assess the fertilizing effects of such products was reported. They demonstrated that DGT extractions of incubated soil/fertilizer mixtures were able to predict the fertilizing effects of the respective products more accurately. Since DGT works with soil/fertilizer mixtures, its interpretation is soil-dependent. Therefore, in order to facilitate its use as a tool to predict fertilizer performance, it needs to be standardized based on a standard substrate. This research aims to develop a standard substrate based on which evaluation categories for the DGT fertilizer extraction can be derived. The substrate composition should allow to vary the most important soil properties determining the plant availability of fertilizer P. It must also be reproducible at any time and any place. Substrate variants with varying proportions of quartz sand, a clay mineral and sphagnum peat were prepared and set to pH-levels 5.5 and 7 by addition of CaCO3. 7 variants were incubated with a set of test fertilizers (2 recycling fertilizers based on sewage sludge ash and 2 conventional mineral fertilizers) for 2 weeks. Substrate/fertilizer mixtures were then extracted with DGT and an ANOVA was performed to test if the DGT extraction was able to depict significant differences between fertilizers and substrate variants. An 8-week pot trial with ryegrass (3 cuts) was set up with the same substrate variants and test fertilizers. P uptake was determined to assess the fertilizing effect and correlated with the results of the DGT extractions. Statistically significant differences were found between DGT results for the various test fertilizers and substrate variants, indicating that DGT is able to differentiate between P solubility of fertilizers in relation to substrate quality. DGT results showed a strong relationship with P uptake, confirming that this method is suitable to predict the fertilizing effect of P fertilizers. Further optimization of substrate composition and tests with a wider variety of crops and fertilizer types are needed, before evaluation categories for DGT values can be derived.
Brazilian farming industry consumed around 2.2 million tons of phosphorus (P) fertilizers in 2016. The agricultural industry depends on imported P fertilizers and over 98% of P fertilizers were used for sugarcane, soybean and maize production.
An alternative is to use P from sugarcane bagasse. Bagasse is the fibrous plant material remaining after extraction of sugarcane juice, and it is combusted for energy production. Remaining ash con-tains up to 0.6 wt% P. The use of bagasse ash (BA) as P fertilizer could decrease the annual import of P fertilizers by 6% of the imported P fertilizer based on 2016 values. Since the bioavailability of P from BA to plants is poorly investigated, this study addresses the effects of (i) gasification tempera-ture (710-849°C), (ii) processing method (gasification vs. combustion), (iii) biomass modifications by co-processing bagasse with chicken manure (BA+CM), and (iv) the soil (Brazilian Oxisol soil vs. nutri-ent poor substrate) on the bioavailability of P from BA to soybeans (Glycine max).
Gasification of BA at 806 °C resulted in significantly highest uptake of P by soybeans and was around 0.33 mmol after 51 days growing. The bioavailability of P significantly increased due to co-gasification of bagasse and chicken manure (BA+CM) and the soybeans took up around 16% more P. Compared to the nutrient-poor substrate, the bioavailability of P in BA+CM ash treated Oxisol soil was signifi-cantly lower by 46% and there was no significant effect of processing method on the bioavailability of P from the BA+CM ash to soybeans. Contrary to the Oxisol soil, the bioavailability of P from co-combusted BA+CM ash was significantly higher compared to co-gasified BA+CM ash. In conclusion, co-processing of bagasse with nutrient rich residues can increase the value of BA as P fertilizer. The bioavailability of P from ash to plants depends on the P forms. Mineralogical analyses of ash P forms by NMR and X-ray diffraction are in progress and will be presented at the conference.
Previous research shows that analytical methods based on Diffusive Gradients in Thin films (DGT) provide very good correlations to the amount of bioavailable nutrients and pollutants in the environmental samples. However, these DGT results do not identify which compound of the specific element has the high bioavailability. Using various spectroscopic techniques (infrared, XANES and NMR spectroscopy) to analyze the dried DGT binding layers after deployment could allow us to determine the specific elements or compounds. Nutrients such as phosphorus and nitrogen are often, together with other elements, present as molecules in the environment. These ions are detectable and distinguishable by infrared and NMR spectroscopy, respectively. In addition, XANES spectroscopy allows for the specification of nutrients and pollutants (e.g. chromium) on the DGT binding layer. Furthermore, microspectroscopic techniques make it also possible to analyze compounds on the DGT binding layer with a lateral resolution down to 5 µm2. Therefore, species of elements and compounds of e.g. a spatial soil segment can be mapped and analyzed, providing valuable insight to understand the dynamics of nutrients and pollutants in the environment. Here we will present the advantages and limitations of this novel combination of techniques.