Identification and evaluation of Escherichia coli strain ATCC 8739 as a surrogate for thermal inactivation of enterohemorrhagic Escherichia coli in fruit nectars: Impact of applied techniques on the decimal reduction time.

The objective of this study was to identify a suitable surrogate for E. coli O157:H7 strain 19685/91 and O113:H21 strain TS18/08, by assessing their thermal resistance at temperatures of 60 °C, 65 °C, and 72 °C in strawberry nectar. The influence of the matrix and the research methodology on the decimal reduction time (D-value) was investigated. Thermal kinetics and safety assessment demonstrated that E. coli ATCC 8739 is a suitable surrogate. The study demonstrated that the presence of fruit particles in the nectar increased thermal resistance of the tested strains. Variations in D-values were observed depending on the research method employed, with D-values in glass capillaries were up to 6.6 times lower compared to larger sample volumes. Encapsulation of E. coli ATCC 8739 exhibited high efficiency of 90.25 ± 0.26% and maintained stable viable counts after 26 days of storage in strawberry nectar at 4 °C. There were no significant differences in thermal resistance between surrogates directly inoculated into strawberry nectar and those encapsulated in alginate beads. Additionally, the encapsulated strains did not migrate outside the beads. Therefore, encapsulated E. coli ATCC 8739 in alginate beads can be effectively utilized in industrial settings to validate thermal treatments as a reliable and safe method.

pH-Responsive Dynaplexes as Potent Apoptosis Inductors by Intracellular Delivery of Survivin siRNA.

Gene knockdown by siRNA offers an unrestricted choice of targets and specificity based on the principle of complementary Watson-Crick base pairing with mRNA. However, the negative charge, large molecular size, and susceptibility to enzymatic degradation of siRNA impede its successful transfection, hence limiting its potential for therapeutic use. The development of efficient and safe siRNA transfection agents is, therefore, critical for siRNA-based therapy. Herein, we developed a protein-based biodynamic polymer (biodynamer) that showed potential as a siRNA transfection vector, owing to its excellent biocompatibility, easy tunability, and dynamic polymerization under acidic environments. The positively charged biodynamers formed stable dynamic nanocomplexes (XL-DPs, hydrodynamic diameter of approximately 104 nm) with siRNA via electrostatic interactions and chemical cross-linking. As a proof of concept, the optimized XL-DPs were stable in physiological conditions with serum proteins and demonstrated significant pH-dependent size change and degradability, as well as siRNA release capability. The minimal cytotoxicity and excellent cellular uptake of XL-DPs effectively supported the intracellular delivery of siRNA. Our study demonstrated that the XL-DPs in survivin siRNA delivery enabled potent knockdown of survivin mRNA and induced notable apoptosis of carcinoma cells (2.2 times higher than a lipid-based transfection agent, Lipofectamine 2000). These findings suggested that our XL-DPs hold immense potential as a promising platform for siRNA delivery and can be considered strong candidates in the advancement of next-generation transfection agents.

Plant variety and soil type influence Escherichia coli O104:H4 strain C227/11ϕcu adherence to and internalization into the roots of lettuce plants.

Human disease outbreaks caused by pathogenic Escherichia coli are increasingly associated with the consumption of contaminated fresh produce. Internalization of enteroaggregative/enterohemorrhagic E. coli (EAEC/EHEC) strains into plant tissues may present a serious threat to public health. In the current study, the ability of the fluorescing Shiga toxin-negative E. coli O104:H4 strain C227/11ϕcu/pKEC2 to adhere to and to internalize into the roots of Lactuca sativa and Valerianella locusta grown in diluvial sand (DS) and alluvial loam (AL) was investigated. In parallel, the soil microbiota was analyzed by partial 16S rRNA gene sequencing. The experiments were performed in a safety level 3 greenhouse to simulate agricultural practice. The adherence of C227/11ϕcu/pKEC2 to the roots of both plant varieties was increased by at least a factor three after incubation in DS compared to AL. Compared to V. locusta, internalization into the roots of L. sativa was increased 12-fold in DS and 108-fold in AL. This demonstrates that the plant variety had an impact on the internalization ability, whereas for a given plant variety the soil type also affected bacterial internalization. In addition, microbiota analysis detected the inoculated strain and showed large differences in the bacterial composition between the soil types.

Differential transcriptome analysis of enterohemorrhagic Escherichia coli strains reveals differences in response to plant-derived compounds.

BACKGROUND: Several serious vegetable-associated outbreaks of enterohemorrhagic Escherichia coli (EHEC) infections have occurred during the last decades. In this context, vegetables have been suggested to function as secondary reservoirs for EHEC strains. Increased knowledge about the interaction of EHEC with plants including gene expression patterns in response to plant-derived compounds is required. In the current study, EHEC O157:H7 strain Sakai, EHEC O157:H- strain 3072/96, and the EHEC/enteroaggregative E. coli (EAEC) hybrid O104:H4 strain C227-11φcu were grown in lamb's lettuce medium and in M9 minimal medium to study the differential transcriptional response of these strains to plant-derived compounds with RNA-Seq technology. RESULTS: Many genes involved in carbohydrate degradation and peptide utilization were similarly upregulated in all three strains, suggesting that the lamb's lettuce medium provides sufficient nutrients for proliferation. In particular, the genes galET and rbsAC involved in galactose metabolism and D-ribose catabolism, respectively, were uniformly upregulated in the investigated strains. The most prominent differences in shared genome transcript levels were observed for genes involved in the expression of flagella. Transcripts of all three classes of the flagellar hierarchy were highly abundant in strain C227-11φcu. Strain Sakai expressed only genes encoding the basal flagellar structure. In addition, both strains showed increased motility in presence of lamb's lettuce extract. Moreover, strain 3072/96 showed increased transcription activity for genes encoding the type III secretion system (T3SS) including effectors, and was identified as a powerful biofilm-producer in M9 minimal medium. CONCLUSION: The current study provides clear evidence that EHEC and EHEC/EAEC strains are able to adjust their gene expression patterns towards metabolization of plant-derived compounds, demonstrating that they may proliferate well in a plant-associated environment. Moreover, we propose that flagella and other surface structures play a fundamental role in the interaction of EHEC and EHEC/EAEC with plants.

Recent advances in cured raw ham manufacture.

Cured raw hams are a valuable and popular group of meat products. The consumption and international trade have increased during the last years, therefore new technologies to accelerate the production process and to increase product quality and safety are needed. In the current review, an overview of European protected cured raw hams is presented. Furthermore, traditional methods for cured raw ham production together with recent advantages in the techniques for pretreatment (trimming, blade tenderization, and freeze-thawing), curing/salting (tumbling, vacuum impregnation, pulsed pressure, ultrasound, pulsed electric fields, simultaneous thawing/salting), drying/ripening (Quick-Dry-Slice-process, oil drop application, high temperature short time process) and postprocessing (vacuum and modified atmosphere packaging, high hydrostatic pressure, high pressure carbon dioxide, high pressure carbon dioxide with ultrasound) are described. Moreover, application techniques and effects of protective cultures and starter cultures, such as molds, yeasts, coagulase-negative staphylococci and lactic acid bacteria, on cured raw ham quality and safety are reviewed.

Adherence factors of enterohemorrhagic Escherichia coli O157:H7 strain Sakai influence its uptake into the roots of Valerianella locusta grown in soil.

Increasing numbers of outbreaks caused by enterohemorrhagic Escherichia coli (EHEC) are associated with the consumption of contaminated fresh produce. The contamination of the plants may occur directly on the field via irrigation water, surface water, manure or fecal contamination. Suggesting a low infectious dose of 10 to 102 cells, internalization of EHEC into plant tissue presents a serious public health threat. Therefore, the ability of EHEC O157:H7 strain Sakai to adhere to and internalize into root tissues of the lamb's lettuce Valerianella locusta was investigated under the environmental conditions of a greenhouse. Moreover, the influence of the two adherence and colonization associated genes hcpA and iha was surveyed regarding their role for attachment and invasion. Upon soil contamination, the number of root-internalized cells of EHEC O157:H7 strain Sakai exceeded 102 cfu/g roots. Deletion of one or both of the adherence factor genes did not alter the overall attachment of EHEC O157:H7 strain Sakai to the roots, but significantly reduced the numbers of internalized bacteria by a factor of between 10 and 30, indicating their importance for invasion of EHEC O157:H7 strain Sakai into plant roots. This study identified intrinsic bacterial factors that play a crucial role during the internalization of EHEC O157:H7 strain Sakai into the roots of Valerianella locusta grown under the growth conditions in a greenhouse.

Modified Bacteriophage S16 Long Tail Fiber Proteins for Rapid and Specific Immobilization and Detection of Salmonella Cells.

Bacteriophage-based assays and biosensors rival traditional antibody-based immunoassays for detection of low-level Salmonella contaminations. In this study, we harnessed the binding specificity of the long tail fiber (LTF) from bacteriophage S16 as an affinity molecule for the immobilization, enrichment, and detection of Salmonella We demonstrate that paramagnetic beads (MBs) coated with recombinant gp37-gp38 LTF complexes (LTF-MBs) are highly effective tools for rapid affinity magnetic separation and enrichment of Salmonella Within 45 min, the LTF-MBs consistently captured over 95% of Salmonella enterica serovar Typhimurium cells from suspensions containing from 10 to 105 CFU · ml-1, and they yielded equivalent recovery rates (93% ± 5%, n = 10) for other Salmonella strains tested. LTF-MBs also captured Salmonella cells from various food sample preenrichments, allowing the detection of initial contaminations of 1 to 10 CFU per 25 g or ml. While plating of bead-captured cells allowed ultrasensitive but time-consuming detection, the integration of LTF-based enrichment into a sandwich assay with horseradish peroxidase-conjugated LTF (HRP-LTF) as a detection probe produced a rapid and easy-to-use Salmonella detection assay. The novel enzyme-linked LTF assay (ELLTA) uses HRP-LTF to label bead-captured Salmonella cells for subsequent identification by HRP-catalyzed conversion of chromogenic 3,3',5,5'-tetramethylbenzidine substrate. The color development was proportional for Salmonella concentrations between 102 and 107 CFU · ml-1 as determined by spectrophotometric quantification. The ELLTA assay took 2 h to complete and detected as few as 102 CFU · ml-1S Typhimurium cells. It positively identified 21 different Salmonella strains, with no cross-reactivity for other bacteria. In conclusion, the phage-based ELLTA represents a rapid, sensitive, and specific diagnostic assay that appears to be superior to other currently available tests.IMPORTANCE The incidence of foodborne diseases has increased over the years, resulting in major global public health issues. Conventional methods for pathogen detection can be laborious and expensive, and they require lengthy preenrichment steps. Rapid enrichment-based diagnostic assays, such as immunomagnetic separation, can reduce detection times while also remaining sensitive and specific. A critical component in these tests is implementing affinity molecules that retain the ability to specifically capture target pathogens over a wide range of in situ applications. The protein complex that forms the distal tip of the bacteriophage S16 long tail fiber is shown here to represent a highly sensitive affinity molecule for the specific enrichment and detection of Salmonella Phage-encoded long tail fibers have huge potential for development as novel affinity molecules for robust and specific diagnostics of a vast spectrum of bacteria.

Surface adhesins and exopolymers of selected foodborne pathogens.

The ability of bacteria to bind different compounds and to adhere to biotic and abiotic surfaces provides them with a range of advantages, such as colonization of various tissues, internalization, avoidance of an immune response, and survival and persistence in the environment. A variety of bacterial surface structures are involved in this process and these promote bacterial adhesion in a more or less specific manner. In this review, we will focus on those surface adhesins and exopolymers in selected foodborne pathogens that are involved mainly in primary adhesion. Their role in biofilm development will also be considered when appropriate. Both the clinical impact and the implications for food safety of such adhesion will be discussed.

Development of a rapid detection system for opportunistic pathogenic Cronobacter spp. in powdered milk products.

Certain species of the genus Cronobacter are considered opportunistic pathogens, but their detection in milk products according to ISO/TS 22964 may take up to six days. The aim of this study was to develop a fast and sensitive PCR-based detection system for these species including enrichment, DNA-isolation and detection by real-time PCR, using the outer membrane protein gene ompA as a target. The assay was successfully validated using type strains of the genus Cronobacter, as well as 18 strains of closely related genera as controls. A total of 40 Cronobacter spp. food isolates yielded positive results, while the food matrix itself did not influence the PCR reaction. An equal detection limit as achieved with the ISO/TS 22964 method was established in this study, when 0.01 CFU Cronobacter sakazakii DSM 4485(T) per gram powdered infant formula were successfully detected after 28 days of storage at ambient temperature. In comparison to the ISO/TS 22964 method, the method described here has an equal detection limit, but offers a specific detection at the genus level in an analysis time of 24 h.

Rapid Acidification and Off-Flavor Reduction of Pea Protein by Fermentation with Lactic Acid Bacteria and Yeasts.

Pea protein is widely used as an alternative protein source in plant-based products. In the current study, we fermented pea protein to reduce off-flavor compounds, such as hexanal, and to produce a suitable fermentate for further processing. Laboratory fermentations using 5% (w/v) pea protein suspension were carried out using four selected lactic acid bacteria (LAB) strains, investigating their growth and acidification capabilities in pea protein. Rapid acidification of pea protein was achieved with Lactococcus lactis subsp. lactis strain LTH 7123. Next, this strain was co-inoculated together with either the yeasts Kluyveromyces lactis LTH 7165, Yarrowia lipolytica LTH 6056, or Kluyveromyces marxianus LTH 6039. Fermentation products of the mixed starter cultures and of the single strains were further analyzed by gas chromatography coupled with mass spectrometry to quantify selected volatile flavor compounds. Fermentation with L. lactis LTH 7123 led to an increase in compounds associated with the "beany" off-flavors of peas, including hexanal. However, significant reduction in those compounds was achieved after fermentation with Y. lipolytica LTH 6056 with or without L. lactis LTH 7123. Thus, fermentation using co-cultures of LAB and yeasts strains could prove to be a valuable method for enhancing quality attributes of pea protein-based products.

Elasticity, an often-overseen parameter in the development of nanoscale drug delivery systems.

Nanoparticles have shown an enormous potential as drug delivery systems in the lab. However, translation to the clinics or even market approval often fails. So far, the reason for this discrepancy is manifold. Physicochemical properties such as size, surface potential, and surface chemistry are in focus of research for many years. Other equally important parameters, influencing whether a successful drug delivery can be achieved, are mechanical properties of nanoparticles. Even though this is often not even considered during formulation development, and it is not requested for approval, an increasing number of studies show that it is important to have knowledge about these characteristics. In this article, we discuss examples highlighting the influence of elasticity in nanoscale biological interactions focusing on mucosal delivery and on tumor targeting. Besides this, we discuss the influence of different measurement settings using atomic force microscopy for the determination of mechanical properties of drug carriers.

The Effect of Elasticity of Gelatin Nanoparticles on the Interaction with Macrophages.

Gelatin is a biocompatible, biodegradable, cheap, and nontoxic material, which is already used for pharmaceutical applications. Nanoparticles from gelatin (GNPs) are considered a promising delivery system for hydrophilic and macromolecular drugs. Mechanical properties of particles are recognized as an important parameter affecting drug carrier interaction with biological systems. GNPs offer the preparation of particles with different stiffness. GNPs were loaded with Fluorescein isothiocyanate-labeled 150 kDa dextran (FITC-dextran) yielding also different elastic properties. GNPs were visualized using atomic force microscopy (AFM), and force-distance curves from the center of the particles were evaluated for Young's modulus calculation. The prepared GNPs have Young's moduli from 4.12 MPa for soft to 9.8 MPa for stiff particles. Furthermore, cytokine release (IL-6 and TNF-α), cell viability, and cell uptake were determined on macrophage cell lines from mouse (RAW 264.7) and human (dTHP-1 cells, differentiated human monocytic THP-1 cells) origin for soft and stiff GNPs. Both particle types showed good cell compatibility and did not induce IL-6 and TNF-α release from RAW 264.7 and dTHP-1 cells. Stiffer GNPs were internalized into cells faster and to a larger extent.

Gelatin nanoparticles with tunable mechanical properties: effect of crosslinking time and loading.

Tuning the elastic properties of nanoparticles intended to be used in drug delivery is of great interest. To this end, different potential formulations are developed since the particle elasticity is affecting the in vitro and in vivo performance of the nanoparticles. Here we present a method to determine the elasticity of single gelatin nanoparticles (GNPs). Furthermore, we introduce the possibility of tuning the elastic properties of gelatin nanoparticles during their preparation through crosslinking time. Young's moduli from 5.48 to 14.26 MPa have been obtained. Additionally, the possibility to measure the elasticity of single nanoparticles revealed the influence of loading a macromolecular model drug (FITC-dextran) on the mechanical properties, which decreased with raising amounts of loaded drug. Loaded particles were significantly softer, with Young's moduli between 1.06 and 5.79 MPa for the same crosslinking time, than the blank GNPs. In contrast to this, lysozyme as a crosslinkable macromolecule did not influence the mechanical properties. A good in vitro cell compatibility was found investigating blank GNPs and FITC-dextran-loaded GNPs in viability assays with the cancer cell line A549 and the human primary cell-derived hAELVi cell line.

Effects of Protein, Calcium, and pH on Gene Transcription, Cell-Envelope Peptidase Activity of Lactococcus lactis Strains, and the Formation of Bitter Peptides.

Calcium- and protein-rich fermented milk products, such as concentrated yoghurts and fresh cheeses, may contain undesired bitter peptides, which are generated by the proteolytic cleavage of casein. Up to now, it is not clear whether this process is caused by endogenous milk enzymes, such as plasmin and cathepsin D, or whether proteolytic enzymes from applied starter cultures, such as the lactococcal cell-envelope peptidase PrtP, are involved. A sensory analysis of fresh cheese products made from milk concentrates fermented with prtP-negative and -positive Lactococcus lactis strains revealed bitterness in the products fermented with prtP-positive L. lactis strains. Two prtP-positive strains, LTH 7122 and LTH 7123, were selected to investigate the effect of increased calcium concentrations (additional 5 mM and 50 mM CaCl2) at neutral (pH 6.6) and acidic (pH 5.5) pH-values on the transcription of the prtP gene and its corresponding PrtP peptidase activity in milk citrate broth (MCB). For both strains, it was shown that prtP transcription was upregulated only under slightly elevated calcium conditions (5 mM CaCl2) after 5 h of growth. In concordance with these findings, PrtP peptidase activity also increased. When higher concentrations of calcium were used (50 mM), prtP expression of both strains decreased strongly by more than 50%. Moreover, PrtP peptidase activity of strain LTH 7123 decreased by 15%, but enzymatic activity of strain LTH 7122 increased slightly during growth under elevated calcium concentrations (50 mM CaCl2). Fermentations of reconstituted casein medium with 3.4% (w/v) and 8.5% (w/v) protein and different calcium concentrations using strain LTH 7122 revealed no clear relationship between prtP transcription and calcium or protein concentration. However, an increase in PrtP peptidase activity under elevated protein and calcium conditions was observed. The activity increase was accompanied by increased levels of bitter peptides derived from different casein fractions. These findings could be a possible explanation for the bitterness in fermented milk concentrates that was detected by a trained bitter panel.

Investigation of factors influencing biogas production in a large-scale thermophilic municipal biogas plant.

A continuously operated, thermophilic, municipal biogas plant was observed over 26 months (sampling twice per month) in regard to a number of physicochemical parameters and the biogas production. Biogas yields were put in correlation to parameters such as the volatile fatty acid concentration, the pH and the ammonium concentration. When the residing microbiota was classified via analysis of the 16S rRNA genes, most bacterial sequences matched with unidentified or uncultured bacteria from similar habitats. Of the archaeal sequences, 78.4% were identified as belonging to the genus Methanoculleus, which has not previously been reported for biogas plants, but is known to efficiently use H(2) and CO(2) produced by the degradation of fatty acids by syntrophic microorganisms. In order to further investigate the influence of varied amounts of ammonia (2-8 g/L) and volatile fatty acids on biogas production and composition (methane/CO(2)), laboratory scale satellite experiments were performed in parallel to the technical plant. Finally, ammonia stripping of the process water of the technical plant was accomplished, a measure through which the ammonia entering the biogas reactor via the mash could be nearly halved, which increased the energy output of the biogas plant by almost 20%.

Combining cryo-TEM and energy-filtered TEM for imaging organic core-shell nanoparticles and defining the polymer distribution.

Nanoparticulate systems intended for the use in drug delivery are getting more and more complex. Composite nanoparticles, such as core-shell particles are designed in order to be used for co-delivery of drugs or a modified release profile. Often the structure can only be postulated by the preparation process, such as surface polymerization, but cannot be experimentally determined due to a lack of appropriate analytical methods. Here a core-shell particle system composed of two biodegradable and biocompatible materials, gelatin and PLGA, is developed. In order to reveal the actual polymer distribution, a combination of cryo-transmission electron microscopy and energy-filtered transmission electron microscopy was established. Using the occurrence of specific elements in combination with degradation kinetics induced by the electron beam allows to conclude on the nanoparticles' architecture. Based on these methods and thus, the particle composition, the drug delivery system can be further developed.

Mechanical properties of gelatin nanoparticles in dependency of crosslinking time and storage.

Mechanical properties of nanoparticles are an important characteristic for drug delivery and therefore, they have gained interest in pharmaceutical research during the last years. Among others, cellular uptake, blood circulation time and accumulation in organs are influenced by the elastic modulus of nanoparticles. Thus, by varying the stiffness of nanoparticles a more specific drug targeting might be achieved. Gelatin nanoparticles (GNPs) show advantageous characteristics in respect to encapsulation and delivery of hydrophilic drugs such as antibodies or other biologicals. Furthermore, the GNPs as hydrogel-nanoparticles offer adjustable elastic behavior. In this study, a method for GNP sample preparation and the determination of the mechanical properties by nanoindentation experiments using atomic force microscopy (AFM) was developed. The obtained force-distance curves were evaluated and fitted with the Hertzian model in order to calculate the Young's modulus. GNPs were crosslinked with glutaraldehyde (GTA) for different incubation times to investigate a possible modification of the Young's modulus. In addition, this study addresses the influence of storage on the mechanical characteristics of GNPs. The results provide first insights about the elastic properties of GNPs and their development over time. In the tested range of crosslinking times no notable differences in the mechanical properties occurred. In turn, the influence of the storage on the mechanical particle properties was observed: particle stiffness raised over time. Furthermore, it could be observed that the cellular uptake in a model cell line (A549) was increased for harder particles.

Diversity of the resident microbiota in a thermophilic municipal biogas plant.

Biogas plants continuously convert biological wastes mainly into a mixture of methane, CO2 and H2O-a conversion that is carried out by a consortium of bacteria and archaea. Especially in the municipal plants dedicated towards waste treatment, the reactor feed may vary considerably, exposing the resident microbiota to a changing variety of substrates. To evaluate how and if such changes influence the microbiology, an established biogas plant (6,600 m3, up to 600 m3 biogas per h) was followed over the course of more than 2 years via polymerase chain reaction-denaturing gradient gel electrophoresis of 16S rRNA genes and subsequent sequencing. Both the bacterial and the archaeal community remained stable over the investigation. Of the bacterial consortium, about half of the sequences were in decreasing order of occurrence: Thermoacetogenium sp., Anaerobaculum mobile, Clostridium ultunense, Petrotoga sp., Lactobacillus hammesii, Butyrivibrio sp., Syntrophococcus sucromutans, Olsenella sp., Tepidanaerobacter sp., Sporanaerobacter acetigenes, Pseudoramibacter alactolyticus, Lactobacillus fuchuensis or Lactobacillus sakei, Lactobacillus parabrevis or Lactobacillus spicheri and Enterococcus faecalis. The other half matched closely to ones from similar habitats (thermophilic anaerobic methanogenic digestion). The archaea consisted of Methanobrevibacter sp., Methanoculleus bourgensis, Methanosphaera stadtmanae, Methanimicrococcus blatticola and uncultured Methanomicrobiales. The role of these species in methane production is discussed.

Photovoltaic cell electrical heating system for removing snow on panel including verification.

Small photovoltaic plants in private ownership are typically rated at 5 kW (peak). The panels are mounted on roofs at a decline angle of 20° to 45°. In winter time, a dense layer of snow at a width of e.g., 10 cm keeps off solar radiation from the photovoltaic cells for weeks under continental climate conditions. Practically, no energy is produced over the time of snow coverage. Only until outside air temperature has risen high enough for a rather long-time interval to allow partial melting of snow; the snow layer rushes down in an avalanche. Following this proposal, snow removal can be arranged electrically at an extremely positive energy balance in a fast way. A photovoltaic cell is a large junction area diode inside with a threshold voltage of about 0.6 to 0.7 V (depending on temperature). This forward voltage drop created by an externally driven current through the modules can be efficiently used to provide well-distributed heat dissipation at the cell and further on at the glass surface of the whole panel. The adhesion of snow on glass is widely reduced through this heating in case a thin water film can be produced by this external short time heating. Laboratory experiments provided a temperature increase through rated panel current of more than 10 °C within about 10 min. This heating can initiate the avalanche for snow removal on intention as described before provided the clamping effect on snow at the edge of the panel frame is overcome by an additional heating foil. Basics of internal cell heat production, heating thermal effects in time course, thermographic measurements on temperature distribution, power circuit opportunities including battery storage elements and snow-removal under practical conditions are described.

Comparison of strategies for the isolation of PCR-compatible, genomic DNA from a municipal biogas plants.

The goal of the project was the extraction of PCR-compatible genomic DNA representative of the entire microbial community from municipal biogas plant samples (mash, bioreactor content, process water, liquid fertilizer). For the initial isolation of representative DNA from the respective lysates, methods were used that employed adsorption, extraction, or precipitation to specifically enrich the DNA. Since no dedicated method for biogas plant samples was available, preference was given to kits/methods suited to samples that resembled either the bioreactor feed, e.g. foodstuffs, or those intended for environmental samples including wastewater. None of the methods succeeded in preparing DNA that was directly PCR-compatible. Instead the DNA was found to still contain considerable amounts of difficult-to-remove enzyme inhibitors (presumably humic acids) that hindered the PCR reaction. Based on the isolation method that gave the highest yield/purity for all sample types, subsequent purification was attempted by agarose gel electrophoresis followed by electroelution, spermine precipitation, or dialysis through nitrocellulose membrane. A combination of phenol/chloroform extraction followed by purification via dialysis constituted the most efficient sample treatment. When such DNA preparations were diluted 1:100 they did no longer inhibit PCR reactions, while they still contained sufficient genomic DNA to allow specific amplification of specific target sequences.