Purification and biochemical characterization of recombinant alcohol dehydrogenase from the psychrophilic bacterium <Emphasis Type="Italic">Pseudomonas</Emphasis><Emphasis Type="Italic">frederiksbergensis</Emphasis>

A gram-negative psychrophilic bacterium, with potential for biodegradation of long-chain n-alkanes was isolated from ice samples collected in Spitzbergen, Denmark. On the basis of results of biochemical and morphological tests and sequence analysis of 16S rRNA, the strain was identified as Pseudomonas frederiksbergensis. In this work, a short-chain NAD⁺-dependent alcohol dehydrogenase (alcDH) (Accession number: AAR13804) from the P. frederiksbergensis was cloned and transformed in E. coli BL21 (3DE) competent cells. The alcDH activity was highest in the crude extract of cells induced with 1.0 mM IPTG. The recombinant alcDH enzyme was purified to 93.4% homogeneity using three consecutive purification steps including ammonium sulphate, Q-Sepharose Fast Flow column and gel filtration chromatography employing Superdex 200 10/30 HR column. Enzyme enrichment and yield levels of 31.4 folds and 25.5%, respectively, were achieved. While the subunit molecular mass of the enzyme was determined on SDS-PAGE to be ~38 kDa, the aggregated native form of the enzyme had a molecular mass of ~238 kDa by gel filtration analysis. Reaction conditions optima for the recombinant alcDH were determined with propan-1-ol as the substrate. While the optimum pH was 9, the optimum temperature was 35 °C. The alcDH enzyme exhibited moderate thermal stability with half-lives of 150 min at 55 °C, 27 min at 65 °C and 8 min at 75 °C. Results for kinetic parameters indicated that the apparent K _m value for alcDH with propan-1-ol as the substrate was found to be 1.42 mM and the V _max value was 0.63 mmol mg⁻¹ min⁻¹. Experimental evidence revealed that the recombinant alcDH exhibited a wide range of substrate specificity, with higher levels of specific activity for aliphatic alcohols as compared to secondary alcohols. Taken together, the present study highlights the potential of alcDH as a member of cold-adapted enzymes in several key biotechnological applications including environmental bioremediation and biotransformations. It is envisaged that, with the ongoing screening of microorganisms and metagenomes, directed evolution approaches and the subsequent overexpression of recombinant proteins, more enzymes will be found that are suitable for bioremediation purposes.     

Impact of cannibalism on predator-prey dynamics: size-structured interactions and apparent mutualism

Direct and indirect interactions between two prey species can strongly alter the dynamics of predator-prey systems. Most predators are cannibalistic, and as a consequence, even systems with only one predator and one prey include two prey types: conspecifics and heterospecifics. The effects of the complex direct and indirect interactions that emerge in such cannibalistic systems are still poorly understood. This study examined how the indirect interaction between conspecific and heterospecific prey affects cannibalism and predation rates and how the direct interactions between both species indirectly alter the effect of the cannibalistic predator. I tested for these effects using larvae of the stream salamanders Eurycea cirrigera (prey) and Pseudotriton ruber (cannibalistic predator) by manipulating the relative densities of the conspecific and heterospecific prey in the presence and absence of the predator in experimental streams. The rates of cannibalism and heterospecific predation were proportional to the respective densities and negatively correlated, indicating a positive indirect interaction between conspecific and heterospecific prey, similar to "apparent mutualism." Direct interactions between prey species did not alter the effect of the predator. Although both types of prey showed a similar 30% reduction in night activity and switch in microhabitat use in response to the presence of the predator, cannibalism rates were three times higher than heterospecific predation rates irrespective of the relative densities of the two types of prey. Cumulative predation risks differed even more due to the 48% lower growth rate of conspecific prey. Detailed laboratory experiments suggest that the 3:1 difference in cannibalism and predation rate was due to the higher efficiency of heterospecific prey in escaping immediate attacks. However, no difference was observed when the predator was a closely related salamander species, Gyrinophilus porphyriticus, indicating that this difference is species specific. This demonstrates that cannibalism can result in the coupling of predator and prey mortality rates that strongly determines the dynamics of predator-prey systems.     

Nativ-basierte Grundöle für die Produktion von Kühlschmierstoffen

Aims and Scope

Compared to mineral oil products, cooling lubricants based on fatty acid esters have technological advantages. Moreover, they are more environmentally compatible and make a contribution to a sustainable economy. Nevertheless, they are rarely applied in practice, because of their comparably high price, that is based on raw-material and synthesis costs. Alternatives concerning raw-materials and synthesis pathways are considered and properties of resulting ester products are compared.

Methods

The investigations aimed at three fatty acid-2-ethyl-hexylesters that were synthesized in an enzymatic-catalytic routine based on peanut oil, suet, and waste edible fat and at esters that were conventionally produced from animal and plant fats and 2-ethyl-1-hexanol. Physical properties, fatty acid patterns, contents of free fatty acids, as well as oxidative and hydrolytic stabilities were determined. Thermolysis experiments were performed.

Results

The physical properties of the seven esters, density, viscosity, pourpoint, and flash point, were comparable and gave reason for the assumption, that the products could be used as basis oils in lubricant formulations. Hydrolysis stabilities of the esters were high and thermal stress did not point at high potential of hazardous compounds formation. Especially the enzymatic-catalytically produced esters showed deficits concerning oxidation stabilities and free fatty acid contents.

Outlook

For practice it will be necessary to define minimum qualities for raw-materials and resulting ester oils to be used as lubricant basis oils. Moreover, processes have to be developed, that yield high quality ester products from waste edible and animal fats without loosing the advantage of low-cost raw-materials by high production and refinement costs. Surely, one contribution is the further development of the energy-saving enzymtic alcoholysis for ester production.     

Review of fly ash inertisation treatments and recycling

Fly ash (FA) is a by-product of power, and incineration plants operated either on coal and biomass, or on municipal solid waste. FA can be divided into coal fly ash, obtained from power plant burning coal, flue gas desulphurisation FA, that is, the by-product generated by the air pollution control equipment in coal-fired power plants to reduce the release of SO₂, biomass FA produced in the plants for thermal conversion of biomass and municipal solid waste incineration (MSWI) FA, that is, the finest residue obtained from the scrubber system in a MSWI plant. Because of the large amount produced in the world, fly ash is now considered the world’s fifth largest material resource. The composition of FA is very variable, depending on its origins; then, also pollutants can be very different. In this frame, it is fundamental to exploit the chemical or physical potentials of FA constituents, thus rendering them second-life functionality. This review paper is addressed to FA typology, composition, treatment, recycling, functional reuse and metal and organic pollutants abatement. Because of the general growing of environmental awareness and increasing energy and material demand, it is expected that increasing recycling rates will reduce the pressure on demand for primary raw materials, help to reuse valuable materials which would otherwise be wasted and reduce energy consumption and greenhouse gas emissions from extraction and processing.