Fate of organo-mineral particles in streams: Microbial degradation by streamwater & biofilm assemblages

Inland waters are of global biogeochemical importance. They receive carbon inputs of ~ 4.8 Pg C/ y of which, 12 % is buried, 18 % transported to the oceans, and 70 % supports aquatic secondary production. However, the mechanisms that determine the fate of organic matter (OM) in these systems are poorly defined. One aspect of this is the formation of organo-mineral complexes in aquatic systems and their potential as a route for OM transport and burial vs. their use as carbon (C) and nitrogen (N) sources within aquatic systems. Organo-mineral particles form by sorption of dissolved OM to freshly eroded mineral surfaces and may contribute to ecosystem-scale particulate OM fluxes. We experimentally tested the availability of mineral-sorbed OM as a C & N source for streamwater microbial assemblages and streambed biofilms. Organo-mineral particles were constructed in vitro by sorption of ¹³C:¹⁵N-labelled amino acids to hydrated kaolin particles, and microbial degradation of these particles compared with equivalent doses of ¹³C:¹⁵N-labelled free amino acids. Experiments were conducted in 120 ml mesocosms over 7 days using biofilms and water sampled from the Oberer Seebach stream (Austria). Each incubation experienced a 16:8 light:dark regime, with metabolism monitored via changes in oxygen concentrations between photoperiods. The relative fate of the organo-mineral particles was quantified by tracing the mineralization of the ¹³C and ¹⁵N labels and their incorporation into microbial biomass. Here we present the initial results of ¹³C-label mineralization, incorporation and retention within dissolved organic carbon pool. The results indicate that 514 (± 219) μmol/ mmol of the ¹³:¹⁵N labeled free amino acids were mineralized over the 7-day incubations. By contrast, 186 (± 97) μmol/ mmol of the mineral-sorbed amino acids were mineralized over a similar period. Thus, organo-mineral complexation reduced amino acid mineralization by ~ 60 %, with no differences observed between the streamwater and biofilm assemblages. Throughout the incubations, biofilms were observed to leach dissolved organic carbon (DOC). However, within the streamwater assemblage the presence of both organo-mineral particles and kaolin particles was associated with significant DOC removal (-1.7 % and -7.5 % respectively). Consequently, the study demonstrates that mineral and organo-mineral particles can limit the availability of DOC in aquatic systems, providing nucleation sites for flocculation and fresh mineral surfaces, which facilitate OM-sorption. The formation of these organo-mineral particles subsequently restricts microbial OM degradation, potentially altering the transport and facilitating the burial of OM within streams.

Measurement of Local Sodium Ion Levels near Micellar Surfaces with Fluorescent PET (Photoinduced Electron Transfer) Sensors

Na+ near membranes controls our nerve signals, besides several other crucial bioprocesses. We demonstrate that fluorescent PET (photoinduced electron transfer) sensor molecules target Na+ in nanospaces near micellar membranes with excellent discrimination against H+. They find that Na+ near anionic micelles is concentrated by factors of upto 160. Sensor molecules which are not held tight to the micelle surface find a Na+ amplification factor of 8 only. These findings are strengthened by the employment of control compounds whose PET processes are permanently ‘on’ or permanently ‘off’.

Highly CO2 sensitive extruded fluorescent plastic indicator film based on HPTS

Highly-sensitive optical fluorescent extruded plastic films are reported for the detection of gaseous and dissolved CO2. The pH-sensitive fluorescent dye used is 8-Hydroxypyrene-1,3,6-trisulfonic acid trisodium salt (HPTS, PTS-) coated on the surface of hydrophilic fumed silica and the base is tetrabutylammonium hydroxide (TBAH). The above components are used to create an HPTS pigment (i.e. HPTS/SiO2/TBAH) with a high CO2 sensitivity (%CO2(S=1/2) = 0.16%) and fast 50% response (t50↓) = 2 s and recovery (t50↑) = 5 s times. Highly CO2-sensitive plastic films are then fabricated, via the extrusion of the HPTS pigment powder in low-density polyethylene (LDPE). As with the HPTS-pigment, the luminescence intensity (at 515 nm) and absorbance (at 475 nm) of the HPTS plastic film decreases as the %CO2 in the ambient gas phase increases. The HPTS plastic film exhibits a high CO2 sensitivity, %CO2(S=1/2), of 0.29%, but a response time ˂2 min and recovery time ˂40 min, which is slower than that of the HPTS pigment. The HPTS plastic film is very stable under ambient conditions, (with a shelf life ˃ six month when stored in the dark but under otherwise ambient conditions). Moreover, the HPTS-film is stable in water, salt solution and even in acid (pH=2), and in each of these media it can be used to detect dissolved CO2.

Studies into the effect of temperature on the impact of model particles in co-melt granulation

In co-melt granulation, collisions occur between the particles to be agglomerated and the binder material. Depending on the stage of granulation, the binder material can be in the solid or liquid phase. The outcome of these collisions controls the dynamics of the granulation process and the fundamental physics of the impacts are of interest. This paper examines the impact of glass beads (model particles) and solid Poly Ethylene Glycol (PEG) flakes on a substrate of PEG as the temperature of the PEG layer is increased from below its melting point to above it. While the layer is in the solid state, the result of the impact can be quantified by the coefficient of restitution. When the layer is in the liquid state, the impact can be quantified by the immersion behaviour. The results obtained show that the coefficient of restitution between either glass beads and PEG flakes and the PEG layer is strongly affected by temperatures. As the PEG layer approaches its melting point, the coefficient of restitution falls to zero. Once the temperature of the PEG layer exceeds the melting point, the impact is characterised by a transient maximum indentation and then rebound to an equilibrium position. These too are strongly dependent on temperature.

Using the fluorescent properties of STO-609 as a tool to assist structure-function analyses of recombinant CaMKK2

Calcium/calmodulin-dependent kinase kinase 2 (CaMKK2) has been implicated in the regulation of metabolic activity in cancer and immune cells, and affects whole-body metabolism by regulating ghrelin-signalling in the hypothalamus. This has led to efforts to develop specific CaMKK2 inhibitors, and STO-609 is the standardly used CaMKK2 inhibitor to date. We have developed a novel fluorescence-based assay by exploiting the intrinsic fluorescence properties of STO-609. Here, we report an in vitro binding constant of KD ∼17 nM between STO-609 and purified CaMKK2 or CaMKK2:Calmodulin complex. Whereas high concentrations of ATP were able to displace STO-609 from the kinase, GTP was unable to achieve this confirming the specificity of this association. Recent structural studies on the kinase domain of CaMKK2 had implicated a number of amino acids involved in the binding of STO-609. Our fluorescent assay enabled us to confirm that Phe(267) is critically important for this association since mutation of this residue to a glycine abolished the binding of STO-609. An ATP replacement assay, as well as the mutation of the 'gatekeeper' amino acid Phe(267)Gly, confirmed the specificity of the assay and once more confirmed the strong binding of STO-609 to the kinase. In further characterising the purified kinase and kinase-calmodulin complex we identified a number of phosphorylation sites some of which corroborated previously reported CaMKK2 phosphorylation and some of which, particularly in the activation segment, were novel phosphorylation events. In conclusion, the intrinsic fluorescent properties of STO-609 provide a great opportunity to utilise this drug to label the ATP-binding pocket and probe the impact of mutations and other regulatory modifications and interactions on the pocket. It is however clear that the number of phosphorylation sites on CaMKK2 will pose a challenge in studying the impact of phosphorylation on the pocket unless the field can develop approaches to control the spectrum of modifications that occur during recombinant protein expression in E. coli.