Sediment Phosphate Release Flux Under Hydraulic Disturbances in the Shallow Lake of Chaohu, China

Quantifying the effect of hydraulic disturbances on sediment phosphate release is a key issue in the water quality assessment of lakes, especially for the shallow lakes which are susceptible to winds and waves. Here, we sampled the original sediment columns from 12 positions in the eastern, central, and western areas of the Chaohu Lake, a representative shallow lake in China, and observed phosphate release under three levels of hydraulic disturbances in the laboratory. When the disturbance was weak and the surface sediment of bottom mud moved individually (the Individual Motion Mode), sediment phosphate release rate was insignificant (0.24 mg/m2/d). When the disturbance was medium and only a small percentage (<16%) of surface sediment started to move (the Small Motion Mode), phosphate release rate sharply increased to 4.81 mg/m2/d. When the disturbance was further strengthened and most (≥16%) of the surface sediment moved (the General Motion Mode), phosphate release rate was more than doubled (10.23 mg/m2/d). With the increase of hydraulic disturbance intensity, the variation range of phosphate release also became wider. Spatial distribution showed that the release rate varies the most in the western area, followed by the eastern and the central areas. By extrapolating the experimental results to the real scale, we found the phosphate release fluxes would probably fall within a wide range between 203.43 kg/d to 7311.01kg/d under different levels of hydrodynamic disturbances with considerably affects phosphate release from shallow lakes. This study also has implications for the pollutant management in other shallow lakes.

Changes in BNR Microbial Community in Response to Different Selection Pressure

This study investigated structural changes in microbial community of biological nutrient removal (BNR) in response to changes in substrate composition (ammonium and phosphate), redox condition, and morphological characteristics (flocs to granules), with a focus on nitrification and phosphate removal. Analyzing treatment performance and 16S rRNA phylogenetic gene sequencing data suggested that heterotrophic nitrification (HN) and autotrophic nitrification (AN) potentially happened in aerobic organic-rich (HN_AS) and aerobic organic-deficient (AN_AS) activated sludge batch reactors, respectively. However, phosphate release and uptake were not observed under alternating anaerobic/aerobic regime. Phosphate release could not be induced even when anaerobic phase was extended, although Accumulibacter existed in the inoculum (5.1% of total bacteria). Some potential HN (e.g., Thauera, Acinetobacter, Flavobacterium), AN (e.g., Nitrosomonas (3.2%) and Nitrospira), and unconventional phosphate-accumulating organisms (PAOs) were identified. Putative HN bacteria (i.e., Thauera (29–36%) and Flavobacterium (18–25%)) were enriched in aerobic granular sludge (AGS) regardless of the granular reactor operation mode. Enrichment of HN organisms in the AGS was suspected to be mainly due to granulation, possibly due to the floc-forming ability of HN species. Thus, HN is likely to play a role in nitrogen removal in AGS reactors. This study is supposed to serve as a starting point for the investigation of the microbial communities of AS- and AGS-based BNR processes. It is recommended that the identified roles for the isolated bacteria are further investigated in future works.

Assaying Paenibacillus alvei CsaB-Catalysed Ketalpyruvyltransfer to Saccharides by Measurement of Phosphate Release

Ketalpyruvyltransferases belong to a widespread but little investigated class of enzymes, which utilise phosphoenolpyruvate (PEP) for the pyruvylation of saccharides. Pyruvylated saccharides play pivotal biological roles, ranging from protein binding to virulence. Limiting factors for the characterisation of ketalpyruvyltransferases are the availability of cognate acceptor substrates and a straightforward enzyme assay. We report on a fast ketalpyruvyltransferase assay based on the colorimetric detection of phosphate released during pyruvyltransfer from PEP onto the acceptor via complexation with Malachite Green and molybdate. To optimise the assay for the model 4,6-ketalpyruvyl::ManNAc-transferase CsaB from Paenibacillus alvei, a β-d-ManNAc-α-d-GlcNAc-diphosphoryl-11-phenoxyundecyl acceptor mimicking an intermediate of the bacterium’s cell wall glycopolymer biosynthesis pathway, upon which CsaB is naturally active, was produced chemo-enzymatically and used together with recombinant CsaB. Optimal assay conditions were 5 min reaction time at 37 °C and pH 7.5, followed by colour development for 1 h at 37 °C and measurement of absorbance at 620 nm. The structure of the generated pyruvylated product was confirmed by NMR spectroscopy. Using the established assay, the first kinetic constants of a 4,6-ketalpyuvyl::ManNAc-transferase could be determined; upon variation of the acceptor and PEP concentrations, a KM, PEP of 19.50 ± 3.50 µM and kcat, PEP of 0.21 ± 0.01 s−1 as well as a KM, Acceptor of 258 ± 38 µM and a kcat, Acceptor of 0.15 ± 0.01 s−1 were revealed. P. alvei CsaB was inactive on synthetic pNP-β-d-ManNAc and β-d-ManNAc-β-d-GlcNAc-1-OMe, supporting the necessity of a complex acceptor substrate.

Key computational findings reveal proton transfer as driving the functional cycle in the phosphate transporter PiPT

Phosphate is an indispensable metabolite in a wide variety of cells and is involved in nucleotide and lipid synthesis, signaling, and chemical energy storage. Proton-coupled phosphate transporters within the major facilitator family are crucial for phosphate uptake in plants and fungi. Similar proton-coupled phosphate transporters have been found in different protozoan parasites that cause human diseases, in breast cancer cells with elevated phosphate demand, in osteoclast-like cells during bone reabsorption, and in human intestinal Caco2BBE cells for phosphate homeostasis. However, the mechanism of proton-driven phosphate transport remains unclear. Here, we demonstrate in a eukaryotic, high-affinity phosphate transporter from Piriformospora indica (PiPT) that deprotonation of aspartate 324 (D324) triggers phosphate release. Quantum mechanics/molecular mechanics molecular dynamics simulations combined with free energy sampling have been employed here to identify the proton transport pathways from D324 upon the transition from the occluded structure to the inward open structure and phosphate release. The computational insights so gained are then corroborated by studies of D45N and D45E amino acid substitutions via mutagenesis experiments. Our findings confirm the function of the structurally predicted cytosolic proton exit tunnel and suggest insights into the role of the titratable phosphate substrate.