Ratiometric Fluorescent Biosensors for Glucose and Lactate Using an Oxygen-Sensing Membrane

In this study, ratiometric fluorescent glucose and lactate biosensors were developed using a ratiometric fluorescent oxygen-sensing membrane immobilized with glucose oxidase (GOD) or lactate oxidase (LOX). Herein, the ratiometric fluorescent oxygen-sensing membrane was fabricated with the ratio of two emission wavelengths of platinum meso-tetra (pentafluorophenyl) porphyrin (PtP) doped in polystyrene particles and coumarin 6 (C6) captured into silica particles. The operation mechanism of the sensing membranes was based on (i) the fluorescence quenching effect of the PtP dye by oxygen molecules, and (ii) the consumption of oxygen levels in the glucose or lactate oxidation reactions under the catalysis of GOD or LOX. The ratiometric fluorescent glucose-sensing membrane showed high sensitivity to glucose in the range of 0.1–2 mM, with a limit of detection (LOD) of 0.031 mM, whereas the ratiometric fluorescent lactate-sensing membrane showed the linear detection range of 0.1–0.8 mM, with an LOD of 0.06 mM. These sensing membranes also showed good selectivity, fast reversibility, and stability over long-term use. They were applied to detect glucose and lactate in artificial human serum, and they provided reliable measurement results.

Pseudodesulfovibrio Alkaliphilus, Sp. Nov., An Alkaliphilic Sulfate-Reducing Bacterium Isolated From a Terrestrial Mud Volcano

The diversity of anaerobic microorganisms in terrestrial mud volcanoes is largely unexplored. Here we report the isolation of a novel sulfate-reducing alkaliphilic bacterium (strain F-1T) from a terrestrial mud volcano located at the Taman peninsula, Russia. Cells of strain F-1T were Gram- -negative motile vibrios with a single polar flagellum; 2.0–4.0 µm in length and 0.5 µm in diameter. The temperature range for growth was 6–37°C, with an optimum at 24°C. The pH range for growth was 7.0–10.5, with an optimum at pH 9.5. Strain F-1T utilized lactate, pyruvate, and molecular hydrogen as electron donors and sulfate, sulfite, thiosulfate, elemental sulfur, fumarate or arsenate as electron acceptors. In the presence of sulfate the end products of lactate oxidation were acetate, H2S and CO2. Lactate and pyruvate could also be fermented. The major product of lactate fermentation was acetate. The main cellular fatty acids were anteiso-С15:0, С16:0, С18:0, and iso-С17:1ω8. Phylogenetic analysis revealed that strain F-1T was most closely related to Pseudodesulfovibrio aespoeensis (98.05% similarity). The total size of the genome of the novel isolate was 3.23Mb and the genomic DNA G + C content was 61.93 mol%. The genome contained all genes essential for dissimilatoty sulfate reduction. We propose to assign strain F-1T to the genus Pseudodesulfovibrio, as a new species, Pseudodesulfovibrio alkaliphilus sp. nov. The type strain is F-1T (= KCTC 15918T = VKM B-3405T).

Three Novel Clostridia Isolates Produce n-Caproate and iso-Butyrate from Lactate: Comparative Genomics of Chain-Elongating Bacteria

The platform chemicals n-caproate and iso-butyrate can be produced by anaerobic fermentation from agro-industrial residues in a process known as microbial chain elongation. Few lactate-consuming chain-elongating species have been isolated and knowledge on their shared genetic features is still limited. Recently we isolated three novel clostridial strains (BL-3, BL-4, and BL-6) that convert lactate to n-caproate and iso-butyrate. Here, we analyzed the genetic background of lactate-based chain elongation in these isolates and other chain-elongating species by comparative genomics. The three strains produced n-caproate, n-butyrate, iso-butyrate, and acetate from lactate, with the highest proportions of n-caproate (18%) for BL-6 and of iso-butyrate (23%) for BL-4 in batch cultivation at pH 5.5. They show high genomic heterogeneity and a relatively small core-genome size. The genomes contain highly conserved genes involved in lactate oxidation, reverse β-oxidation, hydrogen formation and either of two types of energy conservation systems (Rnf and Ech). Including genomes of another eleven experimentally validated chain-elongating strains, we found that the chain elongation-specific core-genome encodes the pathways for reverse β-oxidation, hydrogen formation and energy conservation, while displaying substantial genome heterogeneity. Metabolic features of these isolates are important for biotechnological applications in n-caproate and iso-butyrate production.

Lactate-based microbial chain elongation for n-caproate and iso-butyrate production: genomic and metabolic features of three novel Clostridia isolates

Background The platform chemicals n-caproate and iso-butyrate can be produced by anaerobic fermentation from agro-industrial residues in a process known as microbial chain elongation. A few chain-elongating species have been discovered to utilize lactate and used to study the physiology of lactate-based chain elongation in pure cultures. Recently we isolated three novel clostridial species (strains BL-3, BL-4 and BL-6) that convert lactate to n-caproate and iso-butyrate. Here, we analyzed the genetic background of lactate-based chain elongation in these strains and other chain-elongating species by comparative genomics. Results All three strains produced n-caproate and iso-butyrate from lactate, with the highest proportions of n-caproate (18%) for BL-6 and iso-butyrate (23%) for BL-4 in batch cultivation at pH 5.5. The strains are suggested to represent three novel species based on low similarities with their closest described relatives. The three genomes show low conservation of organization and a relatively small core-genome size (504 out of 6,654 gene families). Including data of another eleven experimentally validated chain-elongating strains, we found that the chain elongation-specific core-genome harbors genes involved in reverse β-oxidation, hydrogen formation and energy conservation, displaying substantial genome heterogeneity. The three new isolates contain the genes for lactate oxidation and a gene cluster encoding enzymes of reverse β-oxidation, including the CoA transferase for the formation of n-caproate. Our analysis gave no hints on the isomerization pathway for iso-butyrate formation. An operon encoding the Rnf complex was found in BL-3 and BL-4 but not in BL-6, which may instead use the Ech hydrogenase complex for energy conservation. BL-3 and BL-6 were predicted to have genes encoding both the BCD/EtfAB complex and the LDH/EtfAB complex for energy coupling. Conclusions The genetic background of lactate-based chain elongation was confirmed in three novel Clostridia species that convert lactate to n-caproate and iso-butyrate. They contain highly conserved genes involved in reverse β-oxidation, hydrogen formation and either of two types of energy conservation systems (Rnf and Ech). Further research is needed to elucidate the mechanism of iso-butyrate formation in these strains. Features of the three isolates may be interesting for further applications in n-caproate and iso-butyrate production.

Increased Glycolysis and Higher Lactate Production in Hyperglycemic Myotubes

Previous studies have shown that chronic hyperglycemia impairs glucose and fatty acid oxidation in cultured human myotubes. To further study the hyperglycemia-induced suppression of oxidation, lactate oxidation, mitochondrial function and glycolytic rate were evaluated. Further, we examined the intracellular content of reactive oxygen species (ROS), production of lactate and conducted pathway-ANOVA analysis on microarray data. In addition, the roles of the pentose phosphate pathway (PPP) and the hexosamine pathway were evaluated. Lactic acid oxidation was suppressed in hyperglycemic versus normoglycaemic myotubes. No changes in mitochondrial function or ROS concentration were observed. Pathway-ANOVA analysis indicated several upregulated pathways in hyperglycemic cells, including glycolysis and PPP. Functional studies showed that glycolysis and lactate production were higher in hyperglycemic than normoglycaemic cells. However, there were no indications of involvement of PPP or the hexosamine pathway. In conclusion, hyperglycemia reduced substrate oxidation while increasing glycolysis and lactate production in cultured human myotubes.

Differentiation but not ALS mutations in FUS rewires motor neuron metabolism

Energy metabolism has been repeatedly linked to amyotrophic lateral sclerosis (ALS). Yet, motor neuron (MN) metabolism remains poorly studied and it is unknown if ALS MNs differ metabolically from healthy MNs. To address this question, we first performed a metabolic characterization of induced pluripotent stem cells (iPSCs) versus iPSC-derived MNs and subsequently compared MNs from ALS patients carrying FUS mutations to their CRISPR/Cas9-corrected counterparts. We discovered that human iPSCs undergo a lactate oxidation-fuelled prooxidative metabolic switch when they differentiate into functional MNs. Simultaneously, they rewire metabolic routes to import pyruvate into the TCA cycle in an energy substrate specific way. By comparing patient-derived MNs and their isogenic controls, we show that ALS-causing mutations in FUS did not affect glycolytic or mitochondrial energy metabolism of human MNs in vitro. These data show that metabolic dysfunction is not the underlying cause of the ALS-related phenotypes previously observed in these MNs.