Evaluation of Electrosorption Process for Phosphate and Nitrate Removal from Wastewater

The present work is dedicated to the preparation and characterization of carbon-based electrodes for the removal of phosphates and nitrate ions from wastewater by CDI method. Carbons obtained from the pyrolysis were used to prepare electrodes and these electrodes were characterized using a number of experimental techniques. Based on the experimental results, the electrodes showed a strong affinity towards the nitrates than phosphates. This was evident from the kinetic constants and significantly higher capacity of electrosorption. At 1mM solutions, representative of a typical wastewater, nitrate exhibited about 3.5 times higher concentration than phosphates on a molar basis. The electrodes were reasonably stable under low concentrations of nitrates. At higher concentrations, the electrodes were not completely regenerable when the desorption step was carried out at 0V. These results are covered in this manuscript.publishedVersio

CAZymes from the thermophilic fungus Thermoascus aurantiacus are induced by C5 and C6 sugars

Background: Filamentous fungi are excellent lignocellulose degraders, which they achieve through producing carbohydrate active enzymes (CAZymes). CAZyme production is highly orchestrated and gene expression analysis has greatly expanded understanding of this important biotechnological process. The thermophilic fungus Thermoascus aurantiacus secretes highly active thermostable enzymes that enable saccharifications at higher temperatures; however, the genome-wide measurements of gene expression in response to CAZyme induction are not understood. Results: A fed-batch system with plant biomass-derived sugars D-xylose, L-arabinose and cellobiose established that these sugars induce CAZyme expression in T. aurantiacus. The C5 sugars induced both cellulases and hemicellulases, while cellobiose specifically induced cellulases. A minimal medium formulation was developed to enable gene expression studies of T. aurantiacus with these inducers. It was found that d-xylose and L-arabinose strongly induced a wide variety of CAZymes, auxiliary activity (AA) enzymes and carbohydrate esterases (CEs), while cellobiose facilitated lower expression of mostly cellulase genes. Furthermore, putative orthologues of different unfolded protein response genes were up-regulated during the C5 sugar feeding together with genes in the C5 sugar assimilation pathways. Conclusion: This work has identified two additional CAZyme inducers for T. aurantiacus, L-arabinose and cellobiose, along with D-xylose. A combination of biochemical assays and RNA-seq measurements established that C5 sugars induce a suite of cellulases and hemicellulases, providing paths to produce broad spectrum thermotolerant enzymatic mixtures

CAZymes from the thermophilic fungus Thermoascus aurantiacus are induced by C5 and C6 sugars.

BackgroundFilamentous fungi are excellent lignocellulose degraders, which they achieve through producing carbohydrate active enzymes (CAZymes). CAZyme production is highly orchestrated and gene expression analysis has greatly expanded understanding of this important biotechnological process. The thermophilic fungus Thermoascus aurantiacus secretes highly active thermostable enzymes that enable saccharifications at higher temperatures; however, the genome-wide measurements of gene expression in response to CAZyme induction are not understood.ResultsA fed-batch system with plant biomass-derived sugars D-xylose, L-arabinose and cellobiose established that these sugars induce CAZyme expression in T. aurantiacus. The C5 sugars induced both cellulases and hemicellulases, while cellobiose specifically induced cellulases. A minimal medium formulation was developed to enable gene expression studies of T. aurantiacus with these inducers. It was found that d-xylose and L-arabinose strongly induced a wide variety of CAZymes, auxiliary activity (AA) enzymes and carbohydrate esterases (CEs), while cellobiose facilitated lower expression of mostly cellulase genes. Furthermore, putative orthologues of different unfolded protein response genes were up-regulated during the C5 sugar feeding together with genes in the C5 sugar assimilation pathways.ConclusionThis work has identified two additional CAZyme inducers for T. aurantiacus, L-arabinose and cellobiose, along with D-xylose. A combination of biochemical assays and RNA-seq measurements established that C5 sugars induce a suite of cellulases and hemicellulases, providing paths to produce broad spectrum thermotolerant enzymatic mixtures

CAZymes from the thermophilic fungus Thermoascus aurantiacus are induced by C5 and C6 sugars

Background Filamentous fungi are excellent lignocellulose degraders, which they achieve through producing carbohydrate active enzymes (CAZymes). CAZyme production is highly orchestrated and gene expression analysis has greatly expanded understanding of this important biotechnological process. The thermophilic fungus Thermoascus aurantiacus secretes highly active thermostable enzymes that enable saccharifications at higher temperatures; however, the genome-wide measurements of gene expression in response to CAZyme induction are not understood. Results A fed-batch system with plant biomass-derived sugars d-xylose, l-arabinose and cellobiose established that these sugars induce CAZyme expression in T. aurantiacus. The C5 sugars induced both cellulases and hemicellulases, while cellobiose specifically induced cellulases. A minimal medium formulation was developed to enable gene expression studies of T. aurantiacus with these inducers. It was found that d-xylose and l-arabinose strongly induced a wide variety of CAZymes, auxiliary activity (AA) enzymes and carbohydrate esterases (CEs), while cellobiose facilitated lower expression of mostly cellulase genes. Furthermore, putative orthologues of different unfolded protein response genes were up-regulated during the C5 sugar feeding together with genes in the C5 sugar assimilation pathways. Conclusion This work has identified two additional CAZyme inducers for T. aurantiacus, l-arabinose and cellobiose, along with d-xylose. A combination of biochemical assays and RNA-seq measurements established that C5 sugars induce a suite of cellulases and hemicellulases, providing paths to produce broad spectrum thermotolerant enzymatic mixtures.ISSN:1754-683

The NDR Kinase Scaffold HYM1/MO25 Is Essential for MAK2 MAP Kinase Signaling in <em>Neurospora crassa</em>

<div><p>Cell communication is essential for eukaryotic development, but our knowledge of molecules and mechanisms required for intercellular communication is fragmentary. In particular, the connection between signal sensing and regulation of cell polarity is poorly understood. In the filamentous ascomycete <em>Neurospora crassa</em>, germinating spores mutually attract each other and subsequently fuse. During these tropic interactions, the two communicating cells rapidly alternate between two different physiological states, probably associated with signal delivery and response. The MAK2 MAP kinase cascade mediates cell–cell signaling. Here, we show that the conserved scaffolding protein HYM1/MO25 controls the cell shape-regulating NDR kinase module as well as the signal-receiving MAP kinase cascade. HYM1 functions as an integral part of the COT1 NDR kinase complex to regulate the interaction with its upstream kinase POD6 and thereby COT1 activity. In addition, HYM1 interacts with NRC1, MEK2, and MAK2, the three kinases of the MAK2 MAP kinase cascade, and co-localizes with MAK2 at the apex of growing cells. During cell fusion, the three kinases of the MAP kinase module as well as HYM1 are recruited to the point of cell–cell contact. <em>hym-1</em> mutants phenocopy all defects observed for MAK2 pathway mutants by abolishing MAK2 activity. An NRC1-MEK2 fusion protein reconstitutes MAK2 signaling in <em>hym-1</em>, while constitutive activation of NRC1 and MEK2 does not. These data identify HYM1 as a novel regulator of the NRC1-MEK2-MAK2 pathway, which may coordinate NDR and MAP kinase signaling during cell polarity and intercellular communication.</p> </div

Phenotypic characteristics of constitutive hyperactive NRC1 and MEK2 variants in the indicated strains.

*<p>in cm/day.</p>**<p>Basal MAK2 activity was determined as described in Material and Methods; MAK2 activity was calculated relative to wild type, whose activity was set to 100% (n≥3).</p

Δ<i>hym-1</i> displays phenotypic characteristics of MAK2 MAP kinase pathway mutants.

<p>(A) Phenotypic characterization of Δ<i>hym-1</i>, Δ<i>mak-2</i> and wild type regarding macroscopic morphology and conidiation pattern (left panel: growth in slants for 5 days on minimal medium), hyphal morphology (upper right panel; bar), and protoperithecia formation (lower right panel). (B) Production of conidiospores was quantified by counting conidia generated in slants grown at room temperature for 5 days (n = 5; standard deviations are indicated as bars). (C) HYM1 and MAK2 are required for vegetative cell fusion. Hyphal (left panel) and germling fusion events (right panel; fusion events are indicated by arrows) were assessed by light microscopy in wild type, Δ<i>hym-1</i> and Δ<i>mak-2</i> cultures. Cell fusion was not observed in Δ<i>hym-1</i> and Δ<i>mak-2</i>. (D) Quantification of cell fusion competence and formation of forced heterokarya that grew on double-selective media supplemented with 150 µg/ml hygromycin and 20 µg/ml nourseothricin. 10⁴ hygromycin-resistant conidiospores of the indicated strains (“wild type” carried an ectopically integrated (EC) hygromycin-resistance cassette, while the hygromycin-resistance cassette was used to replace the two gene deletions in Δ<i>hym-1</i> and Δ<i>mak-2</i>) were plated alone (second column) or in decreasing concentrations together with a second “wild type” strain that carried an ectopically integrated nourseothricin-resistance cassette (column 3–6). Column 1 indicated lack of growth of the “wild type” nat^R control strain. 1% sorbose was added to Vogels minimal medium to restrict radial growth of the forming colonies generated by forced heterokaryons after incubation for 3 days at room temperature. (E) HYM1 is required for MAK2 activity. Total soluble protein was extracted from the indicated strains grown in the presence or absence of 8 mM H₂O₂. Western blot analysis with anti-phospho-p42/p44 antibody detected activated MAK1 and MAK2, respectively. Equal loading was confirmed by re-probing the membrane with anti-tubulin antibody. A typical Western blot is shown. MAK1 and MAK2 activities from 5 independent experiments were quantified for the diagrams.</p

HYM1 is required for signal transduction through the entire MAK2 kinase cascade.

<p>(A) Constitutive hyperactive versions of NRC1 (NRC1(P448S)) and MEK2 (MEK2(S212D;T216D) do not activate MAK2 in a Δ<i>hym-1</i> background. Total soluble protein was extracted from the indicated strains grown at 25°C and Western blot analyzed with anti-phospho-p42/p44 antibody to probe for MAK2 activity. Equal loading was confirmed by re-probing the membrane with anti-FLAG antibody. (B) Expression of a MEK2-NRC1 fusion protein in Δ<i>hym-1</i> resulted in wild type levels of active MAK2 (determined by Western blot experiments with anti-phospho-p42/p44 antibody). Note that the stability of the MEK2-NRC1 fusion protein seems reduced in Δ<i>hym-1</i>, resulting in the consistent appearance of potential degradation products with smaller size (B). An anti-tubulin blot served as loading control to indicate equal protein levels. (C) Expression of a MEK2-NRC1 fusion protein complemented all Δ<i>hym-1</i> defects (panel 1: aerial mycelium formation in slants; panel 2: sexual development/protoperithecia formation; panel 3: germling fusion; panel 4: growth rate).</p