Influence of TIG and Laser Welding Processes of Fe-10Cr-4Al-RE Alloy Cracks Overlayed on 316L Steel Plate.

In this paper, the possibility of applying different welding strategies to overlay an FeCrAl layer against corrosion from heavy liquid metal on a plain plate made of 316L austenitic stainless steel was investigated. This technology could be used in manufacturing the main vessel of CiADS, which may be considered as a more economic and feasible solution than production with the corrosion-resistant FeCrAl alloy directly. The main operational parameters of the laser welding process, including laser power, weld wire feeding speed, diameter of the welding wire, etc., were adjusted correspondingly to the optimized mechanical properties of the welded plate. After performing the standard nuclear-grade bending tests, it can be preliminarily confirmed that the low-power pulse laser with specific operational parameters and an enhanced cooling strategy will be suitable to surface an Fe-10Cr-4Al-RE layer with a thickness of approximately 1 mm on a 40 mm-thick 316L stainless steel plate, thanks to the upgraded mechanical properties incurred by refined grains with a maximum size of around 300 µm in the welded layer.

Production of Recombinant Laccase From Coprinopsis cinerea and Its Effect in Mediator Promoted Lignin Oxidation at Neutral pH.

Laccases are multi-copper oxidases that use molecular oxygen as the electron acceptor to oxidize phenolic and indirectly also non-phenolic substrates by mechanisms involving radicals. Due to their eco-friendliness and broad substrate specificity, laccases span a wide range of biotechnological applications. We have heterologously expressed a laccase from the coprophilic basidiomycete Coprinopsis cinerea (CcLcc9) in the methylotrophic yeast Pichia pastoris. The recombinant CcLcc9 (rCcLcc9) oxidized 2,6-dimethoxyphenol in the neutral pH range, and showed thermostability up to 70°C. The rCcLcc9 efficiently oxidized veratryl alcohol to veratraldehyde in the presence of low molecular weight mediators syringyl nitrile, methyl syringate and violuric acid, which are syringyl-type plant phenolics that have shown potential as natural co-oxidants for lignocellulosic materials. In addition, rCcLcc9 is able to depolymerize biorefinery hardwood lignin in the presence of methyl syringate and syringyl nitrile as indicated by gel permeation chromatography, and infrared spectral and nucleic magnetic resonance analyses. Furthermore, we showed that several added-value aromatic compounds, such as vanillin, vanillic acid, syringaldehyde, syringic acid and p-hydroxybenzoic acid, were formed during sequential biocatalytic chemical degradation of biorefinery lignin, indicating that rCcLcc9 harbors a great potential for sustainable processes of circular economy and modern biorefineries.

Depolymerization of biorefinery lignin by improved laccases of the white-rot fungus Obba rivulosa.

Fungal laccases are attracting enzymes for sustainable valorization of biorefinery lignins. To improve the lignin oxidation capacity of two previously characterized laccase isoenzymes from the white-rot fungus Obba rivulosa, we mutated their substrate-binding site at T1. As a result, the pH optimum of the recombinantly produced laccase variant rOrLcc2-D206N shifted by three units towards neutral pH. O. rivulosa laccase variants with redox mediators oxidized both the dimeric lignin model compound and biorefinery poplar lignin. Significant structural changes, such as selective benzylic α-oxidation, were detected by nuclear magnetic resonance analysis, although no polymerization of lignin was observed by gel permeation chromatography. This suggests that especially rOrLcc2-D206N is a promising candidate for lignin-related applications.

Enhanced Lignocellulolytic Enzyme Activities on Hardwood and Softwood during Interspecific Interactions of White- and Brown-Rot Fungi.

Wood decomposition is a sophisticated process where various biocatalysts act simultaneously and synergistically on biopolymers to efficiently break down plant cell walls. In nature, this process depends on the activities of the wood-inhabiting fungal communities that co-exist and interact during wood decay. Wood-decaying fungal species have traditionally been classified as white-rot and brown-rot fungi, which differ in their decay mechanism and enzyme repertoire. To mimic the species interaction during wood decomposition, we have cultivated the white-rot fungus, Bjerkandera adusta, and two brown-rot fungi, Gloeophyllum sepiarium and Antrodia sinuosa, in single and co-cultivations on softwood and hardwood. We compared their extracellular hydrolytic carbohydrate-active and oxidative lignin-degrading enzyme activities and production profiles. The interaction of white-rot and brown-rot species showed enhanced (hemi)cellulase activities on birch and spruce-supplemented cultivations. Based on the enzyme activity profiles, the combination of B. adusta and G. sepiarium facilitated birch wood degradation, whereas B. adusta and A. sinuosa is a promising combination for efficient degradation of spruce wood, showing synergy in ß-glucosidase (BGL) and α-galactosidase (AGL) activity. Synergistic BGL and AGL activity was also detected on birch during the interaction of brown-rot species. Our findings indicate that fungal interaction on different woody substrates have an impact on both simultaneous and sequential biocatalytic activities.

Analysis of the Accelerator-Driven System Fuel Assembly during the Steam Generator Tube Rupture Accident.

China is developing an ADS (Accelerator-Driven System) research device named the China initiative accelerator-driven system (CiADS). When performing a safety analysis of this new proposed design, the core behavior during the steam generator tube rupture (SGTR) accident has to be investigated. The purpose of our research in this paper is to investigate the impact from different heating conditions and inlet steam contents on steam bubble and coolant temperature distributions in ADS fuel assemblies during a postulated SGTR accident by performing necessary computational fluid dynamics (CFD) simulations. In this research, the open source CFD calculation software OpenFOAM, together with the two-phase VOF (Volume of Fluid) model were used to simulate the steam bubble behavior in heavy liquid metal flow. The model was validated with experimental results published in the open literature. Based on our simulation results, it can be noticed that steam bubbles will accumulate at the periphery region of fuel assemblies, and the maximum temperature in fuel assembly will not overwhelm its working limit during the postulated SGTR accident when the steam content at assembly inlet is less than 15%.

Simultaneous Measurement of Tricarboxylic Acid Cycle Intermediates in Different Biological Matrices Using Liquid Chromatography-Tandem Mass Spectrometry; Quantitation and Comparison of TCA Cycle Intermediates in Human Serum, Plasma, Kasumi-1 Cell and Murine Liver Tissue.

The tricarboxylic acid (TCA) cycle is a central part of carbon and energy metabolism, also connecting to glycolysis, amino acid, and lipid metabolism. The quantitation of the TCA cycle intermediate within one method is lucrative due to the interest in central carbon metabolism profiling in cells and tissues. In addition, TCA cycle intermediates in serum have been discovered to correspond as biomarkers to various underlying pathological conditions. In this work, an Liquid Chromatography-Mass Spectrometry/Mass Spectrometry-based quantification method is developed and validated, which takes advantage of fast, specific, sensitive, and cost-efficient precipitation extraction. Chromatographic separation is achieved while using Atlantis dC18 2.1 mm × 100 mm, particle size 3-µm of Waters column with a gradient elution mobile phase while using formic acid in water (0.1% v/v) and acetonitrile. Linearity was clearly seen over a calibration range of: 6.25 to 6400 ng/mL (r2 > 0.980) for malic acid; 11.72 to 12,000 ng/mL (r2 > 0.980) for cis-aconitic acid and L-aspartic acid; 29.30 to 30,000 ng/mL (r2 > 0.980) for isocitric acid, l-serine, and l-glutamic acid; 122.07 to 125,000 ng/mL (r2 > 0.980) for citric acid, glycine, oxo-glutaric acid, l-alanine, and l-glutamine; 527.34 to 540,000 ng/mL (r2 > 0.980) for l-lactic acid; 976.56 to 1,000,000 ng/mL (r2 > 0.980) for d-glucose; 23.44 to 24,000 ng/mL (r2 > 0.980) for fumaric acid and succinic acid; and, 244.14 to 250,000 ng/mL (r2 > 0.980) for pyruvic acid. Validation was carried out, as per European Medicines Agency (EMA) "guidelines on bioanalytical method validation", for linearity, precision, accuracy, limit of detection (LOD), limit of quantification (LLOQ), recovery, matrix effect, and stability. The recoveries from serum and tissue were 79-119% and 77-223%, respectively. Using this method, we measured TCA intermediates in serum, plasma (NIST 1950 SRM), and in mouse liver samples. The concentration found in NIST SRM 1950 (n = 6) of glycine (246.4 µmol/L), l-alanine (302.4 µmol/L), and serine (92.9 µmol/L).

Bayesian metabolic flux analysis reveals intracellular flux couplings.

MOTIVATION: Metabolic flux balance analysis (FBA) is a standard tool in analyzing metabolic reaction rates compatible with measurements, steady-state and the metabolic reaction network stoichiometry. Flux analysis methods commonly place model assumptions on fluxes due to the convenience of formulating the problem as a linear programing model, while many methods do not consider the inherent uncertainty in flux estimates. RESULTS: We introduce a novel paradigm of Bayesian metabolic flux analysis that models the reactions of the whole genome-scale cellular system in probabilistic terms, and can infer the full flux vector distribution of genome-scale metabolic systems based on exchange and intracellular (e.g. 13C) flux measurements, steady-state assumptions, and objective function assumptions. The Bayesian model couples all fluxes jointly together in a simple truncated multivariate posterior distribution, which reveals informative flux couplings. Our model is a plug-in replacement to conventional metabolic balance methods, such as FBA. Our experiments indicate that we can characterize the genome-scale flux covariances, reveal flux couplings, and determine more intracellular unobserved fluxes in Clostridium acetobutylicum from 13C data than flux variability analysis. AVAILABILITY AND IMPLEMENTATION: The COBRA compatible software is available at github.com/markusheinonen/bamfa. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Carbon 13-Metabolic Flux Analysis derived constraint-based metabolic modelling of Clostridium acetobutylicum in stressed chemostat conditions.

The metabolism of butanol producing bacteria Clostridium acetobutylicum was studied in chemostat with glucose limited conditions, butanol stimulus, and as a reference cultivation. COnstraint-Based Reconstruction and Analysis (COBRA) was applied using additional constraints from (13)C Metabolic Flux Analysis ((13)C-MFA) and experimental measurement results. A model consisting of 451 metabolites and 604 reactions was utilized in flux balance analysis (FBA). The stringency of the flux spaces considering different optimization objectives, i.e. growth rate maximization, ATP maintenance, and NADH/NADPH formation, for flux variance analysis (FVA) was studied in the different modelled conditions. Also a previously uncharacterized exopolysaccharide (EPS) produced by C. acetobutylicum was characterized on monosaccharide level. The major monosaccharide components of the EPS were 40n-% rhamnose, 34n-% glucose, 13n-% mannose, 10n-% galactose, and 2n-% arabinose. The EPS was studied to have butanol adsorbing property, 70(butanol)mg(EPS)g(-1) at 37°C.

Continuous propionic acid production with Propionibacterium acidipropionici immobilized in a novel xylan hydrogel matrix.

The cell immobilization potential of a novel xylan based disulfide-crosslinked hydrogel matrix reinforced with cellulose nanocrystals was studied with continuous cultivation of Propionibacterium acidipropionici using various dilution rates. The cells were immobilized to hydrogel beads suspended freely in the fermentation broth or else packed into a column connected to a stirred tank reactor. The maximum propionic acid productivity for the combined stirred tank and column was 0.88gL(-1)h(-1) and the maximum productivity for the column was determined to be 1.39gL(-1)h(-1). The maximum propionic acid titer for the combined system was 13.9gL(-1) with a dilution rate of 0.06h(-1). Dry cell density of 99.7gL(-1) was obtained within the column packed with hydrogel beads and productivity of 1.02gL(-1)h(-1) was maintained in the column even with the high circulation rate of 3.37h(-1).

Impact of varying lignocellulosic sugars on continuous solvent production in an immobilized column reactor.

The effect of varying glucose, mannose and xylose concentrations on continuous solvent production at various dilution rates was studied by multiple linear regression (MLR) modeling using an immobilized column reactor. The factors affecting the solvent production were dilution rate and concentrations of glucose and mannose. MLR-models also showed a preference of glucose as well as its inhibitory effect on xylose consumption. The fermentation process was studied at bigger scale with a volume factor of 17 with an added recirculation loop in the system. The up-scaled reactor produced 12.5 g/l of acetone-butanol-ethanol (ABE) solvents at a dilution rate of 0.23 h(-1), as compared to 13.4 g/l with a smaller column reactor. The xylose utilization was significantly higher in the modified reactor (73%) as compared to the small scale (43%).

Constraint-based genome-scale metabolic modeling of Clostridium acetobutylicum behavior in an immobilized column.

In this study a step-wise optimization procedure was developed to predict solvent production using continuous ABE fermentation with immobilized cells. The modeling approach presented here utilizes previously published constraint-based metabolic model for Clostridium acetobutylicum without direct flux constraints. A recently developed flux ratio constraint method was adopted for the model. An experimental data set consisting of 25 experiments using different sugar mixtures as substrates and differing dilution rates was simulated successfully with the modeling approach. Converted to end product concentrations the mean absolute error for acetone was 0.31 g/l, for butanol 0.49 g/l, and for ethanol 0.17 g/l. The modeling approach was validated with another data set from similar experimental setup. The model errors for the validation data set was 0.24 g/l, 0.60 g/l, and 0.17 g/l for acetone, butanol, and ethanol, respectively.

The effects of pH oscillation on Lactobacillus rhamnosus batch cultivation.

Inhomogeneous mixing in industrial-sized fermentation processes causes oscillations in process parameters such as temperature or pH value in the cultivation medium, which causes stress to the bacteria being cultivated. In this work, the impact of extracellular pH oscillations on the production of Lactobacillus rhamnosus, a well-studied probiotic bacteria, were investigated by means of a scale-down batch process, simulating inhomogeneous pH values by controlling the pH value of the medium on sinusoidal trajectories. Effects of pH stimulation on the bacteria were assessed by testing storage and freeze-drying stability of harvested cells, two factors relevant for the industrial process. Furthermore, gene expressions of six selected genes, i.e. atpA, fat, cfa, groEL, hrcA, and pstS, known to be related to stress response were monitored. Although storage stability is only slightly negatively affected by pH stimulation of the bacteria, gene expression of four of the studied genes, i.e. fat, hrcA, groEL, and pstS show to correlate with amplitude and frequency of the oscillation.

The effect of temperature and pH gradients on Lactobacillus rhamnosus gene expression of stress-related genes.

In this study, Lactobacillus rhamnosus, a renowned probiotic, was cultivated in fluctuating environment. Base gradients caused by a pH control in an industrial process and temperature gradients caused by uneven heating were simulated with a scale-down method. A pH gradient was created in a plug flow reactor (PFR). Expression of pH stress-related genes (atpA, aldB, cfa, groEL, hrcA and pstS) were studied as a relative gene expression study using ldhD as a reference gene. Expression measurements were carried out with the TRAC method. The responses of groEL, hrcA and atpA genes to temperature and pH changes were observed. The expression of phosphate uptake system-related pstS gene was induced almost linearly in the chemostat cultivation experiments when the base gradient in the PFR was increased. Correlations between the results from gene expression studies and freeze stability or acid stress survival were studied. However, by measuring the expression of these genes, we were not able to predict eventual freeze stability or survival from the acid stress test.