Label-free Quantitative Proteomics for the Extremely Thermophilic Bacterium <i>Caldicellulosiruptor obsidiansis</i> Reveal Distinct Abundance Patterns upon Growth on Cellobiose, Crystalline Cellulose, and Switchgrass

Mass spectrometric analysis of <i>Caldicellulosiruptor obsidiansis</i> cultures grown on four different carbon sources identified 65% of the cells’ predicted proteins in cell lysates and supernatants. Biological and technical replication together with sophisticated statistical analysis were used to reliably quantify protein abundances and their changes as a function of carbon source. Extracellular, multifunctional glycosidases were significantly more abundant on cellobiose than on the crystalline cellulose substrates Avicel and filter paper, indicating either disaccharide induction or constitutive protein expression. Highly abundant flagellar, chemotaxis, and pilus proteins were detected during growth on insoluble substrates, suggesting motility or specific substrate attachment. The highly abundant extracellular binding protein COB47_0549 together with the COB47_1616 ATPase might comprise the primary ABC-transport system for cellooligosaccharides, while COB47_0096 and COB47_0097 could facilitate monosaccharide uptake. Oligosaccharide degradation can occur either via extracellular hydrolysis by a GH1 β-glycosidase or by intracellular phosphorolysis using two GH94 enzymes. When <i>C. obsidiansis</i> was grown on switchgrass, the abundance of hemicellulases (including GH3, GH5, GH51, and GH67 enzymes) and certain sugar transporters increased significantly. Cultivation on biomass also caused a concerted increase in cytosolic enzymes for xylose and arabinose fermentation

Label-free Quantitative Proteomics for the Extremely Thermophilic Bacterium <i>Caldicellulosiruptor obsidiansis</i> Reveal Distinct Abundance Patterns upon Growth on Cellobiose, Crystalline Cellulose, and Switchgrass

Mass spectrometric analysis of <i>Caldicellulosiruptor obsidiansis</i> cultures grown on four different carbon sources identified 65% of the cells’ predicted proteins in cell lysates and supernatants. Biological and technical replication together with sophisticated statistical analysis were used to reliably quantify protein abundances and their changes as a function of carbon source. Extracellular, multifunctional glycosidases were significantly more abundant on cellobiose than on the crystalline cellulose substrates Avicel and filter paper, indicating either disaccharide induction or constitutive protein expression. Highly abundant flagellar, chemotaxis, and pilus proteins were detected during growth on insoluble substrates, suggesting motility or specific substrate attachment. The highly abundant extracellular binding protein COB47_0549 together with the COB47_1616 ATPase might comprise the primary ABC-transport system for cellooligosaccharides, while COB47_0096 and COB47_0097 could facilitate monosaccharide uptake. Oligosaccharide degradation can occur either via extracellular hydrolysis by a GH1 β-glycosidase or by intracellular phosphorolysis using two GH94 enzymes. When <i>C. obsidiansis</i> was grown on switchgrass, the abundance of hemicellulases (including GH3, GH5, GH51, and GH67 enzymes) and certain sugar transporters increased significantly. Cultivation on biomass also caused a concerted increase in cytosolic enzymes for xylose and arabinose fermentation

Label-free Quantitative Proteomics for the Extremely Thermophilic Bacterium <i>Caldicellulosiruptor obsidiansis</i> Reveal Distinct Abundance Patterns upon Growth on Cellobiose, Crystalline Cellulose, and Switchgrass

Mass spectrometric analysis of <i>Caldicellulosiruptor obsidiansis</i> cultures grown on four different carbon sources identified 65% of the cells’ predicted proteins in cell lysates and supernatants. Biological and technical replication together with sophisticated statistical analysis were used to reliably quantify protein abundances and their changes as a function of carbon source. Extracellular, multifunctional glycosidases were significantly more abundant on cellobiose than on the crystalline cellulose substrates Avicel and filter paper, indicating either disaccharide induction or constitutive protein expression. Highly abundant flagellar, chemotaxis, and pilus proteins were detected during growth on insoluble substrates, suggesting motility or specific substrate attachment. The highly abundant extracellular binding protein COB47_0549 together with the COB47_1616 ATPase might comprise the primary ABC-transport system for cellooligosaccharides, while COB47_0096 and COB47_0097 could facilitate monosaccharide uptake. Oligosaccharide degradation can occur either via extracellular hydrolysis by a GH1 β-glycosidase or by intracellular phosphorolysis using two GH94 enzymes. When <i>C. obsidiansis</i> was grown on switchgrass, the abundance of hemicellulases (including GH3, GH5, GH51, and GH67 enzymes) and certain sugar transporters increased significantly. Cultivation on biomass also caused a concerted increase in cytosolic enzymes for xylose and arabinose fermentation

Influence of Structural Defects on Biomineralized ZnS Nanoparticle Dissolution: An in-Situ Electron Microscopy Study

The dissolution of metal sulfides, such as ZnS, is an important biogeochemical process affecting fate and transport of trace metals in the environment. However, current studies of in situ dissolution of metal sulfides and the effects of structural defects on dissolution are lacking. Here we have examined the dissolution behavior of ZnS nanoparticles synthesized via several abiotic and biological pathways. Specifically, we have examined biogenic ZnS nanoparticles produced by an anaerobic, metal-reducing bacterium <i>Thermoanaerobacter</i> sp. X513 in a Zn-amended, thiosulfate-containing growth medium in the presence or absence of silver (Ag), and abiogenic ZnS nanoparticles were produced by mixing an aqueous Zn solution with either H₂S-rich gas or Na₂S solution. The size distribution, crystal structure, aggregation behavior, and internal defects of the synthesized ZnS nanoparticles were examined using high-resolution transmission electron microscopy (TEM) coupled with X-ray energy dispersive spectroscopy. The characterization results show that both the biogenic and abiogenic samples were dominantly composed of sphalerite. In the absence of Ag, the biogenic ZnS nanoparticles were significantly larger (i.e., ∼10 nm) than the abiogenic ones (i.e., ∼3–5 nm) and contained structural defects (e.g., twins and stacking faults). The presence of trace Ag showed a restraining effect on the particle size of the biogenic ZnS, resulting in quantum-dot-sized nanoparticles (i.e., ∼3 nm). In situ dissolution experiments for the synthesized ZnS were conducted with a liquid-cell TEM (LCTEM), and the primary factors (i.e., the presence or absence structural defects) were evaluated for their effects on the dissolution behavior using the biogenic and abiogenic ZnS nanoparticle samples with the largest average particle size. Analysis of the dissolution results (i.e., change in particle radius with time) using the Kelvin equation shows that the defect-bearing biogenic ZnS nanoparticles (γ = 0.799 J/m²) have a significantly higher surface energy than the abiogenic ZnS nanoparticles (γ = 0.277 J/m²). Larger defect-bearing biogenic ZnS nanoparticles were thus more reactive than the smaller quantum-dot-sized ZnS nanoparticles. These findings provide new insight into the factors that affect the dissolution of metal sulfide nanoparticles in relevant natural and engineered scenarios, and have important implications for tracking the fate and transport of sulfide nanoparticles and associated metal ions in the environment. Moreover, our study exemplified the use of an in situ method (i.e., LCTEM) to investigate nanoparticle behavior (e.g., dissolution) in aqueous solutions

Molecular Insights into Arctic Soil Organic Matter Degradation under Warming

Molecular composition of the Arctic soil organic carbon (SOC) and its susceptibility to microbial degradation are uncertain due to heterogeneity and unknown SOC compositions. Using ultrahigh-resolution mass spectrometry, we determined the susceptibility and compositional changes of extractable dissolved organic matter (EDOM) in an anoxic warming incubation experiment (up to 122 days) with a tundra soil from Alaska (United States). EDOM was extracted with 10 mM NH₄HCO₃ from both the organic- and mineral-layer soils during incubation at both −2 and 8 °C. Based on their O:C and H:C ratios, EDOM molecular formulas were qualitatively grouped into nine biochemical classes of compounds, among which lignin-like compounds dominated both the organic and the mineral soils and were the most stable, whereas amino sugars, peptides, and carbohydrate-like compounds were the most biologically labile. These results corresponded with shifts in EDOM elemental composition in which the ratios of O:C and N:C decreased, while the average C content in EDOM, molecular mass, and aromaticity increased after 122 days of incubation. This research demonstrates that certain EDOM components, such as amino sugars, peptides, and carbohydrate-like compounds, are disproportionately more susceptible to microbial degradation than others in the soil, and these results should be considered in SOC degradation models to improve predictions of Arctic climate feedbacks

Indexing Permafrost Soil Organic Matter Degradation Using High-Resolution Mass Spectrometry

<div><p>Microbial degradation of soil organic matter (SOM) is a key process for terrestrial carbon cycling, although the molecular details of these transformations remain unclear. This study reports the application of ultrahigh resolution mass spectrometry to profile the molecular composition of SOM and its degradation during a simulated warming experiment. A soil sample, collected near Barrow, Alaska, USA, was subjected to a 40-day incubation under anoxic conditions and analyzed before and after the incubation to determine changes of SOM composition. A CHO index based on molecular C, H, and O data was utilized to codify SOM components according to their observed degradation potentials. Compounds with a CHO index score between –1 and 0 in a water-soluble fraction (WSF) demonstrated high degradation potential, with a highest shift of CHO index occurred in the N-containing group of compounds, while similar stoichiometries in a base-soluble fraction (BSF) did not. Additionally, compared with the classical H:C vs O:C van Krevelen diagram, CHO index allowed for direct visualization of the distribution of heteroatoms such as N in the identified SOM compounds. We demonstrate that CHO index is useful not only in characterizing arctic SOM at the molecular level but also enabling quantitative description of SOM degradation, thereby facilitating incorporation of the high resolution MS datasets to future mechanistic models of SOM degradation and prediction of greenhouse gas emissions.</p></div

Heatmaps for CHO index as a function of molecular mass of extracted SOM compounds before and after the soil warming experiment.

<p>The color bar represents the relative abundance of compounds identified in each of the SOM extract: <b>(a)</b> WSF0, <b>(b)</b> WSF40, <b>(c)</b> BSF0, and <b>(d)</b> BSF40. A positive correlation between CHO index and mass can be observed for mass > 600 Da.</p

Heatmaps for CHO index as a function of the number of N atoms in extracted SOM compounds identified in (a) WSF0, (b) WSF40, (c) BSF0, and (d) BSF40.

<p>The color bar represents the relative abundance of compounds identified in each of the extract. Note the “island” formations observed for even numbers of N atoms in the BSF samples.</p

Molecular distribution of extracted SOM compounds from a 40-day soil warming incubation experiment.

<p>(a) Box-and-whisker plots of the mass distribution of SOM compounds, including the base-soluble fraction (BSF) at day 0 (BSF0) and day 40 (BSF40) and the water-soluble fraction (WSF) at day 0 (WSF0) and day 40 (WSF40). <b>(b and c)</b> van Krevelen diagram along with CHO index showing the molecular distribution of WSF SOM compounds before (b) and after (c) incubation. <b>(d)</b> Percentages of molecular formulae identified with CHO index values between -2 and 2 before and after soil incubation and are normalized to the total number of formulae displayed in (b) and (c). Compound classes are labeled above colored bars as follows: (A) lipids, (B) unsaturated hydrocarbons, (C) peptides, (D) aminosugars, (E) carbohydrates, (F) lignin, (G) condensed hydrocarbons, (H) tannins.</p

Number weighted (Mean _#) and magnitude-weighted mean (Mean _w) properties for SOM extracts from a simulated soil warming experiment.

<p>The CHO index was calculated as (2×[<i>O</i>]–[<i>H</i>])/[<i>C</i>].</p