Gaseous Signaling Compounds (Hydrogen Sulfide and Nitric Oxide) and Their Relative Roles in Affecting Anaerobic HeLa 229 Cell Viability

Metabolic pathways supporting long-term anaerobic cell viability have not been identified. The effect NO and H2S pathway effectors have on HeLa 229 cell viability was measured after 10 days anaerobic incubation. The addition of arginine or xanthine (NO pathway precursors) consistently increased HeLa cell viability by 13.1- and 4.4-fold, respectively. Allopurinol, a xanthine oxidase inhibitor, also increased viability, as compared to control levels. In contrast, inhibition of iNOS by 1400W increased cell viability by 79-fold. Regarding the H2S pathway, precursor cysteine enhanced viability by 9.8-fold with the greatest number of viable cells measured in response to the presence of a H2S donor (GYY4137), or an inhibitor of glutathione synthesis, propargylglycine (40- and 85-fold, respectively). These results demonstrate that the constitutive level of cell viability after extended (10 days) growth without oxygen can be modulated by affecting NO or H2S generating pathways.

Quantifying the Inhibitory Impact of Soluble Phenolics on Carbon Mineralization from Sphagnum-rich Peatlands

The mechanisms controlling the extraordinarily slow carbon (C) mineralization rates characteristic of Sphagnum -rich peatlands (“bogs”) remain somewhat elusive, despite decades of research on this topic. Soluble phenolic compounds have been invoked as potentially significant contributors to bog peat recalcitrance due to their affinity to slow microbial metabolism and cell growth. Despite this potentially significant role, the effects of soluble phenolic compounds on bog peat C mineralization remain unclear. We analyzed this effect by manipulating the concentration of free soluble phenolics in anaerobic bog peat incubations using water-soluble polyvinylpyrrolidone (PVP), a compound that binds with and inactivates phenolics, preventing phenolic-enzyme interactions. CO 2 and CH 4 production rates (end-products of C mineralization) correlated positively with PVP concentration following Michaelis-Menten (M.M.) saturation functions. Using M.M. parameters, we determined that soluble phenolics inhibit, at minimum, 57 ± 16% of total C (CO 2 +CH 4 ) mineralization in the anaerobic incubation conditions studied. These findings are consistent with other studies that have indicated that soluble phenolics play a significant role in regulating bog peat stability in the face of decomposition.

Optimization of an Assay To Determine Colonization Resistance to Clostridioides difficile in Fecal Samples from Healthy Subjects and Those Treated with Antibiotics

ABSTRACTA healthy, intact gut microbiota is often resistant to colonization by gastrointestinal pathogens. During periods of dysbiosis, however, organisms such as Clostridioides difficile can thrive. We describe an optimized in vitro colonization resistance assay for C. difficile in stool (CRACS) and demonstrate the utility of this assay by assessing changes in colonization resistance following antibiotic exposure. Fecal samples were obtained from healthy volunteers (n = 6) and from healthy subjects receiving 5 days of moxifloxacin (n = 11) or no antibiotics (n = 10). Samples were separated and either not manipulated (raw) or sterilized (autoclaved or filtered) prior to inoculation with C. difficile ribotype 027 spores and anaerobic incubation for 72 h. Different methods of storing fecal samples were also investigated in order to optimize the CRACS. In healthy, raw fecal samples, incubation with spores did not lead to increased C. difficile total viable counts (TVCs) or cytotoxin detection. In contrast, increased C. difficile TVCs and cytotoxin detection occurred in sterilized healthy fecal samples or those from antibiotic-treated individuals. The CRACS was functional with fecal samples stored at either 4°C or −80°C but not with those stored with glycerol (12% or 30% [vol/vol]). Our data show that the CRACS successfully models in vitro the loss of colonization resistance and subsequent C. difficile proliferation and toxin production. The CRACS could be used as a proxy for C. difficile infection in clinical studies or to determine if an individual is at risk of developing C. difficile infection or other potential infections occurring due to a loss of colonization resistance.

Redox Chemistry and Nutrient Release from Organic Amended Terrace Soil under Anaerobic Incubation

Aims: To examine the changes in pH and Eh values of terrace soils during anaerobic incubation when amended with different organic materials, and to study N, P and S release from different manure and bio-slurry in terrace soil under anaerobic condition. Study Design: The experiment was carried out following Complete randomized design (CRD) with two replications. Place and Duration of Study: A laboratory incubation study was conducted in Soil Science Laboratory, Bangladesh Agricultural University, Mymensingh in December 2014 for 98 days. Methodology: The surface (0-15 cm) soil sample was collected from rice growing field of a selected area of Bhaluka, Mymensingh. The incubation study was carried out using four different sources of organic manures with two replications and five treatment combinations. The changes in soil pH, Eh and release pattern of N, P, and S from some organic materials in terrace soil under anaerobic incubation were investigated during December 2014 to April 2015. The soil was amended with all the treatments at 2g 100 g-1 (air dry basis) soil and incubated for 14 weeks at 25° C. The N, P and S release were determined by the measurement of NH4-N, phosphate P and SO4-S on destructive sampling at every two weeks. Results: The pH values increased at initial stage but gradually decreased over time to neutral and the soil became reduced with the advancement of incubation, it varies (1-3 weeks) depending on the quality of organic matter used. The reduction potential showed a significant variation among the treatments. Overall, when the pH values were averaged over the weeks, the highest pH value was measured in poultry bio-slurry (PB) amended soil followed by poultry manure (PM), cow dung bio-slurry (CDB) and cow dung (CD) amended soils and the lowest was in control. In case of Eh, the most negative (-133.08) Eh value was measured in PM amended soil followed by poultry bio-slurry (PB), CD and CDB amended soils. Control soil had comparatively positive Eh value. At the end of incubation, the highest amount of NH4-N found in CDB followed by CD, PM, PB and the lowest was in control; the highest amount of phosphate P found in PB followed by PM, control, CDB and CD; the highest amount of SO4-S found in PB followed by PM, CD, CDB and the lowest was in control. Conclusion: PB is the best source of organic amendment with respect to release of P and S, whereas CDB showed the best performance in release of N. Nutrient release and availability in reduced environment in terrace soil are a function of soil redox chemistry which is influenced by the quality and quantity of organic matter.

Characterization of dissolved organic matter extracted from water treatment sludge

The characteristics of dissolved organic matter (DOM) that formed during the aerobic and anaerobic incubation of drinking water treatment sludge stored at different temperatures (5 °C, 20 °C, 40 °C) for long periods (7, 14, and 21 days) were investigated. Anaerobic incubation at high temperatures with prolonged storage was found to result in higher organic content than aerobic incubation (3.6–6.8 times at 40 °C). The high temperatures caused changes in the DOM fractions, with humic-like substances mainly formed in aerobic incubation and protein-like substances in anaerobic incubation. Results showed that the fluorescence intensity of humic-like and protein-like substances increased by 45% and 22%, respectively, at the end of the anaerobic incubation period. The UV-absorbing DOM constituents in aerobic incubation had lower molecular weights and were more heterogeneous than those in anaerobic incubation.

Impact of Different Parameters upon the Production of Virulence Factors in Escherichia coli Strains Isolated from Marine Water

This study aimed to look into the expression of 10 virulence factors (VF) in 100 Escherichia coli strains harvested from the Black Sea Coast. E. coli strains were grown under different conditions, simulating various environmental stressors. The VF production [esculinase, amylase, pore-forming enzymes (hemolysin - spot and CAMP-like hemolysis; lipase; lecithinase)], lysine-decarboxylase, proteases (caseinase, gelatinase) and DNase was investigated at variable temperatures (4°C, 22°C, 37°C, 44°C and 56°C), NaCl (from 0 to 10%) and glucose concentrations (1.5% and 3%), different pH values (5.0, 7.2 and 9.6) and also in aerobic or anaerobic incubation conditions. The investigation of E. coli strains unraveled their ability to grow at 22°C, 37°C, 44°C, regardless of the salinity, pH, and glucose concentration, both in aerobic and anaerobic incubation conditions. The VF were better expressed at 37°C, followed by 22°C, especially siderophores, amylase, and caseinase production. The expression of different VF was variable at a certain salinity, i.e., at 0% NaCl, only amylase and siderophores production was observed. At 2% and 3%, the amylase was better expressed. The best expression of siderophores and caseinase was at 6% NaCl. At higher salinity, the expression of VF started to decrease. The amylase and caseinase were better expressed at pH 9.6 and siderophores at pH 7.2. Higher glucose concentrations (3%) proved to have an inhibitory effect on amylase expression and caseinase. The aerobic/anaerobic incubation conditions exhibited no significant differences in the VF expression. In conclusion, these outcomes reveal the ability of enterobacterial aquatic strains to survive in the presence of different stressors and maintain the expression of potential VF expression even in extreme environmental conditions.

Paddy soil fertilization, organic and inorganic alternative electron acceptors shape microbial community network and determine competitive pathways of Anaerobic Oxidation of Methane

<p>Anaerobic oxidation of methane (AOM) is a globally important CH<sub>4</sub> sink that is offsetting potential CH<sub>4</sub> emission into the atmosphere. The AOM depends on the availability of the alternative to oxygen electron acceptors (AEAs) which can be of inorganic (e.g. NO<sub>3</sub><sup>-</sup>, Fe<sup>3+</sup>, SO<sub>4</sub><sup>2-</sup>), and organic (e.g. humic acids) origin. Flooded paddy soils are among the ecosystems with pronounced AOM. Due to a variety of fertilization practices, including combinations of mineral (NPK) and organic (pig manure, biochar) fertilizers, there is a range of AEAs available in paddy soil under anaerobic conditions. However, it remains unclear whether (i) AOM has a preferential pathway in paddy soil, and (ii) how do AEAs and fertilization type affect anaerobic microbial interactions. Therefore, we tested the effects of key AEAs – NO<sub>3</sub><sup>-</sup>, Fe<sup>3+</sup>, SO<sub>4</sub><sup>2-</sup>, and humic acids – on bacterial community structure (by 16s rRNA gene sequencing) in paddy soil with ongoing AOM experiment under mineral and organic fertilization. We hypothesized that incorporation of labeled <sup>13</sup>C-CH<sub>4</sub> during AOM into CO<sub>2</sub> and phospholipid fatty acid biomarkers (PLFA) along with co-occurrence bacterial network analysis will reveal the preferential AOM pathway as related to a type of fertilization.</p><p>Bacterial alpha-diversity was significantly increased after 84-day anaerobic incubation. Pig manure significantly increased the microbial biomass as compared with NPK and Biochar, but the AEAs amendment did not affect the biomass. Anaerobic incubation, fertilization treatments specific biochar and NPK, and AEAs amendments specific SO<sub>4</sub><sup>2-</sup> and humic acids were factors contributing to microbiome variation. Network analysis indicated that microbial communities involved in CH<sub>4</sub> cycling (i.e. NC10, sulfate-reducing bacteria, Geobacter, syntrophic bacteria with methanogens and ANME-2) had non-random co-occurrence patterns and was modularized. There were 16 <sup>13</sup>C-enriched PLFA biomarkers confirming the incorporation of C-CH<sub>4</sub> into bacteria. AOM and <sup>13</sup>C-PLFA were significantly higher under Pig manure relative to other fertilizations. AOM was more intensive under NO<sub>3</sub><sup>-</sup> than Fe<sup>3+</sup> and humic acids, but was close to zero under SO<sub>4</sub><sup>2-</sup> amendment. However, the relative abundance of NC10 phylum which includes organisms performing AOM, and sulfate-reducing bacteria were higher under SO<sub>4</sub><sup>2-</sup>. The relative abundance of <em>Geobacter</em> was highest under biochar and NPK fertilization with SO<sub>4</sub><sup>2-</sup> and humic acids amendments. Taken together, NO<sub>3</sub><sup>-</sup>-driven AOM is the most potent AOM pathway in paddy soil, which however co-exists with the AOM pathways via reduction of NO<sub>2</sub><sup>- </sup>by NC10 bacteria and reduction of Fe<sup>3+</sup> and humic acids by consortia of ANME with <em>Geobacter</em>. Consequently, the co-occurrence network and evidence from <sup>13</sup>C incorporation into CO<sub>2</sub> and PLFAs indicate the multiple competitive pathways of AOM in paddy soil.</p>

Effect of Dissimilatory Iron Reduction on the Reduction of CH4Production in Landfill Conditions

A high solid anaerobic incubation system was used to study the effect of dissimilatory iron reduction on the inhibition of methanogenesis in a landfill. Different iron sources including FeC6H5O7, Fe2(SO4)3, Fe2O3, and Fe0were studied. The different iron sources significantly affected the methanogenesis process in the simulated landfill system. FeC6H5O7and Fe2(SO4)3inhibited methanogenesis but Fe0and Fe2O3increased it. The dissimilatory iron reduction with FeC6H5O7as the iron source demonstrates an anaerobic mineralization process, which enhances the biodegradation but inhibits methanogenesis. The highest rate of reduction of CH4production (51.9%) was obtained at a dosage rate of 16,000 mg·kg−1, which corresponded to a reduction of 0.86 g of CH4per kg of organic matrix. Active inhibition by methanogens using both the hydrogenotrophic and acetoclastic pathways is considered to be the main mechanism underlying the reduction of CH4production by dissimilatory iron reduction with FeC6H5O7as the iron source. This is the first report on the effect of different iron forms on the reduction of CH4production during landfilling with organic solid waste.

Reduced Time to Positive Cutibacterium acnes Culture Utilizing a Novel Incubation Technique: A Retrospective Cohort Study

Introduction Cutibacterium acnes ( C. acnes) is a common pathogen in postoperative shoulder infections. The purpose of this study was to evaluate the time to positive cultures for C. acnes and compare our experience before and after implementation of a regulated anaerobic chamber system. We hypothesized that this would reduce the time to identify positive cultures. Methods This was a retrospective review of 34 patients with cultures obtained from the shoulder that were positive for C. acnes. The time until positive result was evaluated before and after implementation of a regulated anaerobic incubation chamber. Results Following implementation of the regulated anaerobic incubation chamber, the time until C. acnes culture growth significantly decreased from 6.5 days (range 3–10 days) to 4.9 days (range 2.75–10 days) (mean difference: 1.6 days, 95% confidence interval: 1.06–2.66 days; P = .002). True infections had a significantly shorter time to positive culture compared to contaminants (5.5 vs 6.8 days, respectively, P = .003). Increased number of positive culture specimens correlated with a shorter time to positivity (Spearman rank = −0.58, P = .007). Conclusion Improved anaerobic culture protocols and techniques may lead to greater accuracy and earlier diagnosis and initiation of treatment of postoperative shoulder infections.