Anaerobic Chamber Research Articles

Soil organic matter affects arsenic and antimony sorption in anaerobic soils

Soil organic matter (SOM) affects arsenic (As) and antimony (Sb) mobility in soils under waterlogged conditions by acting as an electron donor, by catalyzing redox–cycling through electron shuttling and by acting as a competing ligand. This study was set up to disentangle these different effects of SOM towards As and Sb sorption in anaerobic soils. Nine samples were taken at different depths in an agricultural soil profile to collect samples with a natural SOM gradient (<1–40 g soil organic carbon kg−1). The samples were incubated either or not under waterlogged conditions in an anaerobic chamber for 63–70 days, and glucose (5 g C kg−1) was either or not added to the anaerobic incubated samples as an electron donor that neither acts as an electron shuttle nor as a competing ligand. The solid-liquid distribution coefficients (KD) of As and Sb were measured at trace levels. The KD values of As decreased ∼2 orders of magnitude upon waterlogging the SOM rich topsoil, while no additional changes were observed when glucose was added. In contrast, smaller changes in the As KD values were found in the low SOM containing subsoil samples, unless glucose was added that mobilised As. The Sb KD values increased upon reducing conditions up to factor 20, but again only in the high SOM topsoil samples. Surprisingly, the Sb immobilisation during waterlogging only occurred in Sb amended soils whereas the geogenic Sb was mobilised upon reducing conditions, although total dissolved Sb concentrations remained low (<10 nM). The change in As and Sb sorption upon waterlogging was similar in the SOM rich topsoil as in the low SOM subsoil amended with glucose. This suggests that the SOM dependent changes in As and Sb mobility in response to soil waterlogging are primarily determined by the role of SOM as electron donor.

PHLPP2 downregulation protects cardiomyocytes against hypoxia-induced injury through reinforcing Nrf2/ARE antioxidant signaling

Cardiomyocyte injury induced by acute myocardial infarction contributes to myocardial dysfunction. Accumulating evidence has demonstrated that pleckstrin homology domain leucine-rich repeat protein phosphatase 2 (PHLPP2) is a cytoprotective protein that protects against various adverse injuries. However, whether PHLPP2 participates in regulating myocardial-infarction-induced cardiomyocyte injury remains unknown. In the present study, we aimed to investigate the biological role and molecular mechanism of PHLPP2 in regulating hypoxia-induced cardiomyocyte injury. Cardiomyocytes were cultured in an anaerobic chamber for 24 h to induce hypoxic injury in vitro. The expression of PHLPP2 was determined by real-time quantitative PCR and Western blot analysis. Cell viability was measured by MTT assay. Cell apoptosis was assessed by TUNEL and caspase-3 activity assays. Intracellular reactive oxygen species (ROS) levels were measured by DCFH-DA probe. PHLPP2 expression was highly upregulated in hypoxia-injured cardiomyocytes. Inhibition of PHLPP2 by small interfering RNA (siRNA)-mediated gene silencing significantly improved the viability of hypoxia-injured cardiomyocytes and attenuated hypoxia-induced apoptosis and ROS production. In contrast, PHLPP2 overexpression exacerbated hypoxia-induced apoptosis and ROS production in cardiomyocytes. Mechanism research revealed that PHLPP2 silencing increased the phosphorylation of glycogen synthase kinase (GSK)-3β and promoted the nuclear translocation of nuclear factor (erythroid-derived 2)-like 2 (Nrf2). In addition, PHLPP2 inhibition promoted Nrf2/antioxidant response element (ARE) transcriptional activity. However, Nrf2 silencing markedly reversed PHLPP2-inhibition-mediated cardioprotection, while GSK-3β inhibition partially blocked the PHLPP2-overexpression-induced adverse effect. Taken together, these findings demonstrate that PHLPP2 inhibition alleviates hypoxia-induced cardiomyocyte injury by reinforcing Nrf2/ARE antioxidant signaling via inactivating GSK-3β, a pathway that highlights the importance of the PHLPP2/GSK-3β/Nrf2/ARE signaling axis in regulation of cardiomyocyte injury. Our study suggests a potential relevance for PHLPP2 in acute myocardial infarction, and this protein may serve as a promising target for cardioprotection.

186 Anaerobic Versus Aerobically Prepared Fecal Microbiota Transplantation Capsules: A Pilot Comparative Cohort Analysis Using a Novel Sequencing Approach

INTRODUCTION: Fecal microbiota transplantation (FMT) capsules have emerged as a therapeutic option for recurrent C.difficile infection (rCDI). Standard FMT capsules are prepared under aerobic conditions; however, anaerobic processing limits oxygen's impact on healthy anaerobic microbes, may enhance microbial community viability, improve engraftment, and may in turn increase efficacy. Thus, we compared the engraftment profiles of FMT capsules prepared by aerobic (AER) versus an anaerobic process (ANA) in rCDI patients. We applied a novel DNA-sequencing method (PMA-seq) to differentiate living and dead microbes. METHODS: We conducted a prospective cohort study of rCDI patients (3 or more confirmed episodes within 1 year) eligible for standard of care FMT. Subjects were enrolled sequentially to receive either a single administration of 30 capsules of standard AER or ANA. 3 healthy donors were use and stool donation was split; half prepared under anaerobic and half under aerobic conditions. This technique was used to address donor and stool specific confounding effects. All measures were taken to manage oxygen exposure, otherwise the split stool samples were treated identically. ANA preparations occurred inside an anaerobic chamber. Patients were assessed, and stool samples collected at 72 hours, 10 days, 4 weeks and 8 weeks post-FMT. Gut microbial communities were profiled via shotgun metagenomic and 16S sequencing. Additionally, PMA-seq, a novel species-specific live-dead assay was conducted. Pre and post treatment microbiome engraftment outcomes were analyzed. Assessment for clinical cure was done at week 8 [testing via (GDH/EIA)]. RESULTS: A total of 10 recurrent CDI patients were enrolled (5 AER and 5 ANA). Relevant characteristics were similar between the AER [mean age: 73 (SD = 15.9), mean recurrence 3.4] and ANA groups [mean age: 67.1 years (SD = 18.7), mean recurrence 3.0]. Overall, gut microbial community outcomes from ANA and AER, including alpha diversity, beta diversity and relative abundances, did not vary substantially by 16S, shotgun metagenomic sequencing or PMA-seq (Figures 1 and 2). Clinical cure at week 8 for AER and ANA was 100% (5/5 in each group). Both formulations were safe and well-tolerated. CONCLUSION: To our knowledge, this is the first study to compare aerobically versus anaerobically prepared FMT capsules, including assessment of live and dead specific-microbial profiles. This pilot study suggested there were no significant differences in engraftment profiles in rCDI.

PHYLOGENETIC ANALYSIS OF PROTOZOA AND SISTAN COW'S RUMEN BACTERIA FED WITH FORAGE RATIONS BY MOLECULAR AND LABORATORY METHODS

The present study was conducted to investigate the properties of rumen Microbiome in Sistani cattle by performing molecular experiments to clone the 18S rRNA genes of protozoans, culture rumen bacteria and then sequencing their 16S rRNA gene. Rumen liquid samples were collected from 10 Sistani cattle from the Sistan region, which were kept in a similar feeding group and fed on forage rations, through stomach tubes before feeding in the morning. The protozoa genome was extracted by the ASL buffer of the QiaAmp DNA stool kit (Qiagen), and their 18S rRNA gene was amplified and isolated with specific primers by the PCR method. The quantity and quality of DNA extracted with nanodrop 1000 and electrophoresis on 1% agarose gel, respectively, have been in The 18S rRNA protozoan gene was cloned using the T/A cloning technique in the PTZ plasmid and then the recombinant plasmid was transferred to E. coli.vestigated. The bacteria were cultured with the dependent method and then their 16S rRNA gene was sequenced. Rumen bacteria culture was performed in an anaerobic culture medium using an anaerobic chamber with flexible plastic gloves. After purification, the bacteria were partially studied morphologically. Then the bacterial genome was extracted using a kit, and the 16S rRNA gene was propagated by PCR methods. The sequences have been sent to determine the final structure of the gene for sequencing. In the end, the phylogenetic tree is drawn using the MEGAX software.Investigating of the microbial sequences obtained from the rumen of the cattle showed that the results are somewhat consistent with the results of other researchers in other countries and in other animals, but in some cases, there are significant differences. In this study, the Entodinium genus was the dominant protozoan group in the Sistani cattle's rumen. In the library, OTUs had a similarity of over 98.5% with the protozoan sequences identified in the database. The results of the culture of bacteria showed Ruminococcus albus, Ruminococcus flavefaciense, Clostridium colinum, Streptococcus equinus and butyrivibrio genus, which had a high similarity of 96.54% with the bacterial sequences identified in the database. Ruminal microbial ecology is very complex. The real scope of this diversity is determined by the use of molecular identification methods for species. The complexity of this diversity is determined by the use of 16S rRNA genes and 18S rRNA genes. Since each microbial species occupies a small area and is found only in some animals.

Basic Fibroblast Growth Factor Reduces Permeability and Apoptosis of Human Brain Microvascular Endothelial Cells in Response to Oxygen and Glucose Deprivation Followed by Reoxygenation via the Fibroblast Growth Factor Receptor 1 (FGFR1)/ERK Pathway.

BackgroundDisruption of the blood–brain barrier (BBB) is a mechanism in the pathogenesis of traumatic brain injury. Basic fibroblast growth factor (bFGF) is expressed in angiogenesis, neurogenesis, and neuronal survival. This study aimed to investigate the role of bFGF in vitro in human brain microvascular endothelial cells (HBMECs) challenged by oxygen-glucose deprivation/reperfusion (OGD/R).Material/MethodsHBMECs were cultured in glucose-free medium and an environment with <0.5% oxygen in an anaerobic chamber. Immunocytochemistry, Western blot, and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) were used to measure the protein and mRNA expression levels of bFGF, tight junction, adherens junction, apoptotic proteins, and matrix metalloproteinases (MMPs). The effects of bFGF on the viability of HBMECs was evaluated using the cell counting kit-8 (CCK-8) assay. Cell apoptosis was evaluated using the TUNEL assay, and endothelial permeability was quantified using a transwell migration assay with fluorescein isothiocyanate (FITC) conjugated with dextran. The effects of bFGF were evaluated following inhibition of fibroblast growth factor receptor 1 (FGFR1) with PD173074 and inhibition of ERK with PD98059.ResultsFollowing OGD/R of HBMECs, bFGF significantly reduced cell permeability and apoptosis and significantly inhibited the down-regulation of the expressions of proteins associated with tight junctions, adherens junctions, apoptosis and matrix metalloproteinases (MMPs). The effects of bFGF were mediated by the activation of FGFR1 and ERK, as they were blocked by FGFR1 and ERK inhibitors.ConclusionsPermeability and apoptosis of HBMECs challenged by OGD/R were reduced by bFGF by activation of the FGFR1 and the ERK pathway.

Air Abrasion With Bioactive Glass Eradicates Streptococcus mutans Biofilm From a Sandblasted and Acid-Etched Titanium Surface.

Streptococcus mutans is able to form a high-affinity biofilm on material surfaces. S mutans has also been detected around infected implants. Bioactive glasses (BAGs) have been shown to possess antibacterial effects against S mutans and other microorganisms. This in vitro study was performed to investigate the influence of BAG air abrasion on S mutans biofilm on sandblasted and acid-etched titanium surfaces. Sandblasted and acid-etched commercially pure titanium discs were used as substrates for bacteria (n = 107). The discs were immersed in an S mutans solution and incubated for 21 hours to form an S mutans biofilm. Twenty colonized discs were subjected to air abrasion with Bioglass 45S5 (45S5 BAG), experimental zinc oxide containing BAG (Zn4 BAG), and inert glass. After the abrasion, the discs were incubated for 5 hours in an anaerobic chamber followed by an assessment of viable S mutans cells. Surface morphology was evaluation using scanning electron microscopy (n = 12). The thrombogenicity of the glass particle-abraded discs (n = 75) was evaluated spectrophotometrically using whole-blood clotting measurement at predetermined time points. Air abrasion with 45S5 and Zn4 BAG eradicated S mutans biofilm. Significantly fewer viable S mutans cells were found on discs abraded with the 45S5 or Zn4 BAGs compared with the inert glass (P < .001). No significant differences were found in thrombogenicity since blood clotting was achieved for all substrates at 40 minutes. Air abrasion with BAG particles is effective in the eradication of S mutans biofilm from sandblasted and acid-etched titanium surfaces. Zn4 and 45S5 BAGs had similar biofilm-eradicating effects, but Zn4 BAG could be more tissue friendly. In addition, the steady release of zinc ions from Zn4 may enhance bone regeneration around the titanium implant and may thus have the potential to be used in the treatment of peri-implantitis. The use of either BAGs did not enhance the speed of blood coagulation.

Relationship between microbial community and environmental conditions in a constructed wetland system treating greywater

Knowledge of the diversity of microbial communities and their relationship with biogeochemical cycles is a key factor in understanding and improving the performance of constructed wetland (CW) systems, allowing improvements in design and operation methods to reach the desired effluent quality. Assemblies of microbial groups in CWs are driven by several factors, including the environmental conditions of the mesocosm and substrate availability. This study combined physicochemical analyses, high-throughput DNA sequencing, and statistical methods to gain an insight into the influence of operational conditions on the bacteria and archaea communities in a modified constructed wetland system treating raw-light greywater. The EvaTAC system is composed of an upflow evapotranspiration and treatment tank (CEvaT) which has an inbuilt anaerobic chamber (AnC), followed by a horizontal subsurface flow constructed wetland (HSSF-CW). Samples were collected after five years of system operation in both units: sludge from the AnC, coarse gravel from the CEvaT, and fine gravel from the CW. The results showed that the system operated predominantly in anaerobic conditions, with redox potential (Eh) increasing from the inlet (−364.1 mV) to the outlet (−240.4 mV) zone. The Eh was the environmental condition that most influenced the microbial community diversity and richness, and, as well as Eh, increased along the light greywater (LGW) flow. Conversely, the chemical oxygen demand (COD) decreased, thus suggesting a negative correlation among these factors. The clustering analyses of microbial community showed that, besides environmental conditions, the media filter and related depth could also be drivers of microbial community composition. The bottom layer of CEvaT was assigned to the Proteobacteria and Synergistetes phyla indicating the accomplishment of the first steps of anaerobic digestion. The next layer was characterised by the presence of Desulfovibrio, Syntrophobacter, and Methanobacterium, microorganisms responsible for the sulfidogenesis and methanogenesis processes. Finally, in the CW zone, the community composition was dominated by microorganisms involved in the methane oxidation, such as Methylosinus, Bradyrhizobium, and Candidatus Koribacter. This study explored the universe of bacteria and archaea communities, and is an initial step in elucidating microbial diversity conversions in CW systems treating greywater, which is important knowledge to steer the conversion pathway and establish the ideal environmental conditions for the development of desired microbial communities in these systems.

Differences between selected volatile aromatic compound concentrations in sludge samples in various steps of wastewater treatment plant operations

Sewage sludge, one of the main wastes generated during wastewater treatment, constitutes an important source of emissions of volatile chemical compounds such as volatile aromatic compounds These substances may undergo various changes as a result of operations and unit processes, which affects their concentrations in sewage sludge. An important factor determining the potential hazardousness of volatile organic compounds is the quality of wastewater delivered to wastewater treatment plants and the technical and equipment solutions applied to wastewater. In this study, a rapid and sensitive headspace gas chromatography method, coupled with tandem mass spectrometry using the standard addition method, was developed for the determination of selected volatile aromatic compounds in sewage sludge samples collected at different stages from three wastewater treatment plants located in Poland. This study attempted to assess the relationship between differences in the emissions of representative VACs and the given stage of the technological process within three analysed wastewater treatment plants. Toluene was detected with the highest frequency in analysed samples, at concentrations varying from 0.234 ± 0.035 ng/g of sludge to 28.3*102±3.2*102 ng/g of sludge. The highest concentration levels were determined for p-cresol, with concentrations ranging from 44.0*101±5.6*101 ng/g of sludge (sludge from aerobic chamber, wastewater treatment plant no.2) to 47.7*102±6.9*102 ng/g of sludge (sludge from aerobic chamber, wastewater treatment plant no.1), while the lowest concentration levels were observed for chlorobenzene, with concentrations ranging from 0.1300 ± 0.0030 ng/g of sludge (sludge from anaerobic chamber, wastewater treatment plant no.2), to 0.2606 ± 0.0046 ng/g of sludge (primary sludge, wastewater treatment plant no.1). The repeatability of the method was better than 10%, with accuracy levels in the ranges 89%–108%.Wastewater treatment technologies and residual sludge management in the selected wastewater treatment plantsinfluenced volatile aromatic compounds emission. Furthermore, the diversity of the wastewater quality, depending on the catchment area, is also an important factor determining the differentiation in volatile aromatic compounds emission. The microbial composition of raw wastewater highly influenced not only the treatment effectiveness of wastewater treatment plants but also the production of intermediate products, such as volatile aromatic compounds, which may contribute to odour emissions.

Anaerobic growth of Saccharomyces cerevisiae CEN.PK113-7D does not depend on synthesis or supplementation of unsaturated fatty acids.

ABSTRACTIn Saccharomyces cerevisiae, acyl-coenzyme A desaturation by Ole1 requires molecular oxygen. Tween 80, a poly-ethoxylated sorbitan-oleate ester, is therefore routinely included in anaerobic growth media as a source of unsaturated fatty acids (UFAs). During optimization of protocols for anaerobic bioreactor cultivation of this yeast, we consistently observed growth of the laboratory strain S. cerevisiae CEN.PK113-7D in media that contained the anaerobic growth factor ergosterol, but lacked UFAs. To minimize oxygen contamination, additional experiments were performed in an anaerobic chamber. After anaerobic precultivation without ergosterol and Tween 80, strain CEN.PK113-7D and a congenic ole1Δ strain both grew during three consecutive batch-cultivation cycles on medium that contained ergosterol, but not Tween 80. During these three cycles, no UFAs were detected in biomass of cultures grown without Tween 80, while contents of C10 to C14 saturated fatty acids were higher than in biomass from Tween 80-supplemented cultures. In contrast to its UFA-independent anaerobic growth, aerobic growth of the ole1Δ strain strictly depended on Tween 80 supplementation. This study shows that the requirement of anaerobic cultures of S. cerevisiae for UFA supplementation is not absolute and provides a basis for further research on the effects of lipid composition on yeast viability and robustness.

Simultaneous production of bioelectricity and biogas from chicken droppings and dairy industry wastewater employing bioelectrochemical system

Bioelectrochemical systems (BESs) provide an opportunity for COD removal in wastewater while generating electricity simultaneously. Previous studies have shown simultaneously wastewater treatment and hydrogen gas generation through catalytic reactions of microorganism under anaerobic condition. However, in this study, BES is developed to produce biogas instead of hydrogen from different compositions of wastewater while generating electricity. Dual chamber is designed and kept anaerobically over a period of 20 days in a temperature controlled (35 °C) anaerobic chamber. Wastewater of dairy milk industry is used along with chicken droppings as an organic substrate. BES is operated using three different substrate compositions designated as BES-A, BES-B and BES-C. The performance of BES having microbial bio-anode based on a hydrogen-philic methanogenic culture, capable of reducing CO2 and H+ into CH4 by gaining of electrons from cathode is investigated. The highest volume production rate of biogas upto 65 cm3/day in BES-A is obtained as compared to 45 and 37.5 cm3/day in BES-B and BES-C, respectively. Cyclic voltammetry analysis showed that the range of open circuit voltage (−800 to −1225 mV) is highest in BES-A due to the bio-catalytic activities. Moreover, the maximum volume of biogas, electric current, open circuit voltage, current density, power density and COD removal are obtained as 730 cm3, 0.57 mA, −1225 mV, 0.035 mA/cm2 and 43.29 mW/cm2 and 74.19%, respectively in BES-A, after 12 days of hydrolytic retention time. The electricity produced by the cell itself is utilized as cathodic potential for enhancing the efficiency of biogas production. The required potential for the production of biogas is −1400 to −1800 mV. However, the designed BES itself generated −800 to –1225 mV, out of which only −600 mV is supplied through the external source of battery to maintain the required voltage for biogas production. The extra voltage of ∼600 mV can be generated from produced biogas to develop a self-sustained system for wastewater treatment.

Among Live and Dead Bacteria, the Optimization of Sample Collection and Processing Remains Essential in Recovering Gut Microbiota Components.

The human gut microbiota plays an important role in human health. Accessing and culturing the gut microbes remains critical in microbiology. The culturomics approach, combined with sequencing strategies, provides new insights in the study of gut microbiota components. However, we are still far from having described all the microbes. Many factors are involved in recovering as much bacteria as possible. One of the most important factors is sample preparation and conservation. The aim of the present work was to evaluate three different types of stool sample conditioning by mainly studying the effect of atmospheric oxygen on bacterial viability and culturability. Quantitative analysis of fecal samples from eight different healthy individuals was carried out using a culture-independent method (flow cytometry) and a culture-dependent method (plating technique). We found that the cultured bacteria reached a 50% yield when the samples were exposed to oxygen for 120 min without any protectant medium, while the percentage of culturability increased to 67% in the presence of antioxidants. More importantly, when samples were exposed to oxygen for less than 2 min, combined with the work under the anaerobic chamber, no discordance was found between the two counting techniques and the culturability increased to 87%. Our study confirmed the importance of sample conditioning to preserve the bacterial viability in samples, especially for oxygen-sensitive intestinal bacteria.

Inducing in Situ Crystallization of Vivianite in a UCT-MBR System for Enhanced Removal and Possible Recovery of Phosphorus from Sewage.

By mimicking iron(Fe)-based phosphorus (P) immobilization in natural environments, an Fe-retrofitted UCT-MBR involving in situ vivianite crystallization for removing and recovering P from sewage was developed, and its performance was examined in this work. We show that dosing of ferrihydrite, once biological P uptake reached its limit, enabled effective ongoing P removal; whereas conventional conditions in the anaerobic chamber of the University of Cape Town (UCT) system (i.e., a sludge retention time of hours and a completely mixed sludge phase) was insufficient for a satisfactory Fe(III) bioreduction, with the overaccumulation of Fe(III) as fine particles finally resulting in severe membrane fouling and collapse in P removal. The enhancement of reductive conditions in the anaerobic chamber by lowering agitation and adding biocarriers to favor Fe(III) reduction was found to be effective in enabling ongoing P removal and recovery. The average level of effluent P was as low as 0.18 mg/L for a period of 258 d under this condition. Using chemical and spectroscopic methods, the P product was identified as primarily vivianite: Fe3(PO4)2·8H2O. The in situ crystallization of vivianite as a sink for P enabled the UCT-MBR to continuously remove and recover sewage P with no need for sludge discharge.

The Microflora Diversity and Profiles in Dental Plaque Biofilms on Brackets and Tooth Surfaces of Orthodontic Patients

Objective: Fixed orthodontic appliances may influence the oral environment through accumulation of plaque, decreased plaque pH, and increased gingival inflammation. These changes in the oral cavity can potentially lead to periodontal disease, demineralization, and other infectious diseases. Materials and Methods: To investigate the changes in biofilm throughout the initial 2 weeks, we placed a stainless steel bracket on the upper second premolar and collected plaque samples on the bracket and on the tooth surface at different time points (0, 24, 48 h and 1 and 2 weeks) and plated on tryptic soy agar blood agar plate, and kept at 37°C in an anaerobic chamber for 5 days to determine the CFUs of bacteria. At the end of 2 weeks, we removed the bracket and elastomeric module, and we isolated genomic DNA from the bacterial biofilm for identification of bacteria by 16S rRNA PCR analysis. We also analyzed the morphology of biofilm on the bracket by scanning electron microscope. Results: Our results show that the bacterial biofilm was significantly increased on the bracket in all the subjects, whereas on the tooth surface, the CFUs were not significantly increased. PCR assay showed that biofilm on orthodontic brackets from all subjects showed colonization by Streptococcus gordonii, Porphyromonas gingivalis and Streptococcus mutans were observed on some of the subjects after 48 h, whereas Aggregatibacter actinomycetemcomitans biofilm was observed in all the time points except 24 h. Conclusion: This study demonstrated that both periodontal and cariogenic bacterial biofilms were formed on the bracket as early as 24 h.

The response of five intestinal cell lines to anoxic conditions in vitro

In vivo oxygen levels in tissues range from 1% to 15%, while mechanistic cell culture studies employ an atmospheric oxygen level of 21% to grow cells. These oxygen concentrations are therefore not representative for conditions where the cell response is dependent on oxygen partial pressure. In pathological situation, such as (colon) cancer or chronic inflammation, tissue oxygenation is severely affected, and even under physiological conditions a steep oxygen gradient is present in the large intestine, where epithelial cells co-exist with microbial species, resulting in almost anoxia at the midpoint of the lumen. In these situations, a better characterisation of the essential cellular behaviour under hypoxia or anoxia is required. We have characterised the cellular response of commonly used cell cultures for the study of intestinal epithelial processes and colon cancer development (Caco-2, HT-29, SW480, HCT 116 and LoVo) under conventional normoxic conditions (21% O2 ) and in an anoxic (<0.1% O2 ) environment generated in an anaerobic chamber. In general, anoxic conditions led to lower levels of oxidative stress, a reduction in reduced glutathione/oxidised glutathione (GSH/GSSG) ratio, the shift of the redox status to oxidised glutathione levels, reduced cell proliferation, decreased barrier function and higher glycolysis rates at the expense of oxidative respiration. Continuous exposure to anoxic conditions, such as occurring at the host-microbe interface in the intestine, may create an adaptive metabolic cellular response of the cells. Considering adequate oxygen levels is essential for creating more physiologically relevant models for the study of host-microbe interactions and colon cancer development.

DuPont launches with a new take on innovation

Last year, DuPont opened the refurbished Building E353 at its 115-year-old Experimental Station in Wilmington, Delaware. The building serves as the global headquarters of the firm’s industrial bioscience division. The top floor houses executives and other business personnel. Scientists working on enzymes and probiotics occupy the three newly equipped levels below. A high-throughput screening laboratory allows DuPont researchers to rapidly evaluate new enzymes for applications such as laundry detergents. The scientists swap out amino acids in the proteins one by one and set up large arrays to see if they work under the temperature and pH conditions of washing machines. Another lab is equipped with a fluorescence-activated cell sorter for probiotic work and an Illumina DNA sequencer for enzyme and probiotic science. The building has an enormous anaerobic chamber, allowing scientists to study probiotic bacteria without exposing them to oxygen. In another room, scientists examine chicken gizzards brought in from

Isolation and culture of Methanobrevibacter smithii by co-culture with hydrogen-producing bacteria on agar plates

ObjectivesMethanogenic Archaea are considered as extremely oxygen-sensitive organisms, and their culture is fastidious, requiring specific equipment. We report here conditions allowing the cultivation of Methanobrevibacter smithii in an anaerobic chamber without the addition of hydrogen. MethodsWe first enriched the stool sample in an anaerobic liquid medium. To cultivate M. smithii with Bacteroides thetaiotaomicron and other hydrogen-producing bacteria on solid medium in an anaerobic chamber, we divided the agar plates into two compartments and seeded each strain on each compartment. Methane production was assessed by gas chromatography, and the growing colonies were authenticated by MALDI-TOF MS. ResultsWe successfully cultured M. smithii from a liquid culture medium inoculated with stool collected from a healthy donor in an anaerobic chamber. The isolation in pure culture permitted successful culture on agar medium by our performing a co-culture with B. thetaiotaomicron. We also successfully tested the co-cultivation of M. smithii with other known hydrogen-producing bacteria. Gas chromatographic tests showed that these strains produced hydrogen in different amounts. Agar colonies of methanogens were obtained by co-culture with these bacteria, and methane production was detected. ConclusionsWe propose a new approach to isolate and cultivate new strains of M. smithii by using a co-culture–based technique that can facilitate and make available the isolation of new methanogenic Archaea strains in clinical microbiology laboratories.

Growing Threat Increased Carbapenem-Resistance among Klebsiella pneumoniae; Antibiotic Susceptibility Pattern of Klebsiella pneumoniae in a Tertiary Care Hospital

Introduction Ultrasound technology has previously been applied for cataract removal and tennis elbow treatment. Recent data supports the use of ultrasonic debridement in the treatment of diabetic foot ulcers. No data is available concerning the potential antibacterial properties of a clinical grade, lower energy ultrasound probe. We investigated the effect of a ultrasonic probe with respect to P gingivalis bacterial viability. Methods A Tenex Tx1 probe with standard settings for clinical use was used for this study. A Gram negative (Porphyromonasgingivalis) bacteria, known for its pathological activity, was investigated. The bacteria was cultured in an anaerobic broth, re-suspended to achieve a consistent bacterial count, and 5ml of this re-suspension was placed in a test tube for testing. Each tube was sonicated with the Tx1 probe for varying lengths of time (10, 30, 60, 120 seconds). The sonicated was diluted and plated on blood-agar plates, followed by incubation for 48 hours at 37°C in an anaerobic growth chamber. The number of colony forming units were counted, on each plate and the anti-bacterial effect was calculated. A one way analysis of variance was performed for statistical analysis. Results A significant time-dependent antibacterial effect was demonstrated with sonication. When comparing the kill rate between the control and 120 seconds of sonication P Gingivalis had a 64% kill rate. This was the only statistically significant time comparison achieved, although the trend for all the time intervals was a reduction in the colony forming unit counts. Conclusion This study demonstrates that a clinically available ultrasonic probe (Tenex Tx1) has an antibacterial effect against the gram negative anaerobic bacterial species P gingivalis. Complete deactivation was not achieved, and there was a variation in effect dependent on the time of active sonication, with greater sonication times leading to greater kill rates. This data may partially help to explain the ability for ultrasonic debridement to result in the healing of long standing diabetic ulcers, that have been recalcitrant to other forms of treatment.

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

IntroductionCutibacterium 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.MethodsThis 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.ResultsFollowing 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).ConclusionImproved anaerobic culture protocols and techniques may lead to greater accuracy and earlier diagnosis and initiation of treatment of postoperative shoulder infections.

Performing anaerobic stopped-flow spectrophotometry inside of an anaerobic chamber.

The catalytic cycle of most flavin-dependent enzymes can be divided into oxidative and reductive half-reactions. Although some enzymes are oxidized by electron carrier proteins or organic compounds, many use oxygen as the final electron acceptor. In order to properly study the reductive half-reaction of flavin-dependent enzyme that react with oxygen, as in the case of oxidases and monooxygenases, it is necessary to establish anaerobic conditions that will only allow the reduction process to be monitored. The reduced flavoenzyme can be further studied by exposing it to oxygen to monitor the oxidative half-reaction. Anaerobic chambers provide an ideal environment for performing these experiments as they reliably maintain an anaerobic atmosphere inside a large workspace. A common tool used to study flavin-dependent enzymes is the stopped-flow spectrophotometry. This chapter describes methods for performing stopped-flow experiments in an anaerobic chamber. We include information about the chamber components, setting up a stopped-flow spectrophotometer inside of a chamber, preparing anaerobic solutions, and performing experiments to measure the reductive and oxidative half-reactions of flavin-dependent monooxygenases.

COD Removal and Electricity Generation From Domestic Wastewater Using Different Anode Materials in Microbial Fuel Cells

Microbial fuel cells (MFCs) set a new trend of converting chemical energy or bio energy to electricity from wastewater (domestic and industries) at the same time removal of chemical oxygen demand (COD) from the wastewater. Electrical energy generated from microbial fuel cell could be used for small electrical device example biosensors. The main components of MFCs are the anode, and the cathode salt bridge. It contains an anode chamber and a cathode chamber which separate electrodes for the production of electricity, using wastewater in an anaerobic chamber helps grow native microorganisms. Adding substrates increases productivity of the electrons that are moving from the anode chamber to the cathode chamber by help of the salt bridge. Bioreactors based on power generation in MFCs are a new approach to wastewater treatment. Power generation and current is modulated in this system. If it is optimised, MFCs would prove to be new method to offset wastewater treatment plant operating costs.