Anoxic Culture Research Articles

Controlling Pericellular Oxygen Tension in Cell Culture Reveals Distinct Breast Cancer Responses to Low Oxygen Tensions.

In oxygen (O2)-controlled cell culture, an indispensable tool in biological research, it is presumed that the incubator setpoint equals the O2 tension experienced by cells (i.e., pericellular O2). However, it is discovered that physioxic (5% O2) and hypoxic (1% O2) setpoints regularly induce anoxic (0% O2) pericellular tensions in both adherent and suspension cell cultures. Electron transport chain inhibition ablates this effect, indicating that cellular O2 consumption is the driving factor. RNA-seq analysis revealed that primary human hepatocytes cultured in physioxia experience ischemia-reperfusion injury due to cellular O2 consumption. A reaction-diffusion model is developed to predict pericellular O2 tension a priori, demonstrating that the effect of cellular O2 consumption has the greatest impact in smaller volume culture vessels. By controlling pericellular O2 tension in cell culture, it is found that hypoxia vs. anoxia induce distinct breast cancer transcriptomic and translational responses, including modulation of the hypoxia-inducible factor (HIF) pathway and metabolic reprogramming. Collectively, these findings indicate that breast cancer cells respond non-monotonically to low O2, suggesting that anoxic cell culture is not suitable for modeling hypoxia. Furthermore, it is shown that controlling atmospheric O2 tension in cell culture incubators is insufficient to regulate O2 in cell culture, thus introducing the concept of pericellular O2-controlled cell culture.

Biofilm formation and inflammatory potential of Staphylococcus saccharolyticus: A possible cause of orthopedic implant-associated infections.

Staphylococcus saccharolyticus, a coagulase-negative staphylococcal species, has some unusual characteristics for human-associated staphylococci, such as slow growth and its preference for anoxic culture conditions. This species is a relatively abundant member of the human skin microbiota, but its microbiological properties, as well as the pathogenic potential, have scarcely been investigated so far, despite being occasionally isolated from different types of infections including orthopedic implant-associated infections. Here, we investigated the growth and biofilm properties of clinical isolates of S. saccharolyticus and determined host cell responses. Growth assessments in anoxic and oxic conditions revealed strain-dependent outcomes, as some strains can also grow aerobically. All tested strains of S. saccharolyticus were able to form biofilm in a microtiter plate assay. Strain-dependent differences were determined by optical coherence tomography, revealing that medium supplementation with glucose and sodium chloride enhanced biofilm formation. Visualization of the biofilm by confocal laser scanning microscopy revealed the role of extracellular DNA in the biofilm structure. In addition to attached biofilms, S. saccharolyticus also formed bacterial aggregates at an early stage of growth. Transcriptome analysis of biofilm-grown versus planktonic cells revealed a set of upregulated genes in biofilm-embedded cells, including factors involved in adhesion, colonization, and competition such as epidermin, type I toxin-antitoxin system, and phenol-soluble modulins (beta and epsilon). To investigate consequences for the host after encountering S. saccharolyticus, cytokine profiling and host cell viability were assessed by infection experiments with differentiated THP-1 cells. The microorganism strongly triggered the secretion of the tested pro-inflammatory cyto- and chemokines IL-6, IL-8, and TNF-alpha, determined at 24 h post-infection. S. saccharolyticus was less cytotoxic than Staphylococcus aureus. Taken together, the results indicate that S. saccharolyticus has substantial pathogenic potential. Thus, it can be a potential cause of orthopedic implant-associated infections and other types of deep-seated infections.

Long-term evaluation of the effect of peracetic acid on a mixed anoxic culture: Organic matter degradation, denitrification, and microbial community structure

The objective of this study was to assess the long-term effect of peracetic acid (PAA) solution on anoxic organic matter degradation and denitrification. Three bioreactors (R1, R2, and R3) were operated for 166 d, fed with poultry processing wastewater and gradually increased H2O2 (R2) or PAA solution (i.e., PAA + H2O2)(R3). Organic matter degradation, quantified as the extent of COD removal, and denitrification were assessed. COD removal and denitrification were not affected by H2O2 and PAA solutions added to the wastewater before feeding (indirect addition). However, both processes were significantly affected by high levels of H2O2 (i.e., 27 mg/L) and PAA solution (i.e., 60/8.4 mg/L PAA/H2O2) directly added to the reactors, but fast recovery was observed when direct addition of H2O2 and PAA solution ended. The negative impact on denitrification was the combined result of enzyme inhibition and loss of cell viability, both related to increased intracellular ROS levels. Partial acclimation to PAA solution took place during the long-term operation, resulting in higher denitrification activity, higher cell viability and lower intracellular ROS than in a short-term batch assay with one-time PAA addition. Microbiome analysis showed major shift in community composition during the long-term operation with PAA solution addition, but no significant shift after the long-term operation with H2O2 addition. Among the main denitrifying genera, Thauera and unclassified Chitinophagaceae were negatively impacted while Chryseobacterium and unclassified Enterobacteriaceae were enriched during the long-term operation with PAA addition.

The Potential for CH<sub>4</sub> Production by Syntrophic Microbial Communities in Diverse Deep Aquifers Associated with an Accretionary Prism and its Overlying Sedimentary Layers

Accretionary prisms are thick masses of sedimentary material scraped from the oceanic crust and piled up at convergent plate boundaries found across large regions of the world. Large amounts of anoxic groundwater and natural gas, mainly methane (CH4), are contained in deep aquifers associated with these accretionary prisms. To identify the subsurface environments and potential for CH4 production by the microbial communities in deep aquifers, we performed chemical and microbiological assays on groundwater and natural gas derived from deep aquifers associated with an accretionary prism and its overlying sedimentary layers. Physicochemical analyses of groundwater and natural gas suggested wide variations in the features of the six deep aquifers tested. On the other hand, a stable carbon isotope analysis of dissolved inorganic carbon in the groundwater and CH4 in the natural gas showed that the deep aquifers contained CH4 of biogenic or mixed biogenic and thermogenic origins. Live/dead staining of microbial cells contained in the groundwater revealed that the cell density of live microbial cells was in the order of 104 to 106‍ ‍cells‍ ‍mL–1, and cell viability ranged between 7.5 and 38.9%. A DNA analysis and anoxic culture of microorganisms in the groundwater suggested a high potential for CH4 production by a syntrophic consortium of hydrogen (H2)-producing fermentative bacteria and H2-utilizing methanogenic archaea. These results suggest that the biodegradation of organic matter in ancient sediments contributes to CH4 production in the deep aquifers associated with this accretionary prism as well as its overlying sedimentary layers.

Biohydrogen production of obligate anaerobic archaeon Thermococcus onnurineus NA1 under oxic conditions via overexpression of frhAGB-encoding hydrogenase genes

BackgroundThe production of biohydrogen (H2) as a promising future fuel in anaerobic hyperthermophiles has attracted great attention because H2 formation is more thermodynamically feasible at elevated temperatures and fewer undesired side products are produced. However, these microbes require anoxic culture conditions for growth and H2 production, thereby necessitating costly and time-consuming physical or chemical methods to remove molecular oxygen (O2). Therefore, the development of an O2-tolerant strain would be useful for industrial applications.ResultsIn this study, we found that the overexpression of frhAGB-encoding hydrogenase genes in Thermococcus onnurineus NA1, an obligate anaerobic archaeon and robust H2 producer, enhanced O2 tolerance. When the recombinant FO strain was exposed to levels of O2 up to 20% in the headspace of a sealed bottle, it showed significant growth. Whole transcriptome analysis of the FO strain revealed that several genes involved in the stress response such as chaperonin β subunit, universal stress protein, peroxiredoxin, and alkyl hydroperoxide reductase subunit C, were significantly up-regulated. The O2 tolerance of the FO strain enabled it to grow on formate and produce H2 under oxic conditions, where prior O2-removing steps were omitted, such as the addition of reducing agent Na2S, autoclaving, and inert gas purging.ConclusionsVia the overexpression of frhAGB genes, the obligate anaerobic archaeon T. onnurineus NA1 gained the ability to overcome the inhibitory effect of O2. This O2-tolerant property of the strain may provide another advantage to this hyperthermophilic archaeon as a platform for biofuel H2 production.

A Robust Longitudinal Co-culture of Obligate Anaerobic Gut Microbiome With Human Intestinal Epithelium in an Anoxic-Oxic Interface-on-a-Chip

The majority of human gut microbiome is comprised of obligate anaerobic bacteria that exert essential metabolic functions in the human colon. These anaerobic gut bacteria constantly crosstalk with the colonic epithelium in a mucosal anoxic-oxic interface (AOI). However, in vitro recreation of the metabolically mismatched colonic AOI has been technically challenging. Furthermore, stable co-culture of the obligate anaerobic commensal microbiome and epithelial cells in a mechanically dynamic condition is essential for demonstrating the host-gut microbiome crosstalk. Here, we developed an anoxic-oxic interface-on-a-chip (AOI Chip) by leveraging a modified human gut-on-a-chip to demonstrate a controlled oxygen gradient in the lumen-capillary transepithelial interface by flowing anoxic and oxic culture medium at various physiological milieus. Computational simulation and experimental results revealed that the presence of the epithelial cell layer and the flow-dependent conditioning in the lumen microchannel is necessary and sufficient to create the steady-state vertical oxygen gradient in the AOI Chip. We confirmed that the created AOI does not compromise the viability, barrier function, mucin production, and the expression and localization of tight junction proteins in the 3D intestinal epithelial layer. Two obligate anaerobic commensal gut microbiome, Bifidobacterium adolescentis and Eubacterium hallii, that exert metabolic cross-feeding in vivo, were independently co-cultured with epithelial cells in the AOI Chip for up to a week without compromising any cell viability. Our new protocol for creating an AOI in a microfluidic gut-on-a-chip may enable to demonstrate the key physiological interactions of obligate anaerobic gut microbiome with the host cells associated with intestinal metabolism, homeostasis, and immune regulation.

Rumen microbial responses to supplemental nitrate. I. Yeast growth and protozoal chemotaxis in vitro as affected by nitrate and nitrite concentrations

Nitrates have been fed to ruminants, including dairy cows, as an electron sink to mitigate CH4 emissions. In the NO3- reduction process, NO2- can accumulate, which could directly inhibit methanogens and some bacteria. However, little information is available on eukaryotic microbes in the rumen. Protozoa were hypothesized to enhance nitrate reductase but also have more circling swimming behavior, and the yeast Saccharomyces cerevisiae was hypothesized to lessen NO2- accumulation. In the first experiment, a culture of S. cerevisiae strain 1026 was evaluated under 3 growth phases: aerobic, anoxic, or transition to anoxic culture. Each phase was evaluated with a control or 1 of 3 isonitrogenous doses, including NO3-, NO2-, or NH4+ replacing peptone in the medium. Gas head phase, NO3-, or NH4+ did not influence culture growth, but increasing NO2- concentration increasingly inhibited yeast growth. In experiment 2, rumen fluid was harvested and incubated for 3 h in 2 concentrations of NO3-, NO2-, or sodium nitroprusside before assessing chemotaxis of protozoa toward glucose or peptides. Increasing NO2- concentration decreased chemotaxis by isotrichids toward glucose or peptides and decreased chemotaxis by entodiniomorphids but only toward peptides. Live yeast culture was inhibited dose-responsively by NO2- and does not seem to be a viable mechanism to prevent NO2- accumulation in the rumen, whereas a role for protozoal nitrate reductase and NO2- influencing signal transduction requires further research.

Mitofusin-2 mediated mitochondrial Ca2+ uptake 1/2 induced liver injury in rat remote ischemic perconditioning liver transplantation and alpha mouse liver-12 hypoxia cell line models.

AIMTo investigate the protective mechanism of mitofusin-2 (Mfn2) in rat remote ischemic perconditioning (RIC) models and revalidate it in alpha mouse liver-12 (AML-12) hypoxia cell lines.METHODSSprague-Dawley rats were divided into three groups (n = 6 each): sham, orthotopic liver transplantation and RIC. After operation, blood samples were collected to test alanine aminotransferase and aspartate aminotransferase. The liver lobes were harvested for histopathological examination, western blotting (WB) and quantitative real-time (qRT)-PCR. AML-12 cell lines were then subjected to normal culture, anoxic incubator tank culture (hypoxia) and anoxic incubator tank culture with Mfn2 knockdown (hypoxia + Si), and data of qRT-PCR, WB, mitochondrial membrane potential (ΔΨm), apoptosis, endoplasmic reticulum Ca2+ concentrations and mitochondrial Ca2+ concentrations were collected.RESULTSBoth sham and normal culture groups showed no injury during the experiment. The RIC group showed amelioration of liver function compared with the orthotopic liver transplantation group (P < 0.05). qRT-PCR and WB confirmed that Mfn2-mitochondrial Ca2+ uptake 1/2 (MICUs) axis was changed (P < 0.005). In AML-12 cell lines, compared with the hypoxia group, the hypoxia + Si group attenuated the collapse of ΔΨm and apoptosis (P < 0.005). The endoplasmic reticulum Ca2+ decrease and mitochondrial Ca2+ overloading observed in the hypoxia group were also attenuated in the hypoxia + Si group (P < 0.005). Finally, qRT-PCR and WB confirmed the Mfn2-MICUs axis change in all the groups (P < 0.005).CONCLUSIONMfn2 participates in liver injury in rat RIC models and AML-12 hypoxia cell lines by regulating the MICUs pathway.

Self-oxygenating scaffolds for anoxic culture of adipose tissue

Event Abstract Back to Event Self-oxygenating scaffolds for anoxic culture of adipose tissue Huaifa Zhang1, Simon Tran1, Jip Beugels2, Kramer Boris2 and Jake Barralet1, 3 1 McGill University, Faculty of Dentistry, Canada 2 Maastricht University Medical Center, Department of Pediatrics, Netherlands 3 McGill University, Division of Orthopaedics, Department of Surgery, Faculty of Medicine, Canada Introduction: Adipose tissue transplantation is commonly used in reconstructive surgery[1]. However, even vascularized fat flaps undergo considerable resorption after transplantation[2]. Adipocytes can only survive near the surface of the transplant (less than 300 µm in depth). Adipocytes that locate in deeper areas start to die on the first day of transplantation due to ischemia[3]. Oxygen deficiency is one of the factors that jeopardizes viability of many cells after transplantation[4]. We have developed a self-oxygenating scaffold consisting of metal peroxides and hydrogel. In this work, we aimed to examine adipose tissue viability in anoxia with and without our oxygen generating scaffold. Materials and Methods: Oxygen release scaffolds were made from calcium peroxide (CaO2) and calcium cross-linked sodium alginate.Subcutaneous adipose tissue was collected from freshly sacrificed rats and kept on ice during processing. Then the tissue was cut into 3×3 mm pieces and mixed with sodium alginate with or without calcium peroxide suspension. The mixtures were cross-linked by calcium ions to form cylinders 7 mm in diameter and 10 mm in length. The encapsulated adipose tissue was cultured under anoxia (95% N2, 5% CO2, 37 ) for up to seven days. The viability of cells in adipose tissue was inspected by a live/dead assay under a fluorescent microscope. The cultured tissue were also fixed and embedded in paraffin. H&E staining and perilipin staining were performed to examine the tissue structure and live cells, respectively. The scaffolds containing adipose tissue with and without oxygen release material will be further implanted subcutaneously into a rat model. After two and four weeks, the transplants will be retrieved. The transplants will be weighed before and after transplantation to determine the weight changes of the transplanted tissue. The volume of the adipose tissue after transplantation will be assessed by immersion[5]. The size of adipocytes in the transplants will be measured. Cell apoptosis will be examined using a caspase 3/7 assay. In addition, the retrieved transplants will be fixed and embedded in paraffin and histological characterizations will be performed as described previously. Results and Discussion: According to the live/dead assay results (Fig. 1a), adipose tissue preserved with oxygen release material under anoxia maintained high cell viability and scarce red nuclei from dead cells were observed. However, adipose tissue preserved under anoxia without oxygen release material (Fig. 1b) exhibited low cell viability. Our in vitro culture results show that adipose tissue can be preserved by oxygen delivery. Figure 1. Fluorescent images of live/dead assay stained adipose tissue after seven days culture in the absence of oxygen. Live cells were stained by green color. The nuclei of dead cells were stained into red and the nuclei of all the cells blue. Adipose tissue preserved with the oxygen release materials in the scaffold displayed very good viability (a), but adipose tissue preserved under anoxia without oxygen release materials (b) showed poor viability. Conclusion: We successfully preserved adipose tissue by oxygen delivery using a self-oxygenating scaffold for up to seven days under anoxia. Oxygen delivery can be a promising method to maintain cell viability in adipose tissue following transplantation. The Natural Sciences and Engineering Research Council of Canada; China Scholarship Council

Robust software sensor design for the state estimation in a sulfate-reducing bioreactor

The goal of this work is to present a class of state observer in order to infer unavailable concentrations in a sulfate-reducing bioreactor containing an anoxic culture of Desulfovibrio alaskensis 6SR from the measurements of the sulfate concentration. The methodology is applied to the corresponding sulfate-reducing model considering modeling uncertainties. The proposed observer design presents a proportional term plus a sigmoid function structure. The model’s observability was locally analyzed by the observability matrix test, concluding that the sulfate-reducing bioreactor is detectable. A sketch of proof of the observer’s convergence is depicted in order to show the asymptotic convergence characteristics. Numerical experiments provide an overview about the superior performance of the proposed observer methodology compared to a sliding-mode and high order sliding mode observers.

Microbial Decolorization of Reactive Dye Solutions

The most widely used dyes for coloration of cotton textiles are from reactive class. However, the large amount of unfixed dye causes color in the wastewater, which is environmentally unacceptable. Decolorization of various commercially important reactive dyes in aqueous solutions was attempted by microbial treatment using the bacteria Proteus vulgaris and Proteus mirabilis, individually as well as in a consortium. Different parameters such as treatment temperature, pH, dye solution concentration, and the seed age of the bacteria were optimized to achieve maximum decolorization. Although both bacteria displayed good growth in a shake culture, the color removal was better in an anoxic static culture. The selected dyes were based on azo and anthraquinone chromophores and complete decolorization under optimized conditions could be achieved at a concentration of 30 mg/L. Good results were obtained also at dye concentrations of 60 and 90 mg/L.

Simultaneously improving xylose fermentation and tolerance to lignocellulosic inhibitors through evolutionary engineering of recombinant Saccharomyces cerevisiae harbouring xylose isomerase.

BackgroundYeasts tolerant to toxic inhibitors from steam-pretreated lignocellulose with xylose co-fermentation capability represent an appealing approach for 2nd generation ethanol production. Whereas rational engineering, mutagenesis and evolutionary engineering are established techniques for either improved xylose utilisation or enhancing yeast tolerance, this report focuses on the simultaneous enhancement of these attributes through mutagenesis and evolutionary engineering of Saccharomyces cerevisiae harbouring xylose isomerase in anoxic chemostat culture using non-detoxified pretreatment liquor from triticale straw.ResultsFollowing ethyl methanesulfonate (EMS) mutagenesis, Saccharomyces cerevisiae strain D5A+ (ATCC 200062 strain platform), harbouring the xylose isomerase (XI) gene for pentose co-fermentation was grown in anoxic chemostat culture for 100 generations at a dilution rate of 0.10 h-1 in a medium consisting of 60% (v/v) non-detoxified hydrolysate liquor from steam-pretreated triticale straw, supplemented with 20 g/L xylose as carbon source. In semi-aerobic batch cultures in the same medium, the isolated strain D5A+H exhibited a slightly lower maximum specific growth rate (μmax = 0.12 ± 0.01 h-1) than strain TMB3400, with no ethanol production observed by the latter strain. Strain D5A+H also exhibited a shorter lag phase (4 h vs. 30 h) and complete removal of HMF, furfural and acetic acid from the fermentation broth within 24 h, reaching an ethanol concentration of 1.54 g/L at a yield (Yp/s) of 0.06 g/g xylose and a specific productivity of 2.08 g/gh. Evolutionary engineering profoundly affected the yeast metabolism, given that parental strain D5A+ exhibited an oxidative metabolism on xylose prior to strain development.ConclusionsPhysiological adaptations confirm improvements in the resistance to and conversion of inhibitors from pretreatment liquor with simultaneous enhancement of xylose to ethanol fermentation. These data support the sequential application of random mutagenesis followed by continuous culture under simultaneous selective pressure from inhibitors and xylose as primary carbon source.

Validation of the ion-exchange membrane bioreactor concept in a plate-and-frame module configuration

The ion exchange membrane bioreactor (IEMB) is a particular case of a membrane-supported biofilm reactor, in which oxy-anions, used as electron acceptors by an anoxic mixed microbial culture, are removed from a polluted water stream through an anion-exchange membrane. The opposite side of this membrane is used for the development of a biofilm, contacting a biocompartment, to which nutrients and chloride are fed as a source of “driving” counter-ion. The applicability of a plate-and-frame IEMB module configuration, consisting of a series of membranes, for the treatment of drinking water contaminated with nitrate and perchlorate, was evaluated. Permeation of carbon source across the membrane to the treated water stream was avoided by a dedicated start-up procedure involving a gradual increase of ethanol feeding to the IEMB biocompartment. It was demonstrated that the biocompartment pH must be controlled not only to guarantee a complete perchlorate removal, but also to avoid precipitation of struvite on the membrane surface, which provokes membrane scaling and decreases the availability of nutrients for the biofilm. Under these conditions, the IEMB was successfully operated maintaining both nitrate and perchlorate concentrations in the treated water below their recommended levels for drinking water supplies.

Kinetics of nitrate and perchlorate removal and biofilm stratification in an ion exchange membrane bioreactor

The biological degradation of nitrate and perchlorate was investigated in an ion exchange membrane bioreactor (IEMB) using a mixed anoxic microbial culture and ethanol as the carbon source. In this process, a membrane-supported biofilm reduces nitrate and perchlorate delivered through an anion exchange membrane from a polluted water stream, containing 60 mg/L of NO3− and 100 μg/L of ClO4−. Under ammonia limiting conditions, the perchlorate reduction rate decreased by 10%, whereas the nitrate reduction rate was unaffected. Though nitrate and perchlorate accumulated in the bioreactor, their concentrations in the treated water (2.8 ± 0.5 mg/L of NO3− and 7.0 ± 0.8 μg/L of ClO4−, respectively) were always below the drinking water regulatory levels, due to Donnan dialysis control of the ionic transport in the system.Kinetic parameters determined for the mixed microbial culture in suspension showed that the nitrate reduction rate was 35 times higher than the maximum perchlorate reduction rate. It was found that perchlorate reduction was inhibited by nitrate, since after nitrate depletion perchlorate reduction rate increased by 77%. The biofilm developed in the IEMB was cryosectioned and the microbial population was analyzed by fluorescence in situ hybridization (FISH). The results obtained seem to indicate that the kinetic advantage of nitrate reduction favored accumulation of denitrifiers near the membrane, whereas per(chlorate) reducing bacteria were mainly positioned at the biofilm outer surface, contacting the biomedium. As a consequence of the biofilm stratification, the reduction of perchlorate and nitrate occur sequentially in space allowing for the removal of both ions in the IEMB.

Ischemic Preconditioning Augments Survival of Stem Cells via miR-210 Expression by Targeting Caspase-8-associated Protein 2

MicroRNAs (miRs) participate in most cellular functions by posttranscriptional regulation of gene expression albeit with little information regarding their role in ischemic preconditioning (IP) of stem cells. We report that IP of bone marrow-derived mesenchymal stem cells (MSCs) with two cycles of 30-min ischemia/reoxygenation (I/R) supported their survival under subsequent longer exposure to anoxia and following engraftment in the infarcted heart. IP significantly reduced apoptosis in MSCs through activation of Akt (Ser(473)) and ERK1/2 (Thr(202)/Tyr(204)) and nuclear translocation of hypoxia-inducible factor-1alpha (HIF-1alpha). We observed concomitant induction of miR-210 in the preconditioned MSCs ((PC)MSCs). Inhibition of HIF-1alpha or of miR-210 abrogated the cytoprotective effects of preconditioning. Extrapolation of these data to in vivo studies in a rat model of acute myocardial infarction predominantly improved stem cell survival after engraftment with a role for miR-210. Notably, multiple I/R cycles more effectively regulated the miR-210 and hence promoted MSC survival compared with single-cycle hypoxia of an equal duration. Real time PCR array for rat apoptotic genes, computational target gene analyses, and luciferase reporter assay identified FLICE-associated huge protein (FLASH)/caspase-8-associated protein-2 (Casp8ap2) in (PC)MSCs as the target gene of miR-210. Induction of FLASH/CASP8AP2 in miR-210 knocked-down (PC)MSCs resulted in increased cell apoptosis. Taken together, these data demonstrated that cytoprotection afforded by IP was regulated by miR-210 induction via FLASH/Casp8ap2 suppression. These results highlighted that IP by multiple short episodes of I/R is a novel strategy to promote stem cell survival.

Physiological and phylogenetic characterization of a stable benzene-degrading, chlorate-reducing microbial community

A stable anoxic enrichment culture was obtained that degraded benzene with chlorate as an electron acceptor. The benzene degradation rate was 1.65 mM benzene per day, which is similar to reported aerobic benzene degradation rates but 20-1650 times higher than reported for anaerobic benzene degradation. Denaturing gradient gel electrophoresis of part of the 16S rRNA gene, cloning and sequencing showed that the culture had a stable composition after the seventh transfer. Five bacterial clones were further analyzed. Two clones corresponded to bacteria closely related to Alicycliphilus denitrificans K601. The three other clones corresponded to bacteria closely related to Zoogloea resiniphila PIV-3A2w, Mesorhizobium sp. WG and Stenotrophomonas acidaminiphila. DGGE analysis of cultures grown with different electron donors and acceptors indicated that the bacterium related to Alicycliphilus denitrificans K601 is able to degrade benzene coupled to chlorate reduction. The role of the other bacteria could not be conclusively determined. The bacterium related to Mesorhizobium sp. WG can be enriched with benzene and oxygen, but not with acetate and chlorate, while the bacterium related to Stenotrophomonas acidaminophila grows with acetate and chlorate, but not with benzene and oxygen. As oxygen is produced during chlorate reduction, an aerobic pathway of benzene degradation is most likely.

Influence of electron acceptor, carbon, nitrogen, and phosphorus on polyhydroxyalkanoate (PHA) production by Brachymonas sp. P12

Polyhydroxyalkanoates (PHAs), intracellular carbon and energy reserve compounds in many bacteria, have been used extensively in biodegradable plastics. PHA formation is influenced by nutrient limitations and growth conditions. To characterize the PHA accumulation in a new denitrifying phosphorus-removing bacterium Brachymonas sp. P12, batch experiments were conducted in which the electron acceptor (oxygen or nitrate) was varied and different concentrations of carbon (acetate), nitrogen (NH4Cl), and phosphorus (KH2PO4) were used. Polyhydroxybutyrate (PHB) was the dominant product during PHA formation when acetate was the sole carbon source. The PHB content of aerobically growing cells increased from 431 to 636 mg PHB g−1 biomass, but the PHB concentration of an anoxic culture decreased (−218 mg PHB g−1 biomass), when PHB was utilized simultaneously with acetate as an electron donor for anoxic denitrification. The specific PHB production rate of the carbon-limited batch, 158.2 mg PHB g−1 biomass h−1, was much greater than that of batches with normal or excess carbon. The effects of phosphorus and nitrogen concentrations on PHB accumulation were clearly less than the effect of carbon concentration. According to the correlation between the specific PHB production rate and the specific cell growth rate, PHB accumulation by Brachymonas sp. P12 is enhanced by nutrient limitation, is growth-associated, and provides additional energy for the biosynthesis of non-PHB cell constituents to increase the cell growth rate beyond the usual level.

Oxygen-regulated gene expression in bovine blastocysts.

Oxygen concentrations used during in vitro embryo culture can influence embryo development, cell numbers, and gene expression. Here we propose that the preimplantation bovine embryo possesses a molecular mechanism for the detection of, and response to, oxygen, mediated by a family of basic helix-loop-helix transcription factors, the hypoxia-inducible factors (HIFs). Day 5 compacting bovine embryos were cultured under different oxygen tensions (2%, 7%, 20%) and the effect on the expression of oxygen-regulated genes, development, and cell number allocation and HIFalpha protein localization were examined. Bovine in vitro-produced embryos responded to variations in oxygen concentration by altering gene expression. GLUT1 expression was higher following 2% oxygen culture compared with 7% and 20% cultured blastocysts. HIF mRNA expression (HIF1alpha, HIF2alpha) was unaltered by oxygen concentration. HIF2alpha protein was predominantly localized to the nucleus of blastocysts. In contrast, HIF1alpha protein was undetectable at any oxygen concentration or in the presence of the HIF protein stabilizer desferrioxamine (DFO), despite being detectable in cumulus cells following normal maturation conditions, acute anoxic culture, or in the presence of DFO. Oxygen concentration also significantly altered inner cell mass cell proportions at the blastocyst stage. These results suggest that oxygen can influence gene expression in the bovine embryo during postcompaction development and that these effects may be mediated by HIF2alpha.

Decolorization of the textile dyes by newly isolated bacterial strains

Six bacterial strains with the capability of degrading textile dyes were isolated from sludge samples and mud lakes. Aeromonas hydrophila was selected and identified because it exhibited the greatest color removal from various dyes. Although A. hydrophila displayed good growth in aerobic or agitation culture (AGI culture), color removal was the best in anoxic or anaerobic culture (ANA culture). For color removal, the most suitable pH and temperature were pH 5.5–10.0 and 20–35 °C under anoxic culture (ANO culture). More than 90% of RED RBN was reduced in color within 8 days at a dye concentration of 3000 mg l −1. This strain could also decolorize the media containing a mixture of dyes within 2 days of incubation. Nitrogen sources such as yeast extract or peptone could enhance strongly the decolorization efficiency. In contrast to a nitrogen source, glucose inhibited decolorization activity because the consumed glucose was converted to organic acids that might decrease the pH of the culture medium, thus inhibiting the cell growth and decolorization activity. Decolorization appeared to proceed primarily by biological degradation.

Aerobic nitrate respiration by a newly isolated phenol-degrading bacterium, Alcaligenes strain P5

An aerobic, nitrate-respiring bacterium which can degrade phenol under aerobic conditions was isolated and identified as Alcaligenes sp. Under microaerobic culture, the maximum concentration for phenol to be degraded was 0.29 mM in the presence of nitrate/O2 but only 0.16 mM in the presence of O2 alone. Azide (0.1 mM) and Triton X-100 (0.5%) inhibited nitrate reduction and cell growth completely in anoxic culture but had little or no effect on nitrate reduction in aerobic culture.