Metabolic differences inBacillus stearothermophilus grown at 55°C and 37°C

Summary Bacillus stearothermophilus was adapted to grow at 55°C and 37°C in a complex medium with almost equivalent yields in cell mass. In both temperature ranges the maximum specific growth rates (μ_max) were identical. Cellular extracts of this bacterium showed remarkable differences in the activity levels of several enzymes, depending on the respective growth temperature. High activities of glyceraldehyde-3-phosphate dehydrogenase and alcohol dehydrogenase were observed in bacteria from thermophilic cultures (55°C) and the respiratory quotient exceeded 1.0. Under anaerobic conditions at 55°C μ_max was the same as in aerobic cultures. No alcohol dehydrogenase was detected in cells from mesophilic cultures (37°C), however, and the level of glyceraldehyde-3-phosphate dehydrogenase was also extremely low under mesophilic conditions. Succinate dehydrogenase and isocitrate dehydrogenase activity appeared to be higher in bacteria grown at 37°C; the resspiratory quotient was always lower than 1.0. At 37°C, acetoin formation was observed regularly, a fermentation product which was never detected in 55°C-cultures. Under anaerobic conditions at 37°C a very low growth rate was found. When adapted to grow at 37°C or 55°C,B. stearothermophilus is apparently able to use different catabolic systems.     

Phospholipid metabolism in Mycobacterium smegmatis ATCC 607 grown at 37° C and 27° C

The rate of synthesis and degradation of phospholipids in Mycobacterium smegmatis ATCC 607, grown at 27° C and 37° C was studied by incorporation of ³²P into phospholipids and chase of radioactivity of the pulse-labelled phospholipids. A relatively low rate of synthesis and degradation of phospholipids in cells growth at 27° C was observed as compared to those grown at 37° C. Phosphatidylethanolamine (PE) had the maximum turnover at 37° C. However, at 27° C, cardiolipin (CL) showed a turnover rate higher than PE. Phosphatidylinositol mannosides (PIMs) were metabolically more active at 37° C than at 27° C. The differences in metabolic activity of the phospholipids at the two temperatures have been discussed.     

Muscle,Skin and Core Temperature after ?110°C Cold Air and 8°C Water Treatment

The aim of this investigation was to elucidate the reductions in muscle, skin and core temperature following exposure to −110°C whole body cryotherapy (WBC), and compare these to 8°C cold water immersion (CWI). Twenty active male subjects were randomly assigned to a 4-min exposure of WBC or CWI. A minimum of 7 days later subjects were exposed to the other treatment. Muscle temperature in the right vastus lateralis (n = 10); thigh skin (average, maximum and minimum) and rectal temperature (n = 10) were recorded before and 60 min after treatment. The greatest reduction (P<0.05) in muscle (mean ± SD; 1 cm: WBC, 1.6±1.2°C; CWI, 2.0±1.0°C; 2 cm: WBC, 1.2±0.7°C; CWI, 1.7±0.9°C; 3 cm: WBC, 1.6±0.6°C; CWI, 1.7±0.5°C) and rectal temperature (WBC, 0.3±0.2°C; CWI, 0.4±0.2°C) were observed 60 min after treatment. The largest reductions in average (WBC, 12.1±1.0°C; CWI, 8.4±0.7°C), minimum (WBC, 13.2±1.4°C; CWI, 8.7±0.7°C) and maximum (WBC, 8.8±2.0°C; CWI, 7.2±1.9°C) skin temperature occurred immediately after both CWI and WBC (P<0.05). Skin temperature was significantly lower (P<0.05) immediately after WBC compared to CWI. The present study demonstrates that a single WBC exposure decreases muscle and core temperature to a similar level of those experienced after CWI. Although both treatments significantly reduced skin temperature, WBC elicited a greater decrease compared to CWI. These data may provide information to clinicians and researchers attempting to optimise WBC and CWI protocols in a clinical or sporting setting.     

Chloroethene Biodegradation in Sediments at 4°C

Microbial reductive dechlorination of [1,2-¹⁴C]trichloroethene to [¹⁴C]cis-dichloroethene and [¹⁴C]vinyl chloride was observed at 4°C in anoxic microcosms prepared with cold temperature-adapted aquifer and river sediments from Alaska. Microbial anaerobic oxidation of [1,2-¹⁴C]cis-dichloroethene and [1,2-¹⁴C]vinyl chloride to ¹⁴CO₂ also was observed under these conditions.     

Survival of mouse oocytes after being cooled in a vitrification solution to -196°C at 95° to 70,000°C/min and warmed at 610° to 118,000°C/min: A new paradigm for cryopreservation by vitrification

There is great interest in achieving reproducibly high survivals of mammalian oocytes (especially human) after cryopreservation, but the results to date have not matched the interest. A prime cause of cell death is the formation of more than trace amounts of intracellular ice, and one strategy to avoid it is vitrification. In vitrification procedures, cells are loaded with high concentrations of glass-inducing solutes and cooled to −196 °C at rates high enough to presumably induce the glassy state. In the last decade, several devices have been developed to achieve very high cooling rates. Nearly all in the field have assumed that the cooling rate is the critical factor. The purpose of our study was to test that assumption by examining the consequences of cooling mouse oocytes in a vitrification solution at four rates ranging from 95 to 69,250 °C/min to −196 °C and for each cooling rate, subjecting them to five warming rates back above 0 °C at rates ranging from 610 to 118,000 °C/min. In samples warmed at the highest rate (118,000 °C/min), survivals were 70% to 85% regardless of the prior cooling rate. In samples warmed at the lowest rate (610 °C/min), survivals were low regardless of the prior cooling rate, but decreased from 25% to 0% as the cooling rate was increased from 95 to 69,000 °C/min. Intermediate cooling and warming rates gave intermediate survivals. The especially high sensitivity of survival to warming rate suggests that either the crystallization of intracellular glass during warming or the growth by recrystallization of small intracellular ice crystals formed during cooling are responsible for the lethality of slow warming.     

The growth of dermatophytes at 4° C and 37° C; The relation of this character to others

Summary The ability of the generaEpidermophyton, Microsporon andTrichopyton to grow on some media at 4° C and 37° C was studied. It has been shown that specific differences exist among these fungi in the capability or rapidity of the growth at extreme temperatures.There is high positive correlation among perfect state production, isolation from the soil and growth at 4° C (group of characters A) and between pathogenicity and growth at 37° C (group of characters B). Between the groups A and B of characters exists a slighter negative correlation. Some prognosis about the five characters by certain species of dermatophytes may be given.     

Thyroliberin is rapidly transferred to the nucleus of GH3 pituitary cells at both 4°C and 37°C

The time-course of thyroliberin transfer to the nucleus of GH3/B6 rat pituitary prolactin cells was studied by both autoradiography and cell fractionation of intact cells exposed to [³H]thyroliberin at 4°C or 37°C. It was previously shown that thyroliberin is not degraded in these conditions. It is found by autoradiography that [³H]-thyroliberin is transferred to the nucleus of GH3/B6 cells within 5 min at least at both 37° C and 4°C. Consistent results are obtained by fractionation of cells exposed to [³H]thyroliberin at 37°C. However after binding at 4°C 50% of the cell radioactivity is extractible by glutaraldehyde and after fractionation the isolated nuclei retain only 1–1.5% of the cell radioactivity. This suggests the existence of both tightly bound and loosely bound internalized thyroliberin molecules.     

Adaptation of Methanogenic Communities to the Cofermentation of Cattle Excreta and Olive Mill Wastes at 37°C and 55°C

The acclimatization of methanogens to two-phase olive mill wastes (TPOMW) was investigated in pilot fermenters started up with cattle excreta (37°C) and after changing their feed to excreta plus TPOMW (37°C or 55°C) or TPOMW alone (37°C) until a steady state was reached (28 days). Methanogenic diversity was screened using a phylogenetic microarray (AnaeroChip), and positive targets were quantified by real-time PCR. Results revealed high phylogenetic richness, with representatives of three out of the four taxonomic orders found in digesters. Methanosarcina dominated in the starting excreta (>96% of total 16S rRNA gene copies; over 45 times more abundant than any other methanogen) at high acetate (0.21 g liter⁻¹) and ammonia N concentrations (1.3 g liter⁻¹). Codigestion at 37°C induced a 6-fold increase of Methanosarcina numbers, correlated with CH₄ production (r_Pearson = 0.94; P = 0.02). At 55°C, the rise in temperature and H₂ partial pressure induced a burst of Methanobacterium, Methanoculleus, Methanothermobacter, and a group of uncultured archaea. The digestion of excreta alone resulted in low but constant biogas production despite certain oscillations in the methanogenic biomass. Unsuccessful digestion of TPOMW alone was attributed to high Cu levels inducing inhibition of methanogenic activity. In conclusion, the versatile Methanosarcina immediately adapted to the shift from excreta to excreta plus TPOMW and was responsible for the stimulated CH₄ production at 37°C. Higher temperatures (55°C) fostered methanogenic diversity by promoting some H₂ scavengers while yielding the highest CH₄ production. Further testing is needed to find out whether there is a link between increased methanogenic diversity and reactor productivity.Turning residues into energy is a societal and scientific priority due to climate change, fossil fuel exhaustion, and waste accumulation. In 2006, in Europe (EU27), less than 3% of electricity production came from biomass and wastes (11). Biogas plants, which anaerobically treat organic wastes to produce energy, are increasingly promoted in Europe, but their distribution is highly biased (35). While thousands of full- and farm-scale biogas plants are spread over central and northern Europe, anaerobic digestion technology in Mediterranean countries—Portugal, Spain, Italy, Greece, and Turkey—is in its early stages (35). These nations and other circum-Mediterranean countries lead in the production of olive oil and thus in olive mill wastes and wastewaters, which have a huge biogas production potential due to their lipid composition (1). Spain alone generates one-third of the world''s oil production and millions of tons of two-phase olive mill wastes (TPOMW) per year. TPOMW are mostly burned or composted (28), hence releasing methane into the atmosphere. This compels a change in strategy: methane production from TPOMW should be optimized in engineered environments and transformed into energy.TPOMW is a humid residue containing the olive pulp and stone. Its anaerobic digestibility is hampered by its low pH, low ammonia N, and high content in antimicrobial substances (1). However, it has been successfully fermented under laboratory conditions by supplementing it with nutrients and increasing the reactor organic loading rate stepwise (2) or by codigesting it with residues with a high buffering capacity, e.g., cattle excreta (17). These approaches seem to facilitate the adaptation of the methane-producing anaerobic community to the environmental conditions that TPOMW impose.Methanogenic archaea—microbes clustered within five orders of the Euryarchaeota—constitute the last step in the trophic chain of decomposers degrading organic matter in oxygen-free environments (36). Methanogenesis is often the rate-limiting step of anaerobic digestion of organic wastes (3) due to the fast duplication times of bacteria, which generate all substrates for the slow-growing methane-producing archaea. It is also the most sensitive step in processing imbalances (4), likely due to the lack of functional redundancy among methanogens (8). High concentrations of volatile fatty acids, salts, ammonia, and heavy metals can be inhibitory for methanogens (5, 22) and are the most common reasons for reactor failure (3). Our objective was to understand the adaptation of methanogenic communities to TPOMW. We investigated methanogenic diversity and abundance in pilot digesters fed with cattle excreta and after changing their feed to TPOMW or TPOMW plus excreta. We expected that mixing both residues would allow a faster adaptation and more efficient performance of the methanogenic communities in digesting TPOMW. The cofermentation was evaluated at 37°C and 55°C. During an acclimatization period of 28 days, we screened the methanogenic diversity using an in-house-devised phylogenetic microarray, the AnaeroChip (13), and quantified dominant genera by real-time quantitative PCR (qPCR). We have taken primers from the literature, and we present four new sets of genus-specific primers and SYBR green I-optimized assays for quantifying methanogens in anaerobic environments.     

Comparative proteomic analysis reveals differentially expressed proteins in Caenorhabditis elegans pgl-1 mutants grown at 20°C and 25°C

PGL-1 is an RNA-binding protein component of germ granules and essential for fertility in Caenorhabditis elegans. To clarify the molecular function of PGL-1, we performed comparative proteomic analysis using 2-D DIGE and LC-MS/MS. Five groups of synchronized adult hermaphrodites were analyzed: (1) wild-type N2 grown at 20°C, (2) pgl-1(bn101) mutants grown at 20°C, (3) pgl-1(bn101) mutants grown at 20°C then upshifted to 25°C after the L1 stage, (4) pgl-1(ct131) mutants grown at 20°C, and (5) pgl-1(ct131) mutants grown at 20°C then upshifted to 25°C after the L1 stage. The five groups were divided into two experimental sets for 2-D DIGE: set A included N2 and pgl-1(bn101) mutants, and set B included N2 and pgl-1(ct131) mutants. Dunnett's test indicated 90 and 100 specific spots, respectively, with significantly different expression levels from the rest of the experimental set (q≤0.1). Among them, 69 and 58 spots, respectively, were analyzed by LC-MS/MS. Finally, we identified 19 proteins from 24 specific spots common to both the experimental sets. RNAi analysis indicated that decreased eef-1G expression is strongly associated with the temperature-sensitive sterile phenotype of pgl-1. Our results suggest that PGL-1 is closely involved in translational processes during C. elegans germline development.     

Changes in productivity and carbon storage of grasslands in China under future global warming scenarios of 1.5°C and 2°C

Aims The impacts of future global warming of 1.5°C and 2°C on the productivity and carbon (C) storage of grasslands in China are not clear yet, although grasslands in China support ~45 million agricultural populations and more than 238 million livestock populations, and are sensitive to global warming.     

Biosynthetic capacity of hepatocytes after storage at 4°C

Capacity to synthesize glucose, urea, and ketone bodies is well maintained in hepatocytes after storage for at least 24 h at 4 degrees C. Substrates and albumin are the only requirements.     

Microbial metabolism in ice and brine at -5°C

Metabolic activity, but not growth, has been observed in ice at temperatures from -5°C to -32°C. To improve understanding of metabolism in ice, we simultaneously examined various aspects of metabolism ((14) C-acetate utilization, macromolecule syntheses and viability via reduction of CTC) of the glacial isolates Sporosarcina sp. B5 and Chryseobacterium sp. V3519-10 during incubation in nutrient-rich ice and brine at -5°C for 50 days. Measured rates of acetate utilization and macromolecule syntheses were high in the first 20 days suggesting adjustment to the lower temperatures and higher salt concentrations of both the liquid vein network in the ice and the brine. Following this adjustment, reproductive growth of both organisms was evident in brine, and suggested for Sporosarcina sp. B5 in ice by increases in cell numbers and biomass. Chryseobacterium sp. V3519-10 cells incubated in ice remained active. These data indicate that neither low temperature nor high salt concentrations prohibit growth in ice, but some other aspect of living within ice slows growth to within the detection limits of current methodologies. These results imply that microbial growth is plausible in natural ice systems with comparable temperatures and sufficient nutrients, such as debris-rich basal ices of glaciers and ice masses.     

Sodium efflux at 0°C in single barnacle muscle fibers

Summary A study has been made of the efflux of radiosodium in single barnacle muscle fibers cooled to 0°C. Cooling from 24 to 0°C results in a rapid fall in the Na efflux, the magnitude of which averages 81%. Rewarming leads to almost complete restoration of the Na efflux. The Arrhenius plot shows no breaks and gives anE _a value of 14.2 kcal/mol. External application of 10^–4 m-ouabain following cooling to 0°C causes a fall in the residual efflux (8%). Rewarming results in partial restoration of the Na efflux. Lowering external pH (pH _e ) results in a rise in Na efflux at 0°C, which peaks and declines rather slowly. The magnitude of the stimulatory response to acidification is a function of pH _e over the pH _e 7.00–5.8 range, the theoretical threshold being pH _e 7.1. The magnitude of the response to acidification of cooled, ouabain-poisoned fibers suspended in Li-ASW is the same as that of fibers suspended in Na-ASW. Injection of pure protein kinase inhibitor into fibers maintained at 0°C fails to reduce the size of the response to acidification. Benzolamide but not acetazolamide, ethoxzolamide or Cl 13,580 abolishes the response to acidification. It also reverses the response to acidification. SITS is also able to abolish the response to acidification. An additional new observation is that the Na efflux at 0°C is stimulated following the injection of CaCl₂ in a concentration-dependent manner. A similar response is not seen with MgCl₂. Acidification (pH _e 5.8) following peak stimulation by injection of CaCl₂ is without effect. These results add up to a refutation of the concept that the Na efflux at 0°C is wholly passive and that the response to acidification involves Na:Na or Na:Ca exchange. The results also weaken the argument that stimulation of the efflux by acidification is the result of activation of carbonic anhydrase.     

Perfusion of rabbit kidneys with glycerol solutions at 5 °C

The long-term preservation of whole organs will almost certainly require the use of subzero temperatures and cryoprotectants. An essential part of such a technique is the ability to add a cryoprotectant in adequate concentration and subsequently to remove it without damage to the organ. In this study rabbit kidneys have been perfused with solutions containing 3% dextran and 2 m glycerol at 5 °C, and their function has been measured after removal of the glycerol. The assay technique involved the measurement of glomerular filtration rate, protein leakage, and tubular reabsorption of sodium and glucose. The results indicate that the inclusion in the perfusate of an impermeant solute (mannitol) and limitation of the rate of change of glycerol concentration (to 30 mm min^?1) permits rabbit kidneys to retain a degree of function similar to that found in perfused control kidneys, although somewhat reduced in comparison with freshly isolated kidneys.     

L-lysine production at 65°C by auxotrophic-regulatory mutants ofBacillus stearothermophilus

Summary The amino acid L-lysine was produced from auxotrophic-regulatory mutants ofBacillus stearothermophilus at a temperature of 60–65°C. One of the mutants (AEC 12 A5, S-(2-aminoethyl)-cysteine^r, homoserine^–), produced L-lysine at the concentration of 7.5 g/l in shaken flasks in minimal medium containing 5% glucose. Culture conditions for optimizing L-lysine production were not investigated. The aspartokinase activity of the wild strainB. stearothermophilus Zu 183 was inhibited by lysine alone and by threonine plus lysine. AEC resistant mutants showed an aspartokinase activity genetically desensitized to the feedback inhibition. Optimal temperature and pH of aspartokinase were 45°C and 9.5, respectively. The data provide significant evidence that mutants of the speciesB. stearothermophilus have a potential value for amino acid production.     

Dark-recovery of Gamma- and UV-irradiated Saccharomyces carlsbergensis at 4° C

Experiments were carried out to find the effect of dark-holding in distilled water at 4° C on recovery of Gamma- and UV-irradiated cells of a haploid strain ofSaccharomyces carlsbergensis. It was found that there was an appreciable increase in survival of the irradiated cells following 4 to 24 hours' holding while no increase in the number of control cells was observed following similar treatment. It is suggested that some common type of damage induced by both Gamma- and UV-radiations inS. carlsbergensis may be repaired efficiently under metabolic conditions associated with growth at 4° C in distilled water.     

Phase diagrams for dextran-PEG aqueous two-phase systems at 22°C

Summary We have determined phase diagrams at 22°C for the aqueous two-phase systems composed of dextran, polyethylene glycol, and water. The effects of polyethylene glycol and dextran molecular weight on phase separation are reported. These phase diagrams provide more complete data for dextran/PEG/water system, and will be needed for the correlation of biomolecule partitioning.