Metabolic Activity of Fatty Acid-Oxidizing Bacteria and the Contribution of Acetate, Propionate, Butyrate, and CO2 to Methanogenesis in Cattle Waste at 40 and 60°C

The quantitative contribution of fatty acids and CO₂ to methanogenesis was studied by using stirred, 3-liter bench-top digestors fed on a semicontinuous basis with cattle waste. The fermentations were carried out at 40 and 60°C under identical loading conditions (6 g of volatile solids per liter of reactor volume per day, 10-day retention time). In the thermophilic digestor, acetate turnover increased from a prefeeding level of 16 μM/min to a peak (49 μM/min) 1 h after feeding and then gradually decreased. Acetate turnover in the mesophilic digestor increased from 15 to 40 μM/min. Propionate turnover ranged from 2 to 5.2 and 1.5 to 4.5 μM/min in the thermophilic and mesophilic digestors, respectively. Butyrate turnover (0.7 to 1.2 μM/min) was similar in both digestors. The proportion of CH₄ produced via the methyl group of acetate varied with time after feeding and ranged from 72 to 75% in the mesophilic digestor and 75 to 86% in the thermophilic digestor. The contribution from CO₂ reduction was 24 to 29% and 19 to 27%, respectively. Propionate and butyrate turnover accounted for 20% of the total CH₄ produced. Acetate synthesis from CO₂ was greatest shortly after feeding and was higher in the thermophilic digestor (0.5 to 2.4 μM/min) than the mesophilic digestor (0.3 to 0.5 μM/min). Counts of fatty acid-degrading bacteria were related to their turnover activity.     

Differential Expression of Methanogenesis Genes of Methanothermobacter thermoautotrophicus (Formerly Methanobacterium thermoautotrophicum) in Pure Culture and in Cocultures with Fatty Acid-Oxidizing Syntrophs

The expression of genes involved in methanogenesis in a thermophilic hydrogen-utilizing methanogen, Methanothermobacter thermoautotrophicus strain TM, was investigated both in a pure culture sufficiently supplied with H₂ plus CO₂ and in a coculture with an acetate-oxidizing hydrogen-producing bacterium, Thermacetogenium phaeum strain PB, in which hydrogen partial pressure was constantly kept very low (20 to 80 Pa). Northern blot analysis indicated that only the mcr gene, which encodes methyl coenzyme M reductase I (MRI), catalyzing the final step of methanogenesis, was expressed in the coculture, whereas mcr and mrt, which encodes methyl coenzyme M reductase II (MRII), the isofunctional enzyme of MRI, were expressed at the early to late stage of growth in the pure culture. In contrast to these two genes, two isofunctional genes (mtd and mth) for N⁵,N¹⁰-methylene-tetrahydromethanopterin dehydrogenase, which catalyzes the fourth step of methanogenesis, and two hydrogenase genes (frh and mvh) were expressed both in a pure culture and in a coculture at the early and late stages of growth. The same expression pattern was observed for Methanothermobacter thermoautotrophicus strain ΔH cocultured with a thermophilic butyrate-oxidizing syntroph, Syntrophothermus lipocalidus strain TGB-C1. Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole proteins of M. thermoautotrophicus strain TM obtained from a pure culture and a coculture with the acetate-oxidizing syntroph and subsequent N-terminal amino acid sequence analysis confirmed that MRI and MRII were produced in the pure culture, while only MRI was produced in the coculture. These results indicate that under syntrophic growth conditions, the methanogen preferentially utilizes MRI but not MRII. Considering that hydrogenotrophic methanogens are strictly dependent for growth on hydrogen-producing fermentative microbes in the natural environment and that the hydrogen supply occurs constantly at very low concentrations compared with the supply in pure cultures in the laboratory, the results suggest that MRI is an enzyme primarily functioning in natural methanogenic ecosystems.     

Formation of Short-Chain Fatty Acids from H(2) and CO(2) by a Mixed Culture of Bacteria

The biological utilization of CO(2) and H(2) for the formation of short-chain fatty acids was studied by using a mixed culture of bacteria. Optimization of a medium was carried out in continuous culture to identify limiting factors which controlled growth and production of organic acids. The optimal pH for growth and acid production was 7.0 at 37 degrees C; the maximal cell concentration obtained was 5.9 g of cells per liter (dry weight), and the maximal amount of volatile acids formed was 4.7 g/liter, with acetic acid as the predominant acid. With the optimized medium, it was found that the rate of transfer of hydrogen or carbon dioxide, or both, from gas to liquid was the limiting factor which controlled growth and production of acids.     

Catalase Activity of Psychrophilic Bacteria Grown at 2 and 30 C

Catalase activity was measured in resting-cell suspensions of psychrophilic bacteria grown at 2 and at 30 C. Enzyme activity decreased in both cell-suspension types as harvest age increased. At comparable physiological age, cells grown at 2 C had more catalase than cells grown at 30 C.     

Cultured human skin fibroblasts modify their plasma membrane lipid composition and fluidity according to growth temperature suggesting homeoviscous adaptation at hypothermic (30 degrees C) but not at hyperthermic (40 degrees C) temperatures.

Mammalian cell metabolism is responding to changes in temperature. Body temperature is regulated around 37 degrees C, but temperatures of exposed skin areas may vary between 20 degrees C and 40 degrees C for extended periods of time without apparent disturbance of adequate cellular functions. Cellular membrane functions are depending from temperatures but also from their lipid environment, which is a major component of membrane fluidity. Temperature-induced changes of membrane fluidity may be counterbalanced by adaptive modification of membrane lipids. Temperature-dependent changes of whole cell- and of purified membrane lipids and possible homeoviscous adaptation of membrane fluidity have been studied in human skin fibroblasts cultured at 30 degrees C, 37 degrees C, and 40 degrees C for ten days. Membrane anisotropy was measured by polarized fluorescence spectroscopy using TMA-DPH for superficial and DPH for deeper membrane layers. Human fibroblasts were able to adapt themselves to hypothermic temperatures (30 degrees C) by modifying the fluidity of the deeper apolar regions of the plasma membranes as reported by changes of fluorescence anisotropy due to appropriate changes of their plasma membrane lipid composition. This could not be shown for the whole cells. At 40 degrees C growth temperature, adaptive changes of the membrane lipid composition, except for some changes in fatty acid compositions, were not seen. Independent from the changes of the membrane lipid composition, the fluorescence anisotropy of the more superficial membrane layers (TMA-DPH) increased in cells growing at 30 degrees C and decreased in cells growing at 40 degrees C.     

Metabolic and cardiovascular adjustment to work in air and water at 18, 25, and 33 degrees C.

By use of successive increments of discontinuous work with an arm-leg cycle ergometer the VO2, Q, SV, and HR were studied in six male subjects at rest and during exercise in air and in water at 18, 25, and 33 degrees C. The Q values obtained by CO2 rebreathing were reproducible. VO2 was linearly related to work with the plots for air and 33 degrees C water being similar. However, during work in 25 and 18 degrees C water, the VO2 averaged 9.0% (150 ml) and 25.3% (400 ml) higher, respectively, than values observed in 33 degrees C water, with the largest differences observed in leaner subjects. The plot of HR-VO2 was linear and almost identical during work in air and 33 degrees C water, but shifted significantly to the right in cooler water. VO2 averaged 250-700 ml higher in cold water compared to air and 33 degrees C water at a given mean heart rate. The Q vs. VO2 line was similar during work in air and in water with no effect of water or temperature. At similar levels of VO2, SV was significantly larger (P less than 0.05) in 25 and 18 degrees C water than in air or 33 degrees C water. Consequently, the reduction in heart rate during work in cold water was entirely compensated for by a proportionate increase in the SV of the heart. Q was therefore maintained at similar levels of energy expenditure in air and in 18, 25, and 30 degrees C water.     

Fasting reduces plasma leptin-and ghrelin-immunoreactive peptide concentrations of the burbot (Lota lota) at 2 degrees C but not at 10 degrees C

The effects of fasting at two water temperatures (2 and 10 degrees C) on plasma leptin- and ghrelin-immunoreactive peptide concentrations and energy metabolism were investigated in the burbot (Lota lota), a freshwater gadoid, which lives in cold waters and tolerates long periods of fasting. Burbot were assigned to fed and fasted groups at 2 and 10 degrees C. Leptin- and ghrelin-immunoreactive peptides were identified in burbot plasma. Fasting at 2 degrees C reduced the plasma leptin- and ghrelin-immunoreactive peptide concentrations and reduced the relative liver weights and the liver and muscle glycogen concentrations. The concentrations of the leptin- and ghrelin-immunoreactivities correlated positively with each other. At 10 degrees C there were decreased plasma thyroxine levels in both sexes, and slightly lower plasma testosterone concentrations in males. The results support previous studies in stating that immunoreactivities resembling mammalian leptin and ghrelin can be detected in burbot plasma and liver, but the specific functions of these peptides will have to be determined in future studies.     

Structure and mechanical properties of giant lipid (DMPC) vesicle bilayers from 20 degrees C below to 10 degrees C above the liquid crystal-crystalline phase transition at 24 degrees C

We have used micromechanical tests to measure the thermoelastic properties of the liquid and gel phases of dimyristoylphosphatidylcholine (DMPC). We have found that the rippled P beta' phase is only formed when a vesicle is cooled to temperatures below the main acyl chain crystallization transition, Tc, under zero or very low membrane tension. We also found that the P beta' surface ripple or superlattice can be pulled flat under high membrane tension into a planar structure. For a ripple structure formed by acyl chains perpendicular to the projected plane, the projected area change that results from a flattening process is a direct measure of the molecular crystal angle. As such, the crystal angle was found to increase from about 24 degrees just below Tc to about 33 degrees below the pretransition. It was also observed that the P beta' superlattice did not form when annealed L beta' phase vesicles were heated from 5 degrees C to Tc; likewise, ripples did not form when the membrane was held under large tension during freezing from the L alpha phase. Each of these three procedures could be used to create a metastable planar structure which we have termed L*beta' since it is lamellar and plane-crystalline with acyl chains tilted to the bilayer plane. However, we show that this structure is not as condensed as the L beta' phase below 10 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

A novel approach to structure proof of glyceryl ether-containing glycerophospholipids. Base-catalyzed methanolysis of platelet-activating factor (AGEPC) at 60 degrees C

A novel reaction was explored in which synthetic platelet-activating factor, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (AGEPC), upon treatment with 1 N NaOH in methanol at 60 degrees C for 20 min, sequentially released the acetyl group, then the choline moiety with concomitant formation of the monomethyl ester of 1-O-alkyl-glycero-phosphoric acid. A mechanism is proposed in which a transient cyclic phosphate intermediate is formed and then attacked by a CH3O moiety to yield a mixture of the sn-2 and sn-3 methyl esters. Proof of structure of the monomethyl ester derivative was achieved through the use of thin-layer chromatography, aluminum oxide chromatography, and examination of the trimethylsilyl derivative of the monomethyl ester by gas-liquid chromatography-mass spectrometry. Replacement of the acyl group on the 2 position with an ethyl or methyl residue completely prevented any attack by 1 N NaOH in methanol at 60 degrees C. Sphingomyelin was not attacked and only acetate removal was noted with 1-O-alkyl-2-acetyl-sn-glycero-3-phosphoethanolamine under similar conditions. The significance of these findings as they relate to the influence of substituents on the chemical and biological reactivity of AGEPC is discussed.     

Acetate formation from CO and CO2 by cell extracts of Peptostreptococcus productus (strain Marburg)

Cell extracts of Peptostreptococcus productus (strain Marburg) obtained from CO grown cells mediated the synthesis of acetate from CO plus CO₂ at rates of 50 nmol/min × mg of cell protein. ¹⁴CO was specifically incorporated into C₁ of acetate. No label exchange occurred between ¹⁴C₁ of acetyl-CoA and CO, indicating that ¹⁴CO incorporation into acetate was by net synthesis rather than by an exchange reaction. In acetate synthesis from CO plus CO₂ the latter substrate could be replaced to some extent by formate or methyl tetrahydrofolate as the methyl donor. The methyl group of methyl cobalamin was incorporated into acetate ony at very low activities. The cell extracts contained high levels of enzyme activities involved in acetate or cell carbon synthesis from CO₂. The following enzymic activities were detected: CO: methyl viologen oxidoreductase, formate dehydrogenase, formyl tetrahydrofolate synthetase, methenyl tetrahydrofolate cyclohydrolase, methylene tetrahydrofolate dehydrogenase, methylene tetrahydrofolate reductase, phosphate acetyltransferase, acetate kinase, hydrogenase, NADPH: benzyl viologen oxidoreductase, and pyruvate synthase. Some kinetic and other properties were studied.     

Quantum Yields for CO(2) Uptake in C(3) and C(4) Plants: Dependence on Temperature, CO(2), and O(2) Concentration

The quantum yields of C₃ and C₄ plants from a number of genera and families as well as from ecologically diverse habitats were measured in normal air of 21% O₂ and in 2% O₂. At 30 C, the quantum yields of C₃ plants averaged 0.0524 ± 0.0014 mol CO₂/absorbed einstein and 0.0733 ± 0.0008 mol CO₂/absorbed einstein under 21 and 2% O₂. At 30 C, the quantum yields of C₄ plants averaged 0.0534 ± 0.0009 mol CO₂/absorbed einstein and 0.0538 ± 0.0011 mol CO₂/absorbed einstein under 21 and 2% O₂. At 21% O₂, the quantum yield of a C₃ plant is shown to be strongly dependent on both the intercellular CO₂ concentration and leaf temperature. The quantum yield of a C₄ plant, which is independent of the intercellular CO₂ concentration, is shown to be independent of leaf temperature over the ranges measured. The changes in the quantum yields of C₃ plants are due to changes in the O₂ inhibition. The evolutionary significance of the CO₂ dependence of the quantum yield in C₃ plants and the ecological significance of the temperature effects on the quantum yields of C₃ and C₄ plants are discussed.     

Contribution of Metabolites of Photosynthesis to Postillumination CO(2) Assimilation in Response to Lightflects

In the shade plant Alocasia macrorrhiza grown in low light, photosynthetic CO₂ assimilation during a 5 second lightfleck plus postillumination CO₂ assimilation can allow up to 60% more photosynthesis than that which occurs during 5 seconds of steady state light of the same intensity (RL Chazdon, RW Pearcy 1986 Oecologia. 69: 524-531). Metabolites of photosynthesis were measured to determine if the pool of ribulose 1,5-bisphosphate (RuBP) could account for all of the postillumination CO₂ assimilation following a lightfleck in Alocasia. It was found that the pool of triose-P was much larger than that of RuBP and could account for five times more postillumination CO₂ assimilation than could RuBP. The same trend was seen in the sun plant Phaseolus vulgaris when it was grown in the shade. In contrast, sun-grown Alocasia and Phasiolus did not have a large pool of triose-P relative to RuBP following a lightfleck. In sun plants, carbon may rapidly be converted to RuBP in the light whereas in shade plants there may be a restriction in the path between the triose-P and RuBP pools. It is hypothesized that in shade plants the buildup of triose-P rather than RuBP during the lightfleck prevents inhibition of electron transport which may otherwise occur because of competition for ATP between the two kinases of the photosynthetic carbon reduction cycle. Utilization of the triose-P for postillumination CO₂ fixation would require the capacity for significant postillumination ATP synthesis. The extensive grana stacking and large intrathylakoid space which accompanies the high level of chlorophyll in low-light-grown Alocasia could be an important contributing factor to postillumination ATP formation.     

Comparison of formation, activation, and nuclear translocation of receptor . estradiol (R . E2) complex at 0 degrees C and 37 degrees C in intact uterine cells.

The present study was undertaken to establish whether molecular events leading to binding, transformation-activation, and nuclear translocation of cytoplasmic uterine estrogen receptor described for cell-free systems also occur in intact uterine cells. Cell suspensions were incubated at 0 degrees C or 37 degrees C with estradiol (E2) and specific binding to intracellular receptors was measured. The data demonstrate that saturation of specific estrogen binding sites occurs within 60 min at 37 degrees C and within 22 h at 0 degrees C, with a total of approximately 24,000 to 30,000 receptor sites per cell. At equilibrium, the total number and subcellular distribution of receptor . estradiol (R . E2) complexes formed in cells incubated at 0 degrees C or 37 degrees C were identical. Scatchard analysis of the equilibrium binding data yielded the same association constants for cytoplasmic and nuclear R . E2 formed in intact cells incubated at either temperature. Sucrose density gradient analysis of nuclear and cytoplasmic R . E2 formed in intact cells at 0 degrees C or 37 degrees C showed that at both temperatures, the nuclear R . E2 had a 5 S sedimentation coefficient; at both temperatures, a 5 S cytosol R . E2 was detected; only in the 0 degrees C incubation, an additional 4 S cytosol R . E2 was found. These results suggest that the molecular interactions regulating the dynamics of estrogen binding in the intact cell are similar at both physiological and low temperatures.     

Internal conductance to CO(2) diffusion and C(18)OO discrimination in C(3) leaves

(18)O discrimination in CO(2) stems from the oxygen exchange between (18)O-enriched water and CO(2) in the chloroplast, a process catalyzed by carbonic anhydrase (CA). A proportion of this (18)O-labeled CO(2) escapes back to the atmosphere, resulting in an effective discrimination against C(18)OO during photosynthesis (Delta(18)O). By constraining the delta(18)O of chloroplast water (delta(e)) by analysis of transpired water and the extent of CO(2)-H(2)O isotopic equilibrium (theta(eq)) by measurements of CA activity (theta(eq) = 0.75-1.0 for tobacco, soybean, and oak), we could apply measured Delta(18)O in a leaf cuvette attached to a mass spectrometer to derive the CO(2) concentration at the physical limit of CA activity, i.e. the chloroplast surface (c(cs)). From the CO(2) drawdown sequence between stomatal cavities from gas exchange (c(i)), from Delta(18)O (c(cs)), and at Rubisco sites from Delta(13)C (c(c)), the internal CO(2) conductance (g(i)) was partitioned into cell wall (g(w)) and chloroplast (g(ch)) components. The results indicated that g(ch) is variable (0.42-1.13 mol m(-2) s(-1)) and proportional to CA activity. We suggest that the influence of CA activity on the CO(2) assimilation rate should be important mainly in plants with low internal conductances.