Effect of dilution on methanogenesis, hydrogen turnover and interspecies hydrogen transfer in anoxic paddy soil

Abstract Dilution of anoxic slurries of paddy soil resulted in a proportional decrease of the rates of total methanogenesis and the rate constants of H₂ turnover per gram soil. Dilution did not affect the fraction of H₂/CO₂-dependent methanogenesis which made up 22% of total CH₄ production. However, dilution resulted in a ten fold decrease of the H₂ steady state partial pressure from approximately 4 to 0.4 Pa indicating that H₂/CO₂-dependent methanogenesis was more or less independent of the H₂ pool. The rates of H₂ production calculated from the H₂ turnover rate constants and the H₂ steady state partial pressures accounted for only < 5% of H₂/CO₂-dependent methanogenesis in undiluted soil slurries and for even less after dilution. Upon dilution, the Gibbs free energy available for H₂/CO₂-dependent methanogenesis decreased from −28.4 to only −5.6 kJ per mol. The results indicate that methane was mainly produced from interspecies H₂ transfer within syntrophic bacterial associations and was not significantly affected by the outside H₂ pool.     

Hydrogen turnover by psychrotrophic homoacetogenic and mesophilic methanogenic bacteria in anoxic paddy soil and lake sediment

Abstract The effect of temperature on CH₄ production, turnover of dissolved H₂, and enrichment of H₂-utilizing anaerobic bacteria was studied in anoxic paddy soil and sediment of Lake Constance. When anoxic paddy soil was incubated under an atmosphere of H₂/CO₂, rates of CH₄ production increased 25°C, but decreased at temperatures lower than 20°C. Chloroform completely inhibited methano-genesis in anoxic paddy soil and lake sediment, but did not or only partially inhibit the turnover of dissolved H₂, especially at low incubation temperatures. Cultures with H₂ as energy source resulted in the enrichment of chemolithotrophic homoacetogenic bacteria whenever incubation temperatures were lower than 20°C. Hydrogenotrophic methanogens could only be enriched at 30°C from anoxic paddy soil. A homoacetogen     

Confirmation of operative acetate, H2/CO2 and methanol methanogenic pathways in the solid-state refuse fermentation

By use of the radiolabelled substrates sodium [1–¹⁴C] acetate, sodium [2–¹⁴C] acetate, NaH¹⁴CO₃ and ¹⁴CH₃OH, three of the possible methanogenic pathways in fermenting refuse were confirmed. Due to the absence of a methanol pool, however, the relative contribution of each could not be determined. Circumstantial evidence for an operative trimethylamine pathway was gained but not confirmed whilst preliminary attempts to stimulate methanogenesis in refuse by supplementation with mono-and dimethylamine proved unsuccessful.     

Soils contain two different activities for oxidation of hydrogen

Abstract Hydrogen oxidation rates were measured in a neutral compost soil and an acidic sandy loam at H₂ mixing ratios of 0.01 to 5000 ppmv. The kinetics were biphasic showing two different K _m values for H₂, one at about 10–40 nM dissolved H₂, the other at about 1.2–1.4 μM H₂. The low- K _m activity was less sensitive to chloroform fumigation than the high- K _m activity. If sterile soil was amended with Paracoccus denitrificans or a H₂-oxidizing strain isolated from compost soil, it exhibited only a high- K _m (0.7–0.9 μM) activity. It also failed to utilize H₂ mixing ratios below a threshold of 1.6–3.0 ppmv H₂ (160–300 mPa). A similar result was obtained when fresh soil samples were suspended in water, and H₂ oxidation was determined from the decrease of dissolved H₂. However, H₂ was again utilized to mixing ratios lower than 0.05 ppmv, if the supernatant of the soil suspension or the settled soil particles were dried onto sterile soil or purified quarz sand. Obviously, soils contain two different activities for oxidation of H₂: (1) a high- K _m, high-threshold activity which apparently is due to aerobic H₂-oxidizing bacteria, and (2) a low- K _m, low-threshold activity whose origin is unknown but presumably is due to soil enzymes.     

Effect of algal deposition on acetate and methane concentrations in the profundal sediment of a deep lake (Lake Constance)

Abstract In the profundal sediment ot Lake Constance (143 m depth) the temperature is constant at 4 °C. Despite the constant temperature, CH₄ concentrations changed with season between about 120 μM in winter and about 750 μM in summer, measured down to 30 cm depth. The acetate concentration profiles also varied between seasons. In summer, acetate concentration reached a maximum at about 100 μM in 2 or 4 cm depth. In winter, maximal concentrations of about 5 μM were observed over the entire depth. Input of organic material in late spring may be the reason for the seasonal change of both compounds. To simulate such a sedimentation event, intact sediment cores were covered with suspensions of Porphyridium aerugenium or Synechococcus sp. The addition of the phytoplankton material resulted in a drastic increase of acetate concentrations with a maximum at 2 cm depth, similar to in situ acetate concentrations measured in summer. The same applies for CH₄ for which increased concentrations were observed down to 6 cm depth. H₂ concentrations, on the other hand, showed no distinct increase. Treatment of intact sediment cores with ¹⁴C-labeled Synechococcus cells resulted in the formation of ¹⁴C-acetate, ¹⁴CH₄ and ¹⁴CO₂. Maximum concentrations of ¹⁴CH₄ were found at 4 cm depth, i.e. just above the depth to which ¹⁴C-acetate penetrated. The results show that phytoplankton blooms may cause a seasonal variation of acetate and CH₄ in profundal sediments of deep lakes despite the constant low temperature. They also indicate that acetate is the dominant substrate for methanogenic bacteria in the profundal sediments of Lake Constance.     

Assimilation of methane and inorganic carbon by microbial communities mediating the anaerobic oxidation of methane

The anaerobic oxidation of methane (AOM) is a major sink for methane on Earth and is performed by consortia of methanotrophic archaea (ANME) and sulfate-reducing bacteria (SRB). Here we present a comparative study using in vitro stable isotope probing to examine methane and carbon dioxide assimilation into microbial biomass. Three sediment types comprising different methane-oxidizing communities (ANME-1 and -2 mixture from the Black Sea, ANME-2a from Hydrate Ridge and ANME-2c from the Gullfaks oil field) were incubated in replicate flow-through systems with methane-enriched anaerobic seawater medium for 5–6 months amended with either ¹³CH₄ or H¹³CO₃^-. In all three sediment types methane was anaerobically oxidized in a 1:1 stoichiometric ratio compared with sulfate reduction. Similar amounts of ¹³CH₄ or ¹³CO₂ were assimilated into characteristic archaeal lipids, indicating a direct assimilation of both carbon sources into ANME biomass. Specific bacterial fatty acids assigned to the partner SRB were almost exclusively labelled by ¹³CO₂, but only in the presence of methane as energy source and not during control incubations without methane. This indicates an autotrophic growth of the ANME-associated SRB and supports previous hypotheses of an electron shuttle between the consortium partners. Carbon assimilation efficiencies of the methanotrophic consortia were low, with only 0.25–1.3 mol% of the methane oxidized.     

Competition between reductive acetogenesis and methanogenesis in the pig large-intestinal flora

Washed bacterial suspensions obtained from the pig hindgut were incubated under ¹³CO₂ in a buffer containing NaH¹³CO₃ and carbohydrates. Incorporation of ¹³C into short chain fatty acids was assayed by quantitative nuclear magnetic resonance. The effects of different levels of H₂ added to the gas phase (0, 20 and 80% v/v) and of the specific methanogenesis inhibitor 2-bromoethane-sulphonic acid (BES) were determined. In control incubations increasing the concentration of H₂ markedly increased methane production. Single- and double-labelled acetate and butyrate were formed in all incubations. In the absence of BES, increasing H₂ significantly increased the incorporation of ¹³CO² into butyrate and the proportion of double-labelled acetate in total labelled acetate. The addition of BES proved to be very successful as a methane inhibitor and greatly enhanced the amount of mono- and double-labelled acetate, especially at the highest H₂ partial pressure. The results suggest that methanogenesis inhibited both routes of reductive acetogenesis, i.e. the homoacetate fermentation of hexose (represented for the most part by single labelling) and the synthesis of acetate from external CO₂ and H₂ (represented mostly by double labelling). A highly significant interaction between BES and H₂ concentration was observed. At the highest pH2 BES increased the proportion of labelled acetate in total acetate from 17.1% for the control to 50.9%. It was concluded that although acetogenesis and methanogenesis can occur simultaneously in the pig hindgut, reductive acetogenesis may become a significant pathway of acetate formation in the absence of methanogenesis.     

Metabolism of position-labelled glucose in anoxic methanogenic paddy soil and lake sediment

Abstract Turnover times of radioactive glucose were shorter in paddy soil (4–16 min) than in Lake Constance sediment (18–62 min). In the paddy soil, 65–75% of the radioactive glucose was converted to soluble metabolites. In the sediment, only about 25% of the radioactive glucose was converted to soluble metabolites, the rest to particulate material. In anoxic paddy soil, the degradation pattern of position-labelled glucose was largely consistent with glucose degradation via the Embden-Meyerhof-Parnas (EMP) pathway followed by methanogenic acetate cleavage: CO₂ mainly originated from C-3,4, whereas CH₄ mainly originated from C-1 and C-6 of glucose. Acetate-carbon originated from C-1, C-2 and C-6 rather than from C-3,4 of glucose. In both paddy soil and Lake Constance sediment acetate and CO₂ were the most important early metabolites of radioactive glucose. Other early products included propionate, ethanol/butyrate, succinate, and lactate, but accounted each for less than 1–8% of the glucose utilized. The labelling of propionate by [3,4-¹⁴C]glucose suggests that it was mainly produced from glucose or lactate rather than from ethanol. Isopropanol and caproate were also detectable in paddy soil, but were not produced from radioactive glucose. Chloroform inhibited methanogenesis, inhibited the further degradation of radioactive acetate and resulted in the accumulation of H₂, however, did not inhibit glucose degradation. Since acetate was the main soluble fermentation product of glucose and was produced at a relatively high molar acetate: CO₂ ratio (2.5:1), homoacetogenesis appeared to be the most important glucose fermentation pathway.     

Isolation and characterization of a thermophilic, acetate-utilizing methanogenic bacterium

Abstract A thermophilic acetate-decarboxylating methanogenic bacterium was isolated from a laboratory-scale 60°C sludge digestor. Cells form straight filaments with flat to blunted ends normally consisting of 2–3 cells held together by a sheath-like outer cell wall. The organism uses acetate, H₂-CO₂ and formate for methanogenesis and growth. With acetate as the sole methanogenic substrate, almost all of the radioactivity from methyl-labelled acetate appeared as methane. Acetate was converted to methane in equimolar amounts with a doubling time of 3 days.     

Diel variations in carbonate incorporation into otoliths in goldfish

When D-[¹⁴C-U]-glucose was administered intraperitoneally into goldfish Carassius amatus at 20° C and 12L: 12D (dark period 1800–0600 hours) at 0600, 1200, 1800, 2400 and 0600 hours on the following day, glucose was metabolized to release ¹⁴CO₂ and then it was incorporated into otoliths as carbonate. The rate of metabolic activity, judging from the ratio of inorganic to organic radiocarbon in plasma, was low during the dark period. Carbon incorporation into otoliths was also minimized during 1800–2400 h. When fish were exposed to ambient water containing NaH¹⁴CO₃, plasma radioactivity was lowest during 1800–2400 hours, during which time carbon incorporation into otoliths was lowest. Plasma total CO₂ levels markedly increased during the dark period. These results clearly indicate that carbonate formation in otoliths has a diel variation with a nadir lasting 6 h from 1800 to 2400 hours under the photoperiod used.     

Influence of Temperature on Lactate Utilization by Round Spermatids from Rat Testes

Rotenone-sensitive ¹⁴CO₂ formation from [¹⁴C]lactate and oxygen consumption by round spermatids were found to be greater at elevated temperatures than at 34°C. More than 96% of the total radioactivity of the metabolized [¹⁴C]lactate was recovered in the released CO₂ and the acid soluble fraction of the cells. There was practically no incorporation of [¹⁴C]latctate into the lipid, nucleic acid, and protein fractions. Intracellular level of ATP in spermatids was enhanced in the presence of lactate (20 mM) at 34°C (scrotal temperature), whereas it was decrease at 37°C (body temperature). However, this was reversible when the cells were transferred from the elevated temperature to 34°C. It was also found that oxygen consumption and CO₂ production were increased at 34°C by 2, 4-dinitrophenol (DNP), but decreased by oligomycin. On the other hand, oligomycin and DNP had no effect on oxygen consumption and ¹⁴CO₂ formation at the elevated temperature.
These findings provide evidence that lactate utilization by spermatids is coupled with oxidative phosphorylation at scrotal temperature, but becomes uncoupled at elevated temperature, although more lactate is consumed.     

Acetogenesis from H2 and CO2 by methane- and non-methane-producing human colonic bacterial communities

Abstract: The purpose of the study was to define the potential for reductive acetogenesis of colonic microflora from six non-methane- and four methane-excreting human subjects in relation to numbers of the different H₂-utilizing microorganisms. Faecal bacterial suspensions were incubated in the presence of NaH¹³CO₃ and under a gas phase composed of either 100% N₂ (control) or 80% H₂–20% N₂. The effects of a specific methanogenesis inhibitor or of sulfate supplementation were also determined. Quantitative nuclear magnetic resonance showed the presence of both single- and double-labelled acetate in all incubations under hydrogen. H₂/CO₂-acetogenesis appears to be a quantitatively important activity only in the presence of very low numbers of methanogens. Inhibition of methanogenesis induced a large increase in ¹³CO₂ incorporation into acetate in CH₄-producing samples. These results showed that methanogens can efficiently outcompete acetogens in human colonic contents. In contrast, no clear-cut competition for H₂ between acetogenesis and dissimilatory sulfate-reduction could be demonstrated. A slight reduction of the acetogenic activity was only observed at the highest sulfate addition (100 mM).     

Thermodynamics of H2-consuming and H2-producing metabolic reactions in diverse methanogenic environments under in situ conditions

Abstract In situ concentrations of hydrogen and other metabolites involved in H₂-consuming and H₂-producing reactions were measured in anoxic methanogenic lake sediments, sewage sludge and fetid liquid of cottonwood. The data were used to calculate the Gibbs free energies of the metabolic reactions under the conditions prevailing in situ. The thermodynamics of most of the reactions studied were exergonic with Gibbs free energies being more negative for H₂-dependent sulfate reduction methanogenesis acetogenesis and for H₂-producing lactate fermentation ethanol fermentation. Butyrate and propionate fermentation, on the other hand, were endergonic under in situ conditions. This observation is interpreted by suggesting that butyrate and propionate is degraded within microbial clusters which shield the fermentating bacteria from the outside H₂ (and acetate) pool.     

Characterization of populations of aerobic hydrogen-oxidizing soil bacteria

Abstract Freshly isolated soil bacteria were screened for different characteristics of the H₂ metabolism without prior selection for growth on H₂. The bacteria were isolated from different grain size fractions of a neutral meadow cambisol and an acidic forest cambisol, and then tested (1) for the ability to oxidize H₂, (2) for chemolithoautotrophic growth on H₂ as sole electron donor and energy source, (3) for DNA-DNA-hybridization with two hydrogenase gene fragments from Alcaligenes eutrophus and Rhizobium leguminosarum , and (4) for reduction of 2,3,5-triphenyl-2H-tetrazoliumchloride (TTC) in the presence of H₂. Many (65–90%) of the isolates were able to reduce TTC, but only 30–65% were actually able to oxidize H₂ indicating that the TTC test was not a specific characteristic for H₂ oxidation ability. The TTC test was only reliable in pure cultures of known bacteria with optimized test conditions, here shown for Alcaligenes eutrophus, Bradyrhizobium japonicum and Nocardia opaca , but not in mixed cultures of unknown bacteria. Still less (< 30%) of the isolates were able to grow chemolithoautotrophically indicating that culturable aerobic bacteria with the ability for H₂ oxidation are more abundant than bacteria with the ability for chemolithoautotrophic growth. The DNA-DNA-hybridization test failed to detect many of the bacteria with H₂ oxidation activity, probably since the hydrogenase genes present in the isolates were too diverse to be all detected by the DNA probes applied.     

Primary production studies in two linked but contrasting Welsh lakes

SUMMARY. Llyn Padarn and Llyn Peris have distinct phytoplankton populations. During 1975–76, the standing crop measured as chlorophyll- a was 5.5 times greater in Padarn than in Peris and the production rate, determined by the ¹⁴CO₂ method, was faster by 3.4 times. These differences were attributed to the higher concentrations of phosphorus in the lower lake caused by treated sewage effluent. Incident light intensity, which was slightly lower in Peris due to mountain shading, and temperature, which was 1–4°C higher in Padarn, made little significant contribution to these differences during the summer. The reduced transparency of Padarn water, compared with that of Peris, resulted from denser phytopiankton crops in Padarn. During the summer, Padarn exhibited carbon dioxide depletion which correlated with the chlorophyll concentration. Light inhibition at the surfaces of both lakes correlated with solar radiation intensity. However, the relationship between pigment content and maximum photosynthetic rate was poor. Extracellular products accounted for about 16% of the total production in the lakes. Uptake of ¹⁴C-labelled acetate was low compared with that of ¹⁴CO₂ uptake.     

Hydrogen-dependent control of the continuous thermophilic anaerobic digestion process using membrane inlet mass spectrometry

The control of a thermophilic continuous anaerobic digestion system when subjected to potential inhibitory shock loadings was achieved through the regulation of dissolved H₂, monitored using membrane inlet mass spectrometry, by the controlled addition of carbon source. At a feed pump switching threshold equivalent to 1 μmol/1 H₂ a steady state rate of methanogenesis of approximately 40 μmol/1/min was obtained. Higher H₂ thresholds resulted in an inhibition of methanogenesis, but precise control of H₂ concentration was demonstrated with an oscillatory response of period 2·5–5·0 min.     

C4 photosynthesis in Cyperus longus L., a species occurring in temperate climates

Abstract. Cyperus longus L. , which has a widespread but disjunct distribution throughout Europe and extends northwards into Britain, was found to be a C₄ species based upon its Kranz leaf anatomy, low CO₂ compensation point and the labelling of malate as an early product of ¹⁴CO₂ fixation. The photosynthetic characteristics of C. longus are similar to many other C₄ species with a high maximum rate of photosynthesis (> 1.5 mg CO₂ m ⁻² s ⁻¹) and a relatively high temperature optimum (30–35°C), but unlike many C₄ species the rate of photosynthesis does not decline rapidly below the optimum temperature and a substantial rate (0.6 mgCO₂ m⁻²s⁻¹)occursat 15°C. Leaf extension is very slow at 15°C and shows a curvilinear response to temperatures between 15 and 25°C. Leaves extend at a rate of almost 4 cm d⁻¹ at 25°C.     

Effects of Osmotic Pressure on the Oxidative Metabolism of Leishmania major Promastigotes

Leishmania major promastigotes were washed and resuspended in an iso-osmotic buffer. The rate of oxidation of ¹⁴C-labeled substrates was then measured as a function of osmolality. An acute decrease in osmolality (achieved by adding H₂O to the cell suspension) caused an increase in the rates of ¹⁴CO₂ production from [6-¹⁴C]glucose and, to a lesser extent, from [1, (3)-¹⁴C]glycerol. An acute increase in osmolality (achieved by adding NaCl, KCl, or mannitol) strongly inhibited the rates of ¹⁴CO₂ production from [1-: ¹⁴C]alanine, [1-¹⁴C]glutamate, and [1, (3)-¹⁴C]glycerol. The rates of ¹⁴CO₂ formation from [1-¹⁴C]laurate, [1-¹⁴C]acetate, and [2-¹⁴C]glucose (all of which form [1-¹⁴C]acetyl CoA prior to oxidation) were also inhibited, but less strongly, by increasing osmolality. These data suggest that with increasing osmolality there is an inhibition of mitochondrial oxidative capacity, which could facilitate the increase in alanine pool size that occurs in response to hyper-osmotic stress. Similarly, an increase in oxidative capacity would help prevent a rebuild up of the alanine pool after its rapid loss to the medium in response to hypo-osmotic stress.     

Partitioning of CO2 Production Between Glucose and Lactate in Excised Sympathetic Ganglia, with Implications for Brain

Abstract: Chains of lumbar sympathetic ganglia from 15-day-old chicken embryos were incubated for 4 h at 36°C in a bicarbonate-buffered salt solution equilibrated with 5% CO₂-95% O₂. Glucose (1–10 m M ), lactate (1–10 m M ), [U-¹⁴C]glucose, [1-¹⁴C]glucose, [6-¹⁴C]glucose, and [U-¹⁴C]lactate were added as needed. ¹⁴CO₂ output was measured continuously by counting the radioactivity in gas that had passed through the incubation chamber. Lactate reduced the output of CO₂ from [U-¹⁴C]glucose, and glucose reduced that from [U-¹⁴C]lactate. When using uniformly labeled substrates in the presence of 5.5 m M glucose, the output of CO₂ from lactate exceeded that from glucose when the lactate concentration was >2 m M . The combined outputs at each concentration tested were greater than those from either substrate alone. The ¹⁴CO₂ output from [1-¹⁴C]glucose always exceeded that from [6-¹⁴C]glucose, indicating activity of the hexose monophosphate shunt. Lactate reduced both of these outputs, with the maximum difference between them during incubation remaining constant as the lactate concentration was increased, suggesting that lactate may not affect the shunt. Modeling revealed many details of lactate metabolism as a function of its concentration. Addition of a blood-brain barrier to the model suggested that lactate can be a significant metabolite for brain during hyperlactemia, especially at the high levels reached physiologically during exercise.     

Thermophilic methanogens in rice field soil

The soil temperature in flooded Italian rice fields is generally lower than 30°C. However, two temperature optima at ≈ 41°C and 50°C were found when soil slurries were anoxically incubated at a temperature range of 10–80°C. The second temperature optimum indicates the presence of thermophilic methanogens in the rice field soil. Experiments with ¹⁴C-labelled bicarbonate showed that the thermophilic CH₄ was exclusively produced from H₂/CO₂. Terminal restriction fragment length polymorphism (T-RFLP) of archaeal SSU rRNA gene fragments revealed a dramatic change in the archaeal community structure at temperatures > 37°C, with the euryarchaeotal rice cluster I becoming the dominant group (about 80%). A clone library of archaeal SSU rRNA gene fragments generated at 49°C was also dominated (10 out of 11 clones) by rice cluster I. Our results demonstrate that Italian rice field soil contains thermophilic methanogenic activity that was most probably a result of members of the as yet uncultivated euryarchaeotal rice cluster I.