Efficient separation of subfossil Chironomidae from lake sediments

Electron transport system (ETS) activity, CO₂ evolution, O₂ consumption, N₂-fixation (C₂H₂ reduction) and methanogenesis were appropriately measured in aerobic and anaerobically incubated sediment at 4, 10 and 20 ° C to better characterize these activities under different incubation conditions. ETS activity was always higher in the aerobically incubated sediment at all three incubation temperatures, whereas (C₂H₂ reduction was always greater in the anaerobic sediment. Carbon dioxide evolution was detected only in the aerobic sediment at 10 and 20 ° C but not at 4 ° C. Methane evolution in anaerobic sediment increased gradually with an increase in the incubation temperature.     

Anaerobic Oxidation of Acetylene by Estuarine Sediments and Enrichment Cultures

Acetylene disappeared from the gas phase of anaerobically incubated estuarine sediment slurries, and loss was accompanied by increased levels of carbon dioxide. Acetylene loss was inhibited by chloramphenicol, air, and autoclaving. Addition of ¹⁴C₂H₂ to slurries resulted in the formation of ¹⁴CO₂ and the transient appearance of ¹⁴C-soluble intermediates, of which acetate was a major component. Acetylene oxidation stimulated sulfate reduction; however, sulfate reduction was not required for the loss of C₂H₂ to occur. Enrichment cultures were obtained which grew anaerobically at the expense of C₂H₂.     

Effect of Modified Fenton’s Reaction on Microbial Activity and Removal of PAHs in Creosote Oil Contaminated Soil

This study describes the removal of polycyclic aromatic hydrocarbons (PAHs) from creosote oil contaminated soil by modified Fenton’s reaction in laboratory-scale column experiments and subsequent aerobic biodegradation of PAHs by indigenous bacteria during incubation of the soil. The effect of hydrogen peroxide addition for 4 and 10 days and saturation of soil with H₂O₂ on was studied. In both experiments the H₂O₂ dosage was 0.4 g H₂O₂/g soil. In completely H₂O₂−saturated soil the removal of PAHs (44% within 4 days) by modified Fenton reaction was uniform over the entire soil column. In non-uniformly saturated soil, PAH removal was higher in completely saturated soil (52% in 10 days) compared to partially saturated soil, with only 25% in 10 days. The effect of the modified Fenton’s reaction on the microbial activity in the soil was assessed based on toxicity tests towards Vibrio fischeri, enumeration of viable and dead cells, microbial extracellular enzyme activity, and oxygen consumption and carbon dioxide production during soil incubation. During the laboratory-scale column experiments, the toxicity of column leachate towards Vibrio fischeri increased as a result of the modified Fenton’s reaction. The activities of the microbial extracellular enzymes acetate- and acidic phosphomono-esterase were lower in the incubated modified Fenton’s treated soil compared to extracellular enzyme activities in untreated soil. Abundance of viable cells was lower in incubated modified Fenton treated soil than in untreated soil. Incubation of soil in serum bottles at 20 °C resulted in consumption of oxygen and formation of carbon dioxide, indicating aerobic biodegradation of organic compounds. In untreated soil 20–30% of the PAHs were biodegraded during 2 months of incubation. Incubation of chemically treated soil slightly increased PAH-removal compared to PAH-removal in untreated soil.     

Non-symbictic nitrogen fixation in the rhizosphere of Digitaria smutzii stent

Summary The acetylene reduction technique was used to test for the activity of nitrogenase in the rhizosphere of Digitaria smutzii grown in a solodic soil, and in the same soil in the absence of grass. The tests were made in McCartney bottles and regression analysis was used to compare rates of ethylene production. Roots with rhizosphere soil attached, exposed to acetylene and incubated anaerobically for 34 hours produced ethylene at a mean rate of 29 n moles C₂H₄/g root/h. No significant activity was detected under anaerobic conditions in the unplanted soil. Under aerobic conditions, significant but very low rates of ethylene production were observed in both the presence and absence of grass. Temperature treatments within the range 20°–32°C had no significant effect on rates of ethylene production.     

Acetylene Metabolism and Stimulation of Denitrification in an Agricultural Soil

The effects of C₂H₂ metabolism on N₂O production were examined in soil slurries. Enrichment of C₂H₂ consumption activity occurred only in aerobic incubations. Rapid disappearance of subsequent C₂H₂ additions, stimulation of CO₂ production, and most-probable-number enumerations of C₂H₂ utilizers indicated enrichment of the population responsible. During C₂H₂ consumption in slurries incubated statically under air, maximal rates of N₂O evolution were 19 times higher than those in anaerobic incubations. After 20 days of enrichment with C₂H₂, the production of N₂O by slurries supplemented with C₂H₂ and nitrate was 10 times higher than that in the unenriched controls. A Nocardia- or Arthrobacter-like bacterium was isolated that grew on C₂H₂ but did not denitrify. The behavior of soil inoculated with this bacterium became similar to that of C₂H₂-enriched soil incubated aerobically. Ethanol, acetate, and acetaldehyde were identified in enrichment experiments, and denitrification in soil slurries was stimulated by addition of the supernatant from a pure culture grown on mineral medium with C₂H₂. These results indicate that denitrification can be stimulated by the actions of an aerobic, nondenitrifying C₂H₂-metabolizing population. Utilization of intermediate metabolites by denitrifiers and enhanced O₂ consumption are two possible mechanisms for this stimulation.     

Effect of Temperature on Carbon and Electron Flow and on the Archaeal Community in Methanogenic Rice Field Soil

Temperature is an important factor controlling CH₄ production in anoxic rice soils. Soil slurries, prepared from Italian rice field soil, were incubated anaerobically in the dark at six temperatures of between 10 to 37°C or in a temperature gradient block covering the same temperature range at intervals of 1°C. Methane production reached quasi-steady state after 60 to 90 days. Steady-state CH₄ production rates increased with temperature, with an apparent activation energy of 61 kJ mol⁻¹. Steady-state partial pressures of the methanogenic precursor H₂ also increased with increasing temperature from <0.5 to 3.5 Pa, so that the Gibbs free energy change of H₂ plus CO₂-dependent methanogenesis was kept at −20 to −25 kJ mol of CH₄⁻¹ over the whole temperature range. Steady-state concentrations of the methanogenic precursor acetate, on the other hand, increased with decreasing temperature from <5 to 50 μM. Simultaneously, the relative contribution of H₂ as methanogenic precursor decreased, as determined by the conversion of radioactive bicarbonate to ¹⁴CH₄, so that the carbon and electron flow to CH₄ was increasingly dominated by acetate, indicating that psychrotolerant homoacetogenesis was important. The relative composition of the archaeal community was determined by terminal restriction fragment length polymorphism (T-RFLP) analysis of the 16S rRNA genes (16S rDNA). T-RFLP analysis differentiated the archaeal Methanobacteriaceae, Methanomicrobiaceae, Methanosaetaceae, Methanosarcinaceae, and Rice clusters I, III, IV, V, and VI, which were all present in the rice field soil incubated at different temperatures. The 16S rRNA genes of Rice cluster I and Methanosaetaceae were the most frequent methanogenic groups. The relative abundance of Rice cluster I decreased with temperature. The substrates used by this microbial cluster, and thus its function in the microbial community, are unknown. The relative abundance of acetoclastic methanogens, on the other hand, was consistent with their physiology and the acetate concentrations observed at the different temperatures, i.e., the high-acetate-requiring Methanosarcinaceae decreased and the more modest Methanosaetaceae increased with increasing temperature. Our results demonstrate that temperature not only affected the activity but also changed the structure and the function (carbon and electron flow) of a complex methanogenic system.     

Production and Consumption of Hydrogen in a Eutrophic Lake

The vertical distribution of hydrogen was measured in the Loclat, a eutrophic and holomictic lake near Neuchâtel, Switzerland, before and during summer stratification. H₂ concentrations decreased with depth in the anaerobic hypolimnion and were often below the detection limit (2.5 nl of H₂ liter⁻¹) in the water adjacent to the lake sediment. H₂ was apparently not released from the lake sediment. The highest H₂ concentrations (>4 μl of H₂ liter⁻¹) were observed in the aerobic water of the epilimnion and metalimnion. There, the H₂ concentrations changed with time, indicating a turnover of H₂. The H₂ production processes could not be studied in the laboratory since incubation of water samples in light or darkness did not result in H₂ production but rather always in H₂ consumption. The possible role of cyanobacteria and algae for H₂ production is discussed. Aerobic or anaerobic H₂ consumption activities were observed at all depths of the water column, with highest activities in the hypolimnion. Aerobic H₂ consumption activity was insensitive to azide inhibition, but sensitive to heat, mercuric chloride, or cyanide. It was restricted to a particle fraction of 0.2 to 3.0 μm in size, so that it must be due to single bacterial cells. Aerobic hydrogen bacteria, on the other hand, occurred in clusters of >3.0 μm. Therefore, the hydrogen bacteria could not have caused the H₂ consumption in lake water. The aerobic H₂ consumption activity followed Michaelis-Menten kinetics, with a K_m of 67 nM H₂. This is an exceptionally low value compared with K_m values of hydrogenases in hydrogen bacteria and other species, but is similar to that for H₂-decomposing abiontic soil hydrogenases.     

Carbon monoxide-dependent evolution of hydrogen by the homoacetate-fermenting bacteriumClostridium thermoaceticum

Clostridium thermoaceticum was found to form H₂ when cultivated heterotrophically on dextrose under a carbon monoxide (CO) gas phase. In contrast, when cultivated under CO₂, only minimal levels of hydrogen were detected. Resting cells from the CO-grown cultures also formed H₂ when incubated under CO with dextrose, while a comparative study with resting cells from CO₂-grown cultures demonstrated that the CO₂-grown cells were not competent in H₂ formation when incubated under CO. When dextrose was deleted, CO-cultivated resting cells did not form H₂ when incubated under CO.     

Soil H2-uptake in relation to soil properties and rhizobial H2-production

Summary The biological nature of soil H₂-consumption has been investigated. Soil microorganisms were capable to remove H₂ present in the gas phase at concentrations in the range of 200 ppm at rates varying between 0.2 and 1.0 l.min^–1. 100 g^–1. Free soil enzymes did not contribute significantly at the H₂ concentrations tested. Oxygen seemed to be the predominant electron acceptor. The influence of microbiological and physical soil properties on the H₂-uptake activity was examined for 38 soils.A highly significant correlation between biomass-C and H₂-uptake rate of the soil was noted, suggesting that the latter parameter might be useful as an indirect estimation of soil microbial biomass. The correlation was however not applicable for soils recently grown with legumes. Indeed, soya plants nodulated with aRhizobium strain with a weak hydrogen uptake capability, strongly increased the hydrogen oxidizing capability of the surrounding soil.     

Hydrogen production from aromatic acids byRhodopseudomonas palustris

The capability of five strains of the phototrophic bacteriumRhodopseudomonas palustris to produce molecular hydrogen (H₂) from the aromatic acids benzoate,p-hydroxybenzoate, cinnamate and D- and L-mandelate was investigated. Optimal H₂ production was achieved when the strains were grown anaerobically in the light at 10,000 lx under nitrogen (N) limitation using 1 mM L-glutamate as an N source. In the presence of 2 mM benzoate or L-mandelate as carbon and electron sources, strain DSM 131 produced 45% H₂ of the maximal theoretical value and strain F2 32%, respectively. Increased H₂ production correlated with increased nitrogenase activities, but H₂ formation was not further stimulated by inhibition of the H₂ uptake (hup) hydrogenase with ethylenediaminetetraacetic acid (EDTA).     

A study on electron transport-driven proton translocation in Desulfovibrio desulfuricans

Proton translocation by washed cells of the sulfate-reducing bacterium Desulfovibrio desulfuricans strain Essex 6 was studied by means of pH and sulfide electrodes. Reversible extrusion of protons could be induced either by addition of electron acceptors to cells incubated under hydrogen, or by addition of hydrogen to cells incubated in the presence of an appropriate electron acceptor. Proton translocation was increased in the presence of ionophores that dissipate the membrane potential (thiocyanate, methyl triphenylphosphonium cation, but not valinomycin) and was sensitive to the uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP). Upon micromolar additions of H₂, usually sulfide was formed in stoichiometric amounts, and extrapolated H⁺/H₂ ratios were 1.8±0.5 with sulfate, 2.3±0.3 with sulfite and 0.5±0.1 with thiosulfate. In several experiments hydrogen pulses caused increased proton extrusion not associated with sulfide production. This was a hint that sulfite might be reduced via intermediates. In the absence of H₂S formation, extrapolated H⁺/H₂ ratios were 3.1±0.8 with sulfate, 3.4±1.1 with sulfite, 4.4±0.8 with thiosulfate and 6.3±1.2 with oxygen. Micromolar pulses of electron acceptors to cells incubated under H₂ caused less proton translocation than H₂ pulses in presence of excess of electron acceptor; extrapolated H⁺/H₂ ratios were 1.3±0.4 with sulfite, 3.3±0.9 with nitrite and 4.2±0.5 with oxygen. No proton translocation was observed after micromolar pulses of sulfate, thiosulfate or nitrate to cells incubated under hydrogen in the presence of thiocyanate. Inhibition experiments with CO and CuCl₂ revealed that the hydrogenase activity was localized in the intracellular space, and that no periplasmic hydrogenase was present. The results indicate that D. desulfuricans can generate a proton gradient by pumping protons across the cytoplasmic membrane.Abbreviations APS adenosine 5-phosphosulfate - CCCP carbonyl cyanide m-chlorophenylhydrazone - MTTP⁺ methyl triphenylphosphonium cation     

Effect of oxygen partial pressure on nitrogen fixation and acetylene reduction in a sandy loam soil amended with glucose

Summary Small samples of soil amended with 2% (w/w) of glucose were preincubated either aerobically or anaerobically and then assayed (N₂ ¹⁵ and C₂H₂-C₂H₄) either aerobically or anaerobically for different time periods. One-hour C₂H₂-C₂H₄ assays showed greatest activity when anaerobic assay followed anaerobic preincubation. During the anaerobic preincubation a lag of 12–24 h occurred before rapid increase in one-hour assay activity was observed. When aerobic assay followed aerobic preincubation a longer lag was observed and lower activities were obtained. When anaerobic assay followed aerobic preincubation (orvice versa) negligible activities were observed in short assays, and longer assays showed increasing activity related to changes in atmosphere and/or microbial population in the closed system. Preincubation of soil on a diffusion gradient at a series of different partial pressures of oxygen confirmed the above pattern and showed that as preincubation pO₂ increased, the anaerobic assay activity rapidly decreased. As preincubation pO₂ decreased from 0.2 atm the aerobic assay activity decreased but less rapidly. The activities observed were related to the sizes of the Azotobacter and Clostridium populations. There was no evidence of aerobic or anaerobic C₂H₂ reduction in any cultures of ‘oligonitrophiles’ isolated. Incorporation of N₂ ¹⁵ was related to C₂H₂ reduction activity in the soil system studied. However, observed C₂H₄/N₂ molar ratios ranged from 10 to 22 and appeared to be highest in samples which were preincubated anaerobically. Issued as Macdonald College Journal Series No.618 and as Canadian IBP contribution No.84.     

The leucine operon carrying theleu-500 promoter mutation is expressed under anaerobic conditions

TheSalmonella typhimurium leu-500 auxotrophic mutant grew when cultivated in minimal medium anaerobically, but not aerobically. This mutant carries an AT CG mutation in the Pribnow box of the promoter region of the leucine operon and was found to be suppressible by anaerobic conditions. Analysis of the anaerobic gases revealed that hydrogen in the anaerobic gas mixture (85% N₂, 10% CO₂, 5% H₂) is essential for the suppression of theleu-500 mutation. Whenleu-500 mutant cells were incubated in the presence of the hydrogen gas, the synthetic rates for the first and last gene products of theleu-500 operon were similar to those of the wild-type cells. It was concluded that the entire leucine operon was efficiently expressed inleu-500 when the cells were grown under the hydrogen gas-containing anaerobic environment. Thus, theleu-500 promoter mutant is a model system for regulation of gene expression by a specific atmospheric environment, i.e., hydrogen gas found in the anaerobic environment.     

Emission of hydrogen from deep and shallow freshwater environments

In-situ partial pressures of hydrogen in anoxic profundal lake sediments reached values of up to 5 Pa which were more than 5 orders of magnitude lower than the partial pressures of methane. Analysis of gas bubbles collected from anoxic submerged paddy soil showed H₂ partial pressures in the range of 1.8 ± 1.3 Pa being ca. 4 orders of magnitude lower than the CH₄ partial pressures. H₂ emission rates, on the other hand, were less than 3 orders of magnitude lower than the CH₄ emission rates indicating that H₂ and CH₄ were oxidized to a different extent in the rhizosphere of the soil before they reached the atmosphere, or that H₂ was produced by the plants. More than 70% of the emitted H₂ reached the atmosphere via plant-mediated flux. The rest was emitted via ebullition from the anoxic soil and, in addition, was produced in the paddy water. A significant amount of H₂ was indeed found to be produced in the water under conditions where thallic algae and submerged parts of the rice plants produced oxygen by photosynthesis. Very little H₂ was emitted via molecular diffusion through the paddy water; in addition, this amount was less than expected from the degree of supersaturation and the diffusional emission rate of CH₄ indicating a relatively high rate of H₂ consumption in the surface film of the paddy water. The total H₂ source strength of rice paddies and other freshwater environments was estimated to be less than 1 Tg yr^-1, being negligible in the atmospheric budget of H₂.     

Temperature responses of carbon monoxide and hydrogen uptake by vegetated and unvegetated volcanic cinders

Ecosystem succession on a large deposit of volcanic cinders emplaced on Kilauea Volcano in 1959 has resulted in a mosaic of closed-canopy forested patches and contiguous unvegetated patches. Unvegetated and unshaded surface cinders (Bare) experience substantial diurnal temperature oscillations ranging from moderate (16 °C) to extreme (55 °C) conditions. The surface material of adjacent vegetated patches (Canopy) experiences much smaller fluctuations (14–25 °C) due to shading. To determine whether surface material from these sites showed adaptations by carbon monoxide (CO) and hydrogen (H₂) consumption to changes in ambient temperature regimes accompanying succession, we measured responses of CO and H₂ uptake to short-term variations in temperature and long-term incubations at elevated temperature. Based on its broader temperature optimum and lower activation energy, Canopy H₂ uptake was less sensitive than Bare H₂ uptake to temperature changes. In contrast, Bare and Canopy CO uptake responded similarly to temperature during short-term incubations, indicating no differences in temperature sensitivity. However, during extended incubations at 55 °C, CO uptake increased for Canopy but not Bare material, which indicated that the former was capable of thermal adaptation. H₂ uptake for material from both sites was completely inhibited at 55 °C throughout extended incubations. These results indicated that plant development during succession did not elicit differences in short-term temperature responses for Bare and Canopy CO uptake, in spite of previously reported differences in CO oxidizer community composition, and differences in average daily and extreme temperatures. Differences associated with vegetation due to succession did, however, lead to a notable capacity for thermophilic CO uptake by Canopy but not Bare material.     

Acetylene reduction byDaviesia mimosoides under Eucalyptus

Summary Daviesia mimosoides is a common understorey legume in Eucalyptus forests of the Brindabella Range in southeastern Australia, capable of fixing atmospheric nitrogen. Rates of N fixation were measured by the acetylene-reduction technique over a growing season in the field. Pot trials under controlled conditions were also carried out to elucidate effects of soil moisture, temperature, and light. Average rates in the field varied from about 1–5 μ mol C₂H₄/g/h (wet weight of nodule), but rates up to 14 μ mol C₂H₄/g/h were measured in optimum controlled conditions. Annual N-fixation rates approximate 4.5–7.0 kg/ha. In pot trials, rate of acetylene reduction decreased with soil moisture to about−10 MPa tension, with a marked depression at about−6 MPa, but within the normal field range of soil moisture there was little correlation of moisture with average acetylene reduction rate. Rates were similar in the temperature range of 20–30°C, but were depressed by either low or high temperature (<10 or >30°C). Diurnal fluctuations in acetylene reduction rates were not correlated with solar radiation, but rates were limited by high mid-day temperatures.     

Methanogenesis at low temperatures by microflora of tundra wetland soil

Active methanogenesis from organic matter contained in soil samples from tundra wetland occurred even at 6 °C. Methane was the only end product in balanced microbial community with H₂/CO₂ as a substrate, besides acetate was produced as an intermediate at temperatures below 10°C. The activity of different microbial groups of methanogenic community in the temperature range of 6–28 °C was investigated using 5% of tundra soil as inoculum. Anaerobic microflora of tundra wetland fermented different organic compounds with formation of hydrogen, volatile fatty acids (VFA) and alcohols. Methane was produced at the second step. Homoacetogenic and methanogenic bacteria competed for such substrates as hydrogen, formate, carbon monoxide and methanol. Acetogens out competed methanogens in an excess of substrate and low density of microbial population. Kinetic analysis of the results confirmed the prevalence of hydrogen acetogenesis on methanogenesis. Pure culture of acetogenic bacteria was isolated at 6 °C. Dilution of tundra soil and supply with the excess of substrate disbalanced the methanoigenic microbial community. It resulted in accumulation of acetate and other VFA. In balanced microbial community obviously autotrophic methanogens keep hydrogen concentration below a threshold for syntrophic degradation of VFA. Accumulation of acetate- and H₂/CO₂-utilising methanogens should be very important in methanogenic microbial community operating at low temperatures.     

Valinomycin inhibited methane synthesis in Methanobacteriumthermoautotrophicum

$\text{Methanobacterium}\text{thermoautotrophicum}$ cells, incubated anaerobically under H₂ in 0.1 M KCl or 0.1 M NaCl, above pH 7.5, are interior acid with respect to the incubation medium. The pH gradient thus established can be discharged by either carbonyl cyanide m-chlorophenylhydrazone or valinomycin at high concentration (17μM). In these cells, which actively synthesize CH₄ from CO₂ and H₂, methanogenesis is strongly inhibited when the pH gradient is discharged.     

The fermentation stoichiometry of Thermotoga neapolitana and influence of temperature,oxygen, and pH on hydrogen production

The hyperthermophilic bacterium, Thermotoga neapolitana, has potential for use in biological hydrogen (H₂) production. The objectives of this study were to (1) determine the fermentation stoichiometry of Thermotoga neapolitana and examine H₂ production at various growth temperatures, (2) investigate the effect of oxygen (O₂) on H₂ production, and (3) determine the cause of glucose consumption inhibition. Batch fermentation experiments were conducted at temperatures of 60, 65, 70, 77, and 85°C to determine product yield coefficients and volumetric productivity rates. Yield coefficients did not show significant changes with respect to growth temperature and the rate of H₂ production reached maximum levels in both the 77°C and 85°C experiments. The fermentation stoichiometry for T. neapolitana at 85°C was 3.8 mol H₂, 2 mol CO₂, 1.8 mol acetate, and 0.1 mol lactate produced per mol of glucose consumed. Under microaerobic conditions H₂ production did not increase when compared to anaerobic conditions, which supports other evidence in the literature that T. neapolitana does not produce H₂ through microaerobic metabolism. Glucose consumption was inhibited by a decrease in pH. When pH was adjusted with buffer addition cultures completely consumed available glucose. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Conservation in Soil of H2 Liberated from N2 Fixation by Hup- Nodules

Pigeon peas (Cajanus cajan) were grown in large soil columns (90-cm length by 30-cm diameter) and inoculated with four different strains of cowpea rhizobia, which varied with respect to hydrogen uptake activity (Hup). Despite the profuse liberation of H₂ from Hup^- nodules in vitro, H₂ gas was not detected in any of the soil columns. When H₂ was injected into the columns, the rates of consumption were highest in the treatments (including control) containing Hup^- nodules (218 and 177 nmol · h⁻¹ · cm⁻²) and lowest in the Hup⁺ treatments (158, 92, and 64 nmoles · h⁻¹ · cm⁻²). In situ H₂ uptake rates in small soil cores at fixed distances from the nodules decreased exponentially with distance from the nodule (R² = 0.99). This decrease in H₂ consumption was associated with a similar decrease in numbers of H₂-oxidizing chemolithotrophic bacteria as determined by the most-probable-number method. On the basis of two equations derived separately upon diffusive theory (Fix's Law) and kinetic theory (Michaelis-Menten), the empirically derived rate constants and coefficients indicated that all of the H₂ emitted from Hup^- nodules would be consumed by H₂-oxidizing bacteria within a 3- to 4.5-cm radius of the nodule surface. It is concluded that H₂ is not lost from the soil-plant ecosystem during N₂ fixation in C. cajan but is conserved by H₂-oxidizing bacteria.