Production and consumption of ethylene in temperate volcanic forest surface soils

To date our knowledge is limited with regard to the cycling of ethylene (C₂H₄) in temperate forest soils containing volcanic ash, and the effect of forest‐to‐orchard conversion on its cycling. We studied ethylene accumulation in such forest soils by oxic and anoxic incubations, along with the stimulatory effect of glucose addition on soil C₂H₄ accumulation. We also studied the effect of antibiotics and autoclaving on C₂H₄ production and consumption by volcanic forest soils, and the cycling of C₂H₄ and CH₄ in surface soils after conversion of a Japanese cedar forest to an orchard. Ethylene production and consumption by forest surface soils results from a microbial process, and soil streptomycin‐sensitive bacteria make a minor contribution. Soil C₂H₄ accumulation was much larger during anoxic than during oxic incubation, which indicates that anoxic conditions can induce C₂H₄ accumulation in forest soils. Glucose addition as a carbon source can sharply increase C₂H₄ accumulation rates in the anoxic and oxic forest soils during the first week of incubation. However, there was no difference in total C₂H₄ accumulation in the amended and non‐treated soils after 35 days of anoxic incubation. Ethylene production of the 0–5 cm and 5–10 cm soils beneath forest and orchard showed the greatest rate after 2 weeks of anoxic incubation when soil CH₄ production started to increase sharply, and later it was strongly suppressed. The forest‐to‐orchard conversion showed little influence on the CH₄ production of surface soils during short‐term anoxic incubation, but significantly reduced soil C₂H₄ production. The conversion also significantly decreased the consumption of soil CH₄ and C₂H₄, the former more than the latter. Soil properties such as total C, water‐soluble organic C and pH contribute to the consumption and production of C₂H₄ in the 0–5 cm and 5–10 cm soils, and there are the parallels between CH₄ and C₂H₄ consumption in soils, which suggests the presence of similar microorganisms. Long‐term anoxic conditions of in situ surface upland soils are normally not prevalent, so it can be reasonably concluded that there is a larger C₂H₄ accumulation rather than CH₄ accumulation in surface soils beneath forest and orchard after heavy rainfall, especially beneath forest.     

Responses of ethylene and methane consumption to temperature and pH in temperate volcanic forest soils

There is limited knowledge about the consumption and interaction of methane (CH₄) and ethylene (C₂H₄) in forest soils under disturbances of temperature and acidification. Temperate volcanic forest topsoils (0‐5 cm) sampled under different tree species (e.g. Pinus sylvestris, Cryptomeria japonica and Quercus serrata) were used to study the capacities for CH₄ and C₂H₄ consumption and their sensitivity to temperature and pH. We also studied the responses of soil nitrogen (N) transformations to temperature and relationships to consumption of both CH₄ and C₂H₄. The C₂H₄ consumption rates increased with temperature up to 35^oC, whereas the optimum temperature for CH₄ consumption rates was approximately 25^oC. Both Q₁₀ values and activation energies for CH₄ consumption rates over the range 5 to 25^oC were larger than corresponding values for C₂H₄ consumption rates. The rates of nitrous oxide (N₂O) and nitric oxide (NO) evolution and net N mineralization in the soils increased exponentially with temperature up to 35^oC, with relatively large Q₁₀ values and activation energies for NO evolution. In these forest topsoils, rates of CH₄ and C₂H₄ consumption at pH < 4.0 were negligible, and the pH optimum for both consumptions varied from 5.5 to 6.2. Most of the tested forest soils had an optimum pH for CH₄ and C₂H₄ consumption that was above natural pH values, which indicated that soil acidification would inhibit CH₄ and C₂H₄ consumption in situ. There was a high rate of net C₂H₄ evolution from forest soils acidified experimentally to pH < 4.0, particularly from Cryptomeria japonica forest soil, and 67% of the variation in C₂H₄ evolution rates could be accounted for by the increase in soil water‐soluble organic carbon concentrations. Previous studies have shown that addition of C₂H₄ in headspace gases can inhibit atmospheric CH₄ consumption in such forest soils. Hence, the evolution of C₂H₄ from temperate volcanic forest soils at decreasing pH can exacerbate inhibition of the soil atmospheric CH₄ consumption in situ.     

Microbial ethylene production and inhibition of methanotrophic activity in a deciduous forest soil

Production of C₂H₄, but not of CH₄, was observed in anoxically incubated soil samples (cambisol on loamy sand) from a deciduous forest. Ethylene production was prevented by autoclaving, indicating its microbial origin. Ethylene production gradually decreased from 4 to 12 cm soil depth and was not affected by moisture or addition of methionine, a possible precursor of C₂H₄. Oxidation of atmospheric CH₄ in soil samples was inhibited by C₂H₄. Ethylene concentrations of 3, 6 and 10 μl l⁻¹ decreased CH₄ uptake by 21, 63 and 98%, respectively. Methionine and methanethiol, a possible product of methionine degradation, also inhibited CH₄ oxidation. Under oxic conditions, C₂H₄ was consumed in the soil samples. Ethylene oxidation kinetics exhibited two apparent K_m values of 40 μl l⁻¹ and 12,600 μl l⁻¹ suggesting the presence of two different types of C₂H₄-oxidizing microorganisms. Methanotrophic bacteria were most probably not responsible for C₂H₄ oxidation, since the maximum of C₂H₄ oxidation activity was localized in soil layers (2-8 cm depth) above those (8-10 cm depth) of CH₄ oxidation activity. Our observations suggest that C₂H₄ production in the upper soil layers inhibits CH₄ oxidation, thus being one reason for the localization of methanotrophic activity in deeper soil layers.     

Ethylene oxidation,atmospheric methane consumption,and ammonium oxidation in temperate volcanic forest soils

Temperate volcanic forest surface soils under different forest stands (e.g., Pinus sylvestris L., Cryptomeria japonica, and Quercus serrata) were sampled to study the kinetics of ethylene (C₂H₄) oxidation and the C₂H₄ concentrations that effectively inhibit oxidation of atmospheric methane (CH₄) and nitrification. The kinetics of C₂H₄ oxidation in temperate volcanic forest soils was biphasic, indicating that at least two different microbial populations, one with low and another with high apparent K _m values, were responsible for ethylene oxidation. Methane consumption activity and ammonium oxidation of soil were inhibited by adding ethylene. Added C₂H₄ at concentrations of 3, 10, and 20 μl C₂H₄ per liter in the headspace gas respectively reduced by 20%, 50%, and 100% atmospheric CH₄ consumption by soil, and these values were much smaller than those inhibiting ammonium oxidation in these forest soils; thus, the CH₄ consumption activity was more sensitive to the addition of C₂H₄ than ammonium oxidation. Previous studies have shown that accumulation of C₂H₄ in such volcanic forest soils within 3 days of aerobic and anaerobic incubations can reach a range from 0.2 to 0.3 and from 1.0 to 3.0 μl C₂H₄ per liter in the headspace gas, respectively. It is suggested that C₂H₄ production beneath forest floors, particularly after heavy rain, can to some extent affect the capacity of forest surface soils to consume atmospheric CH₄, but probably, it has no impact on ammonium oxidation.     

Efficient Photocatalytic Reduction of CO<Subscript>2</Subscript> Present in Seawater into Methanol over Cu/C-Co-Doped TiO<Subscript>2</Subscript> Nanocatalyst Under UV and Natural Sunlight

Photocatalytic reduction of CO₂ in seawater into chemical fuel, methanol (CH₃OH), was achieved over Cu/C-co-doped TiO₂ nanoparticles under UV and natural sunlight. Photocatalysts with different Cu loadings (0, 0.5, 1, 3, 5, and 7 wt%) were synthesized by the sol–gel method and were characterized by XRD, SEM, UV–Vis, FTIR, and XPS. Co-doping with C and Cu into TiO₂ remarkably promoted the photocatalytic production of CH₃OH. This improvement was attributed to lowering of bandgap energy, specific catalytic effect of Cu for CH₃OH formation, and the minimization of photo-generated carrier recombination. Co-doped TiO₂ with 3.0 wt% Cu was found to be the most active catalyst, giving a maximum methanol yield rate of 577 μmol g-cat^?1 h^?1 under illumination of UV light, which is 5.3-fold higher than the production rate over C-TiO₂ and 7.4 times the amount produced using Degussa P25 TiO₂. Under natural sunlight, the maximum rate of the photocatalytic production of CH₃OH using 3.0 wt% Cu/C-TiO₂ was found to be 188 μmol g-cat^?1 h^?1, which is 2.24 times higher than that of C-TiO₂, whereas, no CH₃OH was observed for P25.     

石灰性土壤交换性钙和镁测定方法的研究

石灰性土壤交换性盐基组成的测定,通行的方法是采用70%乙醇溶液反复洗盐,再经pH 8.50.1 mol L-1氯化铵-70%乙醇(CH3CH2OH)溶液进行多次交换处理,测定交换液中的K+、Na+、Ca2+、Mg2+浓度。但此方法常常受操作步骤繁琐,以及土壤中碳酸盐的溶解量因多次浸提而增加的困扰,最终导致测定结果偏高。基于上述原因,选择不同浓度、不同pH的NH4OAc和NH4Cl 10种交换剂,对比分析10种交换剂中的碳酸盐溶解度和土壤交换性钙镁含量。结果表明,pH=8.5 1 mol L-1氯化铵-70%乙醇(CH3CH2OH)溶液较适合石灰性土壤交换性盐基的测定。此新方法是先经70%乙醇(CH3CH2OH)溶液洗盐,再用pH8.5 1 mol L-1氯化铵(NH4Cl)-70%乙醇(CH3CH2OH)溶液进行一次性交换处理,然后测定交换液的K+、Na+、Ca2+、Mg2+浓度,简化了操作程序的同时有效抑制了土壤碳酸盐的溶解,降低了测定结果的偏差。     

Diffusion probe for gas sampling in undisturbed soil

Soil‐atmosphere fluxes of trace gases such as methane (CH₄) and nitrous oxide (N₂O) are determined by complex interactions between biological activity and soil conditions. Soil gas concentration profiles may, in combination with other information about soil conditions, help us to understand emission controls. This paper describes a simple and robust diffusion probe for soil gas sampling as part of flux monitoring programmes. It can be deployed with minimum disturbance of in‐situ conditions, and also at sites with a high or fluctuating water table. Separate probes are used for each sampling depth, in this study ranging from 5 to 100 cm. The probe has a 10‐ml diffusion cell with a 3‐mm diameter opening covered by a 0.5‐mm silicone membrane. At sampling the diffusion cell is flushed with 10 ml N₂ containing 50 µl l^?1 ethylene (C₂H₄) as a tracer; tracer recovery is used to calculate sample concentrations. Ethylene is immediately removed by flushing with unamended N₂. Equations are presented to correct for dead volumes of connecting tubing and valves. Laboratory tests evaluated recovery of CH₄, N₂O and carbon dioxide (CO₂), removal of C₂H₄ and equilibration of CH₄, N₂O and CO₂ in air and water. Field tests on peat soils used for grazing showed soil gas concentrations of CH₄ and N₂O as influenced by topography, site conditions and season. The applicability of the diffusion probe for trace gas monitoring is discussed.     

Metal ion interaction with the hydrous oxides of aluminium

Suspensions of Al(OH)3 gel, gibbsite or alumina were loaded with varying amounts of Cu, Cd, Zn, or Pb ions by varying the system pH. A complex relationship between metal uptake and equilibrium pH was noted (due to substrate buffering) but total loss of metal ion from solution was observed at pH > 6.5. The pre-loaded particles were back-extracted with fifteen different chemical solutions and the percentage of sorbed ion retrieved generally varied along the sequence NaCl, CaCl₂ < MgCl₂, NH₄NO₃ < CH₃OOONH₄, Na citrate, Na₄P₂O₇, EDTA, DTPA ≈ CH₃OOOH, H₂C₂O₄, HCI, HN0₃. The recovery value varied with initial surface loading and an observed minimum around 1 gruel M²⁺ per 20 mg solid is considered to reflect changes in metal species nature (e.g., bonded M²⁺, MOH⁺, precipitated M(OH)₂) and substrate surface charge. In the ‘minima’ region less than 10% of metal ion was displaced by many reagents. With different loadings up to 40% was displaceable by salts (i.e., weakly sorbed) while acids or complex formers at times released over 90 % of the pre-sorbed metal species. It was concluded that the degree of metal ion interaction varied with the initial system pH, with retention being due to a combination of weak adsorption, occlusion in gels, chemi-sorption and precipitation of M(OH)₂.     

Acetylene inhibition of nitrous oxide reduction and measurement of denitrification and nitrogen fixation in soil

Reduction of N₂O in moist soil was inhibited completely by 10^?2 atm C₂H₂ and partially by 10^?5 atm C₂H₂. The effect of C₂H₄ was 10⁴ times less than that of C₂H₂. Denitrification of NO^?₃ occurred in anaerobically or aerobically incubated waterlogged soil and in anaerobic but not in aerobic moist soil. In the absence of C₂H₂ there was transient accumulation of N₂O. In the presence of C₂H₂ there was stoichiometric conversion of NO^?₃ to N₂O. Some kinetics of the reduction of N₂O and of NO^?₃ to N₂O are presented. Denitrification of 1 μg added NO^?₃-N.g^? could be measured within 1 h. Stoichiometries of production of N₂O from NO^?₂ and NO^?₃, respectively, and production of CO₂ attributable to denitrification were consistent with reported energy yields. Reduction of C₂H₂ to C₂H₄ occurred immediately following complete denitrification of added NO^?₃. The incubation of soil in the presence and in the absence of C₂H₂ thus permits assay of both denitrification and N₂ fixation and provides information on the mole fraction of N₂O in the products of denitrification.     

Effect of long-term field exposure of soil to acetylene on nitrification,denitrification, and acetylene consumption

Coated CaC₂ is a newly developed product which can supply nitrification-inhibiting quantities of C₂H₂ (1–10 Pa) to the soil, throughout a cropping season. This method of applying C₂H₂ to the soil maintains C₂H₂ in the soil continuously for several months. It is not know whether these low C₂H₂ concentrations alter soil microbial processes. A field study was initiated to determine the effect of supplying C₂H₂ to a clay soil, using coated CaC₂, on soil respiration, denitrification, nitrification, and C₂H₂ consumption. The C₂H₂ consumption rate increased with length of soil exposure to C₂H₂ (r ²=0.59). The rates of CO₂ production (r ²=0.88) and denitrification (r ²=0.86) were both highly correlated with the C₂H₂ consumption rates. The nitrifier potential decreased to a minimum of 21% of the control after 3 months of C₂H₂ treatment. After this time, nitrifier activity increased to 41% of the control after 11 months of treatment. This increase was due to increased C₂H₂ consumption in the soil. After 3 months of continuous application of C₂H₂ to the soil, the C₂H₂ concentrations were generally below that necessary to inhibit nitrification. No adaptation to the C₂H₂ by nitrifiers was found. Repeating these measurements 1 year later showed that soils previously exposed to C₂H₂ retained their enhanced C₂H₂ oxidation capacity and the capacity to use C₂H₂ to increase denitrification. Nitrification potentials remained about 50% lower in soils exposed to C₂H₂ a year earlier compared to soils not previously exposed to C₂H₂.     

Methanogenesis during rice growth as related to the water regime between crop seasons

In a long-term pot experiment with paddy rice, the effect of a wet or dry fallow on methanogenesis was studied during the ninth season. At times of CH₄ measurement, the oxidation of CH₄ was suppressed by C₂H₂. Methanogenesis started earlier in continuously flooded soil and was higher during the entire rice-growing period than in soil kept dry during the fallow and rewetted again before transplanting the rice seedlings. Increased CH₄ emission from the wet fallow treatment was, in contrast to the dry fallow treatment, associated with constantly low redox potentials. The experiment shows that the water regime during the fallow period is important to methanogenesis during the growth of rice plants.     

Side Effects of Acetylene on the Conversion of Nitrate in Soil

Laboratory studies were conducted to determine if acetylene affects the rate of NO₃^? reduction to N₂O and N₂, if C₂H₂ ist anaerobically metabolized in the presence of nitrate, and if C₂H₂ affects soil carbon metabolism. These studies in water saturated soil, incubated under air or N₂ atmosphere, with or without acetone-free acetylene, show that C₂H₂ can accelerate NO₃^? reduction. Acetylene inhibited carbon mineralization when NO₃^? was limited but accelerated it when sufficient NO₃^? was available. After three days, only two per cent or less of the added C₂H₂ was directly oxidized to CO₂, however, up to 28 % of the added C₂H₂ carbon remained in the soil. The residual C₂H₂ carbon was oxidized aerobically and anaerobically when NO₃^? was added. These data suggest that when C₂H₂ is used in denitrification studies, the results must be carefully scrutinized. Once a soil is exposed to C₂H₂ it should not be used again soon to assess denitrification.     

Inhibitory effect of low partial pressures of acetylene on nitrification

The inhibiting effect of C₂H₂ on nitrification was investigated in two agricultural soils. Nitrification was totally inhibited at 10 Pa partial pressure of C₂H₂ which is lower than previously reported for soils. There were no differences in rates of nitrate production between flasks without C₂H₂ and flasks with C₂H₂ at 0.01 Pa, while there was an effect on nitrification at 0.1 PaC₂H₂. At 0.1 Pa no inhibition was noted during the first 3 days; after this period nitrification was partially inhibited. The inhibiting effect did not cease until 7 days after removal of C₂H₂. The sensitivity of nitrification to low concentrations of C₂H₂ should be noted when denitrification rates are determined by the use of the acetylene inhibition method (usually C₂H₂ at 10kPa).     

Acetylene reduction (N2-fixation) by nodules of Acacia cyanophylla

The specific C₂H₂ reducing activity of excised nodules of Acacia cyanophylla was found to be 0.442 nmoles C₂H₄ produced min^?1 mg dry wt nodule^?1 and the apparent Km value for C₂H₂ between 8.5–11.0 × 10^?3atm. Subjection of plants of A. cyanophylla to drought, waterlogging, shading and defoliation led to drastic reduction or complete cessation of the ability of their nodules to reduce C₂H₂ to C₂H₄ When water-stressed and waterlogged plants were returned to a regular watering regime and the shaded plants to normal daylight the ability of their nodules to reduce C₂H₂ to C₂H₄ was partially or totally restored.     

The influence of moisture level,light, aeration and glucose upon acetylene reduction by a black earth soil

The influence of moisture level, light, aeration, and glucose upon C₂H₂ reduction by a clay soil was studied. C₂H₂ reduction was greater in 15 g samples of the air-dried soil moistened with 9 ml water than in samples moistened with 5 ml water. Illumination of the soil led to significantly more C₂H₂ reduction than when the soil samples were incubated in the dark. The addition of glucose to the soil increased C₂H₂ reduction. C₂H₂ reduction by soil samples incubated under an aerobic gas phase (either Ar + O₂, 80; 20; or air) was not significantly different from that of samples incubated under an anaerobic gas phase (Ar). Several questions regarding the use of the C₂H₂ reduction technique in assessing nitrogen fixation by natural ecosystems are raised.     

The sulphur-mercury(II) system in natural waters

Sulphur is an essential element for aquatic biosystems, the life processes of which lead to the formation of low molecular weight S compounds in the water. The results of our calculations indicate a pronounced tendency for Hg(II) to form HgS (or HgOHSH) and Hg(SR)₂ complexes in the presence of H₂S and thiols. Likewise, McHg will form CH₃HgSH and CH₃HgSR complexes, but in this case the chloride complex will dominate at low concentrations of H₂S and thiols. In acidic low salinity water, CH₃HgCl is the dominant McHg species at the lowest concentration of sulphide/thiols (0.1 nM), whereas a hundredfold increase of the sulphide/thiol concentration, or an increase of the pH to neutral or slightly alkaline conditions, will result in a total dominance for CH₃HgSH and CH₃HgSR.     

Substrate-dependent biosynthesis of ethylene by rhizosphere soil fungi and its influence on etiolated pea seedlings

Several fungi were isolated from rhizosphere soils of various crops through enrichment on l-methionine (l-MET) as a sole C and N source. These fungi were examined for their C₂H₄ production potential in vitro. GC-FID analysis indicated that 78%, 83%, 89% and 72% of fungi isolated from rhizosphere soil of wheat, maize, potato and tomato, respectively, produced C₂H₄ from substrate l-MET. Eight of the most efficient C₂H₄-producing fungal isolates (two from each crop) were further tested for their ability to produce C₂H₄ from 2-keto-4-methylthiobutyric acid (KMBA) and α-ketoglutaric acid (α-KGA). Interestingly, all eight fungi produced C₂H₄ from KMBA, but none used α-KGA as C₂H₄ precursor. This result implies that the MET→KMBA→C₂H₄ pathway was most likely operating in these fungi. The most efficient C₂H₄ producer fungus (isolated from maize rhizosphere soil) was identified as Aspergillus terreus; this isolate was further studied to determine the optimal conditions for C₂H₄ production from l-MET. It was observed that a substrate (l-MET) concentration of 10 mmol l⁻¹, a glucose (C-source) concentration of 6.0 g L⁻¹, no nitrogen, a pH of 6.0, an incubation temperature of 30 °C and an incubation time of 72 h were optimal conditions for l-MET-derived C₂H₄ biosynthesis by Aspergillus terreus. In addition, C₂H₄ released from precursor (l-MET) by Aspergillus terreus significantly affected the seedling length and stem diameter of etiolated pea seedlings in both sterilized and non-sterilized soils compared to a control. C₂H₄-producing microflora may be ubiquitous in soil, and the availability of a substrate like l-MET could enhance C₂H₄ synthesis in the rhizosphere of a plant, in turn evoking a physiological response. As far as we know, this study represents the first comprehensive screening of rhizosphere fungi for their ability to produce C₂H₄ from l-MET and KMBA.     

Critical evaluation of the acetylene reduction test for estimating the activity of nitrogen-fixing bacteria associated with the roots of wheat and barley

The reliability of the C₂H₂ reduction test for estimating the activity of N₂-fixing bacteria associated with the roots of cereals has been evaluated in Scottish soils. Six wheat cultivars, including two chromosome substitution lines, and five barley cultivars were grown in a glasshouse in nine soils from the North East of Scotland. All the soils exhibited C₂H₄ oxidase activity which was completely inhibited by 0.0001–0.1 atm C₂H₂. Over-estimation of C₂H₂ reduction, resulting from the accumulation of endogenous C₂H₄, could, therefore, occur in assays of undisturbed plants, with the real possibility of deducing the existence of N₂-fixation where none existed. However, radiolabelled C₂H₂ reduction tests on undisturbed plants producing 2.4–18.0 μmol C₂H₄ day^?1, showed that all the C₂H₄ had been derived from the C₂H₂. With less active plants, the source of the C₂H₄ could not be accurately determined by this tracer method. These low rates of C₂H₄ production (< 2.4 μmol C₂H₄ day^?1), referred to as apparent C₂H₂ reduction, should, therefore, not be considered proof of N₂-fixation.The highest C₂H₂ reduction activities were observed in soils at maximum water holding capacity (MWHC). Roots removed from these soils reduced C₂H₂ immediately, if the initial partial pressure of O₂ (pO₂) was < 0.1 atm. Roots washed free of soil did not oxidize C₂H₄ during the 8 h assay. The C₂H₂ reduction activities of these excised roots could not be related to the activity of plants in soil for three reasons. (1) Development of C₂H₂ reduction was dependent on protein synthesis (inhibited by chloramphenicol), indicating re-establishment of activity destroyed by exposure to atmospheric pO₂, rather than continuation of the activity of undisturbed roots. (2) A lag period, dependent on the volume of the incubation vessel, was observed, indicating the involvement of root respiration in the assay. (3) Growth of N₂-fixing bacteria on compounds released from the roots (reducing sugars, amino acids and α-keto acids) occurred during the assay.Even with the possibility of over-estimation of N₂-fixation, the C₂H₂ reduction activities measured were considered to be too low to contribute significantly to the nitrogen requirement of the cereals grown under field conditions in Scotland.Some guidelines for screening programmes of N₂-fixation associated with the roots of grasses are suggested.     

Availability and extractability of soil manganese in a liming experiment

Abstract

The availability of soil Mn to corn in relation to extractability of soil Mn by EDTA, Mg(NO₃)₂, CH₃COONH₄, hydroquinone, H₃PO₄, and NH₄H₂PO₄ as affected by liming was evaluated under field conditions on a single soil type. EDTA, Mg(NO₃)₂ and CH₃COONH₄‐extractable Mn were related inversely to available Mn. No useful relationships were found between hydroquinone, H₃PO₄, and NH₄H₂PO₄‐extractable soil Mn and Mn uptake by sweet corn.     

Factors regulating acetylene reduction assay for measuring heterotrophic nitrogen fixation in water-logged soils

In the C₂H₂-C₂H₄ assay for measurement of heterotrophic N₂ fixation in water-logged soils, the diffusion of C₂H₂ into the soil and the recovery of C₂H₄ from it are critical factors regulating the assay result. To establish an C₂H₂-C₂H₄ assay technique suitable for waterlogged soils, the C₂H₂-reducing activities (ARA), assayed by varying the method of assay gas filling, the pC₂H₂ of the assay gas, the duration of assay incubation and of soil vibration before the gas sampling, were compared.

A maximum ARA was measured when the following set of procedures were applied to the soil sample in assay flasks: 1) a 4-fold repetition of I-min evacuation under 0.01 atmospheric pressure and the subsequent I-min filling under 1 atmospheric pressure with assay gas at pC₂H₂ of 0.1 atm, 2) an assay incubation for 3 hr, and 3) a sampling of an aliquot of the headspace gas after strongly vibrating the flask for 1 min.

The ARA measured by this technique was several times larger than those measured by the techniques hitherto applied, and corresponded to an almost 80% of the V _max of the sample. This technique was, therefore, proposed for the assay of heterotrophic N₂ fixation in waterlogged soils.

A striking depression of ARA in the soil sample prepared with agitation indicated that a microbial ecosystem established in the soil should be kept as undisturbed as possible throughout the C₂H₂-C₂H₄ assay.