(GWC―gravimetric water content, WHC―water holding capacity, dm―dry mass).

Our procedure was based essentially on Grace et al., Priha and Smolander [20] [21] with minor modifications [18] . GWC and other contents are given as ratios, but not %. Dry mass was determined by drying at 110˚C to constant weight.

SOM was estimated as loss of weight in ignition to constant weight (LOI) at 500˚C on selected samples. Mean densities of SOM (ρ = 1.3) and of the mineral fraction of dry weight (ρ = 2.65), are from Ilsted et al. [4] and many others.

Organic matter = LOI was between 0.45 and 0.50.

ρ(soil) = (LOI * 1.30 + (1 − LOI) * 2.65 = 1.96.

(OCD―oxygen consumption, PCO₂―production of CO₂, RQ―respiratory quotient).

We used the volumetric method with the Scholander type respirometer to get both the oxygen consumption and carbon dioxide production. The capacity of its cylindrical flasks is 50 ml (inside radius r = 2 cm, V = 4 cm²) and we refer data as μl∙g⁻¹(dry mass)∙h⁻¹. Each experimental series consisted of two parts. In the first, without hydroxide in the flasks, changes in the volumes were recorded mostly for 5 hours, this gave the sum of CO₂ changes and O₂ consumption, in the second part after placing filter paper soaked with NaOH solution into the respirometric chamber, we recorded the oxygen consumption (OCD) for another 5 hours interval, readings each 30 minutes. This involves assumption of slow rates of metabolic changes. The bath temperature was 17.5˚C. Sensitivity of the volumetric respirometer is effectively not much worse (<0.1 μl gas∙g⁻¹∙hour⁻¹) than of the IR meters, though rates of providing data are not comparable.

While data of the second part were the OCD, PCO₂ was calculated as first reading (without hydroxide) + second reading (with hydroxide), respecting the signs in readings and calculations. We then plotted the results against time of readings, the slope coefficient of the regression (β₁) was the rate (O₂ consumption-OCD or PCO₂); the respective regression coefficients were all r > 0.95, they were later corrected for pressure and temperature, multiplied by the calibration factor and related per gram dry soil and hour. Weights of respiration vials with soil were taken regularly; tare was subtracted to get rates of drying.

Later in Results and discussion we will consider to what extent the data measured might be biased by diffusion or other transport problems within the soil items, the measured data may then be classified as apparent data (AOCD, APCO₂ and ARQ).

Wet soil was gently pressed before start, there were no fissures between soil and wall of respirometric flask, they appeared however later during drying. We measured them by a series of blades (used in fine mechanics to measure narrow crevices, range 0.05 till 2 mm) on four sides of the vial, their mean was subtracted from r = 2 cm to get the radius of the base. The height (he) of the cylinders was estimated using a plate put on the top of the open vial and fixed to center by 4 screws projecting down on the outside of the flask, there were 5 evenly distributed holes on the plate, through which the distance from the top of the plate to soil top was measured by a rod, read by caliper and subtracted from the distance bottom of flask-top of plate. Mean of the 5 readings was height of the soil (he). Final calculation as cylinder volume:

Ws is mass of wet soil.

Relationships of gas exchange (OCD, PCO₂) with time and GWC are more complex, the respective regression coefficients are lower compared to regressions of GWC and time; the overall tendency of respiration (OCD and PCO₂) is a decrease with progressing drying as water is a primary essential-even substrate-of life (Table 5, Figure 1). Data on respiration, both OCD and PCO₂, follow common trends: high values immediately at the begin-

For MS experiments coefficients are for all data and also for dates after d2 and d3 due to the delayed Birch effect.

ning of experiments in both LS, or after three or five days in the MS series. There is a more or less stable respiration in the central part of the course in MS1 and MS2 (Table 2 and Table 4). Basic statistics of respiration data show small variance, se’s in all presented and vast majority of data not shown are within the point icons. The data in figures are of similar magnitude with those published by selected authors for forest soils considering problems in recalculation of area related data to mass related. Butler et al. [8] consider “rainfall as main driver of soil CO₂ efflux and its components”.

Distribution coefficient between gas and water for CO₂ is very close to one at the temperature of the experiments, storage capacity for CO₂ in soil water is therefore equal to GWC * dry mass of soil. It is the volume of water in respirometer (microcosm), about 20 ml in the first days of the MS experiments or 5 ml in the both LS; apparent production in two or three days.

Another estimate of CO₂ stored may result from the difference between AOCD and APCO₂ during the flush of respiration when soil is still wet, in MS1 days 3 to12. Assuming real RQ = 1.0 it is 2.81 ml CO₂ stored in 28.8 ml H₂O, the storage capacity is thus used in less than 10%.

Similar data for the MS2 experiment is for days 5 to 12 and show 2.79 ml CO₂ stored in 12.7 ml water, storage capacity used in approximately 22%. Assuming real RQ = 0.7 result in 1.97 ml CO₂ stored in MS1 and 1.95 ml CO₂ in MS2 respectively. Details of calculations are not shown.

The storage capacity for CO₂ was falling in experiments with progressing drying. Angert et al. [23] compared measured data of RQ (ARQ) with soil air analysis showing relatively low pCO₂. Their assumption on equilibrium between headspace (soil air) and wet acidic soil might not be fulfilled; there might be physical separation between the sections of the soil gas and the watered section, where CO₂ is primarily produced. Also a careful acidification and mixing of a tightly closed dilute solution of bicarbonate without gas phase need not result in production of gas bubbles. This may apply to a wet soil, where CO₂ is produced by microorganisms. ARQ data of Angert et al. [23] agree with our RQ in the respiration budget, there is also agreement on denial of carbonate chemistry as cause in the background of RQ < 1, we propose oxidation of methane and particularly ethanol produced in fermentation as hypothetic substrates in respiration with RQ < 1. We also seem to agree in accent on processes in studies. Sierra and Renault [14] conclude, that production is negligible compared to dissolved CO₂, this does not apply to acidic soils (pH < 4), where bicarbonate is practically missing. Therefore we did not consider bicarbonate storage, as estimates in our soil were always pH < 4.0. In acid soils however even a small volume stored may result in serious shifts in RQ.

To show gas exchange in strictly anoxic soil we placed 21 g fresh collected sieved wet soil (7.3 g dm) into each of four respirometers. Two of the respirometers were flushed with pure dinitrogen 50 ml∙min⁻¹, for 10 minutes; we registered O₂ consumption and CO₂ production [with and without NaOH trap inside]. The other pair of respirometers was a control to see regular soil respiration. There were 3.83 and 3.89 μl∙g⁻¹(dm)∙h⁻¹ respectively gas produced in the N₂ flushed respirometers without the hydroxide trap. There was however no gas production, nor consumption when hydroxide trap was placed in both respirometers; this also shows that the gas produced without the trap is absorbed by hydroxide, it is most probably CO₂ and not CH₄. RQ cannot be determined as shown in Introduction. The regular respiratory control in the second pair of respirometers provided 8.92 and 8.56 μl O₂∙g⁻¹(dm)∙h⁻¹ respectively and 4.51 μl CO₂∙g⁻¹(dm)∙h⁻¹ in both respirometers, this is consistent with data in Figure 1.

Another pilot experiment aimed to differences of respiration in highly dried soils as “mud pies” or small sandy like aggregates. In the “mud pies” both OCD and PCO₂ are some 20% lower than in the sandy sieved small aggregates. Fast drops of simultaneous decrease of OCD and PCO₂ by approximately 0.6 - 0.8 μl∙g⁻¹∙h⁻¹ occurred at 0.63 GWC in “mud pies”, at 0.43 in “sand”. Drying in “mud pies” and associated decrease of respiration is slower in “mud pies”. There is no contradiction to Figure 1.

High respiration data occurring after changed conditions (sieving, watering…) are referred as the Birch effect [24] . In our experiments it occurred in LS1 on d1, in MS1 on d 5 - 14 and also at lower GWC = 1.41 starting value in LS2 and MS2 thus at a somewhat lower moisture throughout the drying experiment (Figure 1). The delayed respiration flush in both MS experiments resulted in distorsions of the relationships of respiration to GWC, this was overcome by skipping the d 1 or d 2 data from the relationships in MS2 and MS1 (Table 6). The delay of flush in both MS experiments was probably caused by pressing the greater amount of soil at start resulting in formation of solid “mud pies”, while the small amounts of soil in both LS resulted in a thin layer of soil with minor limitation in gas traffic in the first days. The slightly smaller humidity in MS2 compared to MS1 was of minor importance in this range. We however feel that the impression of extraordinarily of high starting RQ’s in MS2 is partly misleading, it comes rather from general characteristics of ratios; OCD and PCO₂ data are of similar magnitude (1.78 and 1.07 respectively), not strikingly different indeed.

High flushes of respiration following change of conditions are debated for last 50 years [24] . So far they were attributed to increased availability of respiration substrates from organisms which died in preceding dries, even if the drop in water content was small [25] or to repeated drying and rewetting (e.g. [26] ) or simply to watering of soil (e.g. [13] ). Even the detailed study by Unger [27] which includes interaction of soil and the vegetation using highly advanced methodology does not seem to be the last word in the debate. Flush took place in all four experiments in fresh and sieved samples, but not watered, of course sampling was at the very beginning (Figure 1, Table 5, first section of the course). Respiration decreased dramatically in the next readings. Therefore we want to propose a hypothesis not relying primarily on the experimental conditions, but rather on the organisms themselves: Before the collection of soil organisms were adapted to low respiration rates in their natural settings, supplemented by fermentation. Sieving (or in other papers published-water added) allows more air to enter the soil; previously accumulated CO₂ is released to air or absorbed in water. The overall tendency during the experiments is decrease of AOCD and APCO₂ with decreasing GWC, the data of the first part of experiments are however influenced by CO₂ produced and stored in soil water and Va available, at this stage it was not included in the changes of gas contents measured. Some loss of humidity (GWC) in sieving is highly probable. Soil biota may require time to adapt to available dioxygen, presumably by activation/reconstruction of respiratory mechanisms (e.g. enzyme complexes of oxidative phosphorylation pathway) which appear to be of small use in more enzyme copies in wet soils in natural settings with limited oxygen availability.

Summed OCD and PCO₂ for the whole drying experiment showed in all four experiments RQ around 0.7 (Table 6). ΣRQ is somewhat higher in the faster drying experiments (LS), suggesting less hypoxia. This may support the hypothesis presented in introduction on a mixed status of organisms in wet soils, respiration contemporarily with fermentation and use of the fermentation products (e.g. ethanol) as substrates in respiration when O₂ becomes available.

Studies of some process which take into account all data, characterizing the variable soil condition during a process may provide more information, than short measurements or flashes of soil metabolism in ± constant conditions. Type of instrumentation may be of secondary importance only?modern methods of O₂ estimation are used only exceptionally [27] .

Nevertheless if there is O₂ consumption, anoxia is not in the total mass of soil, possibly in a limited area in center of the soil bloc.

There is a small, but significant peak of respiration in at the beginning of the decreasing phase at d 26 in MS1; in MS2 at d 26. A similar peak was registered by Thomson et al. [10] on the 5-th day of their 7-days experiment,

ΣRQ is the ratio of the two sums. Duration of experiments in Table 3.

which did not continue to a respiration stop. We propose to interpret as increased respiration due to increasing access of air to the drying soil, in our experiments shortly overrun but proceeding desiccation of the soil and confirmed by fast drops afterwards.

All four series (including the prematurely finished LS1) end up with RQ = 1 when GWC is approximately = 1 in LS experiments, but GWC ≈ 0.6 in MS (Figure 1).

CO₂ retained in the first section of experiments in water is completely aerated from the soil towards the end when soil is very dry; when respiration seizes at GWC ca 0.15, there is no measurable PCO₂, as is no OCD.

All drying experiments were terminated when GWC dropped to 0.15 and respiration stopped. The experiment LS1 was in fact finished prematurely, nevertheless its final RQ = 1. Also in a preliminary experiment not reported here (summer 2014) respiration (both OCD and PCO₂) seized at GWC = 0.15 and RQ = 1. The respiration limit at GWC = 0.15 need not be a universal value for many soils, Chowdhury et al. [13] reported respiration in Australian soils at recalculated GWC = 0.025.