Soil samples were collected from mixed forest situated in central Poland, (52˚20'212N, 020˚51'260E), at elevation of 81 m above sea level. The climate of the area is mild, changing in particular years between Atlantic and more continental type. Mean annual temperature for year 2016 was 9.18˚C, and annual precipitation was 529 mm (Polish meteo service: www.weatheronline.pl). Soil samples were collected in a mature mixed stand with canopy closure of 100%. Dominant tree species there were: alder―Alnusglutinosa (L.), oak―Quercu- srobur L., horn beam―Carpinusbetulus L., pine―Pinussylvestris L., birch― Betula pendula Roth., maple―Acer platanoides L. The soil type there is a histosol. The site was described in detail by Blažka and Fischer [10] . This soil was used as the material for laboratory experiments, not as the soil characteristic for a specific site. Soil samples were taken from the soil A1 layer, at a depth of about 10 cm, sieved through a 5 mm sieve, following suggestions by Carter and Gregorich [11] . The soil was highly organic, nearly 50% LOI, sand constituting the majority of mineral fraction.

There were three key experiments in the study (Mc, Ma, Ms). The experiments differed in: 1) the initial moisture content, 2) the air supply (unrestricted and anoxic), 3) the watering pattern (one-time or repeated).

Initial moisture content. Two different levels of initial soil moisture were used in the study: the first was the actual moisture of the soil directly after taking it from the field, so the moisture content values varied in separate experiments from GWC 0.46 to 0.78. The second initial moisture level was that of the desiccated soil, where GWC < 0.1 and respiration was equal to 0.

Air supply. Air supply to the soil was assumed to be good when the container was filled up to 1/3 of the volume with the soil and it was not compressed. Dry mass of the sample ranged from 4.23 to 6.38 g, depending on moisture content. Oxygen supply was assumed to be in deficiency when the whole container was filled up with the soil and the soil was maximally compressed. Dry mass of these soil samples ranged from 32.8 to 36.6 g.

Wattering. The soil was watered once or continuously during the experiment. The same amount of water was supplied every 4 days in continuous watering. Respirometric measurements were taken a day before each supplementation of the water. In one-time watering pattern respirometric containers were filled up with dried or wet soil, depending on the experiments, then samples were watered up to desired moisture level, and measurements were performed on the fourth day.

Table 1 shows the scheme of conditions applied in particular experiments. In the series of MC and MS trials, GWC was increased by about 0.5, following each watering while in the MA series approximately by 0.2. In MS and MA trials,

respiration was additionally measured in 24 hours after supplementation of the water to confirm the stabilization of respiration rate.

GWC and WHC were determined after each trial. Respiration rate was determined in 6 replicates.

Means (m), standard deviations (s), coefficients of variation (cv) and standard errors of the mean (se) were determined for all data series. T-test for equality of means was used to determine the statistical significance of mean differences.

The MC and MS trial, as described in the methods (Table 1) was carried out on uncompressed soil, only in partially filled up respirometric containers. Sieving, necessary to uniform soil samples, facilitates an influx of greater amounts of air into the soil, the release of CO₂ previously accumulated into the atmosphere [1] or even a change in soil granulometry [15] . Sieved soil placed into containers exposed to the air can be compared to surface layers of the soil.

The analysis of results of Mc experiment in which continuous watering was applied to samples with two different levels of moisture content (GWC 0.78 and GWC 0.1) revealed, that oxygen consumption of the desiccated soil (GWC 0.1) was higher than that of the non-desiccated soil over the whole period of the experiment (36 days) (Table 2). Only after the soil reached its moisture content (GWC) equal to 2.09, during the highest intensity of metabolism, differences between the series were statistically insignificant. Carbon dioxide emission was also higher in B series (Table 2), but the differences were not always statistically significant. The RQ index did not differ in both series in most of moisture regimes. This implies that supplementation of the water to the desiccated (partially dead) soil created better conditions for metabolic processes than in an active wet soil. This result was attributed to the Birch effect [2] [16] . Despite a regular (every 4 days) watering and the soil becoming increasingly wetter the effect of its initial dessication was still visible (Table 2). This result supports the theory that a spontaneous microbial and fungal hyphae biomass growth takes place after desiccation [16] . Additionally, it is also possible that wetting of desiccated soil

makes previously inaccessible substrates available. An increase in metabolic activity may be related to both the multiplication of organisms and the restoration/activation of respiratory mechanisms in the organisms previously adapted to drought.

WHC (water holding capacity) measurements were taken at the end of the experiment. The measurements revealed the significance of WHC mean differences in the desiccated and non-desiccated soil series at the level (t-Test groups) of p = 0.034. This may suggest that desiccation affects the quality of organic substances in the soil [10] .

The comparison of metabolism in one time watered, dessicated and naturally wet soils (Ms trials, Table 3) yielded similar results, i.e. a higher respiration of the desiccated soil. This suggests that irrespectively of whether water is supplemented once or continuously, it is the level of desiccation of the soil that matters in the course of metabolism.

Following the supplementation of subsequent amounts of water i.e. during reaching higher levels of soil moisture, its respiration increased in Ms A series, while the respiration of desiccated soil (Ms B) decreased. If respiration of non- desiccated soil is assumed as 100%, the oxygen consumption of desiccated soil equaled to 209.90% of such value in the non-desiccated soil at the lowest moisture level, and only 51% at the highest. Carbon dioxide emission equaled to 191.92% and 111% at the lowest and the highest moisture levels, respectively. Its consequence was the increase of the RQ index up to 1 in the desiccated soil, while in the non-desiccated soil it ranged from 0.7 to 0.8 (Table 3).

Hence, one can hypothesize that desiccation primarily stimulates aerobic respiration. Non-desiccated soil reacts to supplementation of the water with increased respiration. With increasing moisture, aerobic respiration is suplemented by fermentation. At low levels of moisture, i.e. when the soil is well-ae- rated and aerobic respiration optimized, desiccation is the strongest determinant of respiration. The higher water content in the soil, and thereby a restricted supply of oxygen for aerobic respiration, the less pronounced is the Birch effect’s impact.

In conclusion, it should be stated that not only the final moisture content but

also an initial state of desiccation of the soil that is supplemented with water, are important for metabolic processes of the surface, to some extent aerated, layer of the soil. Desiccation increases soil respiration rates so substantially that respiration is statistically higher than of the non-desiccated soil even after 30 days of constant watering. The obtained results indicate that the increase of the respiration rate after watering of desiccated soil can be primarily related to aerobic respiration, which suggests that it results not from the change in moisture level but from the alteration of soil aeration.

As it was mentioned in the Introduction, mainly because of methodological difficulties, the studies concerning the metabolism of deeper soil layers being under anoxia are unique. Such studies have been rather focused on a variety of pro- cesses under anoxia e.g. in peatlands. The method applied in the present study is an attempt of oxygen deficiency simulation, i.e. the conditions similar to those of soil layers below its surface. The results of the measurements which were conducted under oxygen deficiency (experiment Ma) differ significantly from the results obtained under unrestricted air-supply conditions (Table 4). Respiration measured under unrestricted oxygen-supply conditions has always been higher than under oxygen deficiency. It is understandable, since respiration is more effective than fermentation, which is a dominating process under anoxia. Under oxygen deficiency, at low moisture levels (GWC < 1), when some amounts of air in the soil remain not replaced yet by the water, RQ is below 0.5. CO₂ emission may result partially from respiration and partially from fermentation, e.g. the transformation of lactic acid to ethanol. At higher moisture levels (GWC > 1), RQ value increases sharply which is the result of a drastic decrease in the quantity of oxygen used. At the moisture level of GWC ranging from 1.17 to 1.22, under the oxygen deficiency, the amount of oxygen used then equals to only 6.6% of the oxygen used under unrestricted air-supply conditions, while the amount of produced carbon dioxide equals to 16.9%. This suggests an enhancement of fermentation processes, which do not use oxygen. Those processes in the soil have a character of chain reaction and they are not necessarily directly reflected in gaseous processes. Fermentation may neither induce a production of carbon

dioxide nor an oxygen consumption. Respiration decreases to zero under oxygen deficiency conditions, at the moisture of the soil at the level GWC 1.5 and higher.

The respiration rate of previously desiccated soil is different under oxygen deficiency (Table 5). Desiccation affects respiration, however, in a different way compared with the soil having good access to oxygen. At the lowest moisture levels of 0.79 - 0.86, the desiccated soil has a significantly lower oxygen consumption than the non-desiccated soil i.e. inversely to the aerated soil (Table 3). The other results are not significantly different. Although the comparison of the data was only possible at a small moisture gradient, but within this range RQ value for desiccated soil always equalled to 1. RQ 1 value may appear in various situations, whereby this index reflects the total effect, resulting from a number of intermediate stages including fermentation. Perhaps the desiccation changes the pathways of fermentation processes and induces soil activity even at the moisture level at which the non-desiccated soil does not respire yet (Table 5). Probably, the difference between the desiccated and non-desiccated soil respiration rate being in conditions of oxygen deficiency consists indeed of an alteration of fermentation processes.

To sum up, we can state that under the conditions of oxygen deficiency soil respiration is significantly lower than in aerated soil. Desiccation of soil being in anoxia does not increase the use of oxygen, nor carbon dioxide emission, however the value of RQ index remains 1. Probably the desiccation alters fermentation processes.