An Influence of Multiple Drying/Rewetting Cycles upon the Respiration of Organic Forest Soil

It is already known that repeating cycles of drying and rewetting decrease the metabolic activity of the soil. The aim of this paper is to explain on the basis of a laboratory experiment how the respiratory processes of organic soil collected from the forest ecosystem typical for a moderate climate are changing during ten consecutive events of watering, and how alters the relationship between changing humidity of the soil and oxygen consumption/carbon dioxide emission. After 10 cycles, the respiration decreases by 2.4 times however amounts of excreted carbon dioxide and consumed oxygen do not differ between cycles 9 and 10. In successive DRW cycles also the relationship between oxygen consumption/carbon dioxide excretion and humidity level changes. This relationship is logarithmic and the analysis of subsequent regressions indicates the direction of those changes. In successive cycles the value of β coefficient (slope) decreases, and both the values of β and coefficient R are always higher for oxygen consumption that for carbon dioxide excretion. This indicates that processes involving oxygen consumption are always more sensitive to fluctuations of humidity than processes producing carbon dioxide. The optimum of respiration declines in successive DRW cycles.


Introduction
Climate changes occurring in last decades are undisputable, and their consequences include changes in the humidity of upper soil and fluctuations of the level of ground water. Changes in soil humidity are a significant variable in cli-matic system [1], but also influence the functioning of whole ecosystems. Particularly drastic is the current decrease of soil humidity in the northern hemisphere [2]. According to predictions of climatic models, we may expect further intensification of such hydrological cycles in the future i.e. longer dry periods and more intensive rainfalls [3].
A consequence of such hydrological cycles is variable in time amounts of water available for the soil but also changes in the rhythm of its supply. Reaction of soil processes towards fluctuations of soil humidity is very complicated and their coincidence influences whole ecosystems. Therefore the importance of cyclic changes of soil humidity for the regulation of ecosystem functioning may become more important than the total input of water to the soil. Changes in soil humidity affect not only the amount of CO 2 emitted from the soil but also other components of carbon cycle in ecosystems [4]. Significant also is the fact that for many forest ecosystems of moderate zone in particular; such meteorological situation is a novelty, since in the past, hydrological cycles were more balanced there. Until now, studies on this problem were mostly concentrated upon arid ecosystems [5] [6]. However it should be taken into account, that climate changes apply as well to ecosystems so far considered as humid, and that so-called "water history" of the soil is a significant factor for the functioning of the whole system [7] [8] [9]. Just because of "water history" moist mixed forests may be more threatened by negative effects of cyclically repeating dry periods [10]. Hence, considering world trends of changes in soil humidity [11], their dangerous effect on forest ecosystems in contemporary zone of moderate climate should be assumed.
In the majority of ecosystems, upper soils are subject from time to time to longer or shorter drying followed by moistening. Always this is a stress for microorganisms inhabiting such soils. They are forced then for the regulation of changing osmotic pressure [8], which is connected with higher energy expenditure and disturbance of normal energy budget. Important may be also the lysis of their cells as a result of a thorough drying up, which would supply then an additional amount of dead organic matter. However, considering the ability of assemblages of microorganisms for survival and adaptation to extreme circumstances like freezing or high temperatures, it can be expected that in conditions of repeatable drying up there is possible some kind of adaptation for cycles of drying and watering [8]. According to Fierer [10] and Evans [8], fluctuations in humidity level may cause a change in taxons of soil microorganisms which would result in simultaneous change of relations between environmental factors and functioning of soil microorganisms. This in turn would influence both: the rate of oxygen consumption and production of carbon dioxide. Emission of CO 2 from the soil is a key component of global carbon cycle and equals to 40 -70 Pg annually [9]. Similarly, the oxygen consumption by soil microorganisms is also an important part of the global cycle of this element.
While there were numerous papers on the subject of Birch effect and discus- is the knowledge not only on CO 2 emission but also on oxygen consumption.
There is only few data on this aspect. Nevertheless, both parameters are extremely important for the functioning of the whole ecosystem. An important issue seems to be also an explanation to what extend declines the intensity of metabolic processes in the soil following repeated fluctuations of soil humidity.
The aim of this paper is to answer the question how the metabolic activity of organic soil collected from the ecosystem typical for a moderate climate is changing during ten consecutive events of watering, and how is the relationship between changing humidity of the soil and oxygen consumption/carbon dioxide emission in the course of subsequent cycles.

The Site and Soil Sampling
The study was performed with the soil sampled in September 2018 at a mixed The applied procedure was based essentially on Grace et al., Priha and Smolander [17] [18] with minor modifications [19]. GWC and other contents are given as ratios, not as percentage. Dry mass was determined by drying soil samples at 110˚C to constant weight.

Soil Organic Matter (SOM)
SOM of soil samples was estimated as loss of weight in ignition to constant weight (LOI) at 500˚C. Organic matter = LOI was between 0.19 and 0.20.

Respiration
(OCD-oxygen consumption, PCO 2 -production of CO 2 , RQ-respiratory quotient) To estimate the oxygen consumption and carbon dioxide production, the classic volumetric method using the Scholander type respirometer was applied with minor modifications which are described below in details. The capacity of its cylindrical flasks was 50 ml (inside radius r = 2 cm, V = 4 cm 2 ) and the unit of obtained data was 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 CO 2 changes and O 2 consumption, in the second part after placing filter paper soaked with NaOH solution into the respirometric chamber, recorded was the oxygen consumption (OCD) for another 5 hours interval, with readings each 30 minutes. Such procedure allows for recording even small changes in respiration. The water 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. Values of OCD were obtained in the second reading (with the use of hydroxide) while PCO 2 was calculated as the sum of readings with and without hydroxide, respecting the signs. Then the results were plotted against time of readings, the slope coefficient of the regression (β) was the rate (O 2 consumption-OCD or PCO 2 ); the respective regression coefficients were all r > 0.95, subsequently corrected for pressure and temperature, multiplied by the calibration factor and related per gram of dry soil and hour. Weights of respiration vials with soil were taken regularly; tare was subtracted to get rates of drying.

The Experiment
The aim of the experiment was a comparison of soil metabolism during 10 consecutive cycles of watering and drying (DRW). Every cycle lasted for 10 days.

Statistics
Means (m), standard deviations (s), coefficients of variation (cv) and standard errors of the mean (se) were determined for all data series. A T-test for equality of means was used to determine the statistical significance of mean differences. All calculations were done with PS Imago Pro 5.1 statistics software.

Results and Discussion
DRW cycles change the circulation of elements in ecosystems in a drastic way [8] [20] [21] [22], and soil microorganisms both participate in those changes and respond to them [8] [10] [23]. Those reactions depend on the type of the ecosystem, the duration of wet and dry periods, temperature, the volume of precipitation in a studied period and the climatic history of a given region [8] [9] [24] [25] [26]. Numerous studies indicate that assemblages of microorganisms change during repeated DRW cycles and therefore modify their reaction towards changes of humidity [8] [23] [27]. From such broad problem, with a number of mutually conditioned aspects, two issues were selected for the analysis: potential stabilisation of carbon dioxide excretion and oxygen consumption during subsequent DRW cycles and respirometric reaction of the soil towards changes of humidity during DRW cycles. The laboratory experiment was conducted with organic forest soil, without considerable, frequent dry periods in its history, so according to Fierer apparently sensitive to DRW cycles [10]. Despite the fact that the experiment was conducted with a soil concoction from formerly sieved soil, hence with disturbed diffusive structure, so because the soil was not moved during the whole experiment (120 days), obtained results allow for drawing a conclusion about the reaction of the assemblage of microorganisms towards DRW cycles within the range of GWC from the full saturation with water (GWC > 1) until drying (GWC < 0.3).  Table 2. According to both Figure 2 and Table 1   should be assumed that the divergence between presented results and data of Meisner results from the fact, that Meisner conducted his experiments during first hours after watering of the dry soil while in this experiment, first 72 hrs were neglected. In the beginning and the end of the experiment also the organic matter contents and WHC value were determined. Applied methodology did not found changes in the organic matter contents but this does not mean that there was no alteration of the composition of organic matter. The WHC value did not significantly change.

An Influence of Repeating Cycles of Watering and Drying (DRW) upon the Oxygen Consumption and Excretion of Carbon Dioxide from the Soil
Therefore presumably, that if during a hot summer torment rainfalls occur, the intensity of metabolism of soil microorganisms significantly decreases and become stable at low level. The decline of both: oxygen consumption and carbon dioxide excretion indicates very serious disturbance in the activity of destruents, which must result in alteration of carbon cycle in the whole ecosystem.

A Relationship between Changing Humidity of the Soil and Oxygen Consumption/Carbon Dioxide Excretion during Subsequent Cycles of Drying/Watering
The reaction of metabolic activity of soil microorganisms for changes in soil  [29]. Considering intensifying conditions of water deficit, water holding capacity (WHC) is closely connected with that issue and not less important. This parameter to a great extend is decisive regarding the availability of water and air to soil organisms and is changing seasonally since its value depends on the inflow of organic matter and the system of humidification of the soil [19].
The WHC of soil used for this experiment was not high (WHC = 1.1723; sd = 0.0823; se = 0.02744 p = 0.000). For such forests this value is rather low. In 2014 the maximal WHC value for soils sampled in the same site ranged between 0.5 and >4 [19].
It was demonstrated that under an influence of DRW cycles, assemblages of microorganisms change their associations and also display various reactions towards humidity of the soil [10] [29]. The relationship between decreasing humidity and carbon dioxide excretion/oxygen consumption is the most similar to sinusoid, assuming that initial humidity considerably exceeds the maximal value.
Theoretically it is accepted that the optimum of aerobic respiration occurs at humidity of about 0.6 GWC which results from diffusive properties of the soil, which influence availability of water and oxygen for living organisms [30] [31].   changes react with delayed activity, the cycles A and B should be regarded as that of resistant assemblage because they react for increased humidity quickly with intensified metabolic activity. The cycle C shows the optimum of respiration at humidity of about 0.6 while the next cycle D at humidity of about 0.2. Hence in subsequent cycles of drying, the optimum of respiration occurs in decreasing humidity. The C DRW cycle seems to be the transitional one, in following cycles the coefficient "β" decreases, and the reaction of respiration for successive drying becomes weaker and more stable. The respiration in two last cycles is practically no different. Assuming that coefficient "β" describes the rate of reaction of microbial assemblage for humidity changes, it is possible to say that in all DRW cycles the excretion of carbon dioxide always reacts slower for changes of humidity than oxygen consumption. Also the determination coefficient R 2 is always lower in regressions concerning excretion of carbon dioxide. Considering results presented in Table 2 it is possible to conclude, that the process of oxygen consumption is more sensitive for humidity changes than the production of carbon dioxide. Recently frequent torment rains in Central Europe, during high temperatures moisten mostly the upper layer of the soil, i.e. the layer where oxygen processes dominate and which is the most sensitive to changes of humidity. This layer though is responsible for the circulation of gasses in the soil and therefore for processes regulating the carbon cycle in ecosystems.

Conclusions
A laboratory experiment with organic forest soil from Central Europe has proved that both the excretion of carbon dioxide and oxygen consumption decreases significantly under an influence of cycles of drying and subsequent rapid rewetting (Table 2). After 10 cycles, the respiration decreases by 2.4 times however amounts of excreted carbon dioxide and consumed oxygen do not differ between cycles 9 and 10.
In successive DRW cycles also changes the relationship between oxygen consumption/carbon dioxide excretion and humidity level. This relationship is logarithmic and the analysis of subsequent regressions indicates the direction of those changes. During first cycles of rewetting the respiration responds with a sharp slope of the curve (β coefficient for OCD = 1.73; 1.03; for CO 2 = 1.12; 0.50). In successive cycles the value of β coefficient (slope) decreases and both: the values of β and coefficient R 2 are always higher for oxygen consumption that for carbon dioxide excretion. This indicates that processes involving oxygen consumption are always more sensitive than processes producing carbon dioxide. The optimum of respiration declines in successive DRW cycles.
Obtained results provide evidence that increasing hot periods with accompanying torment rains may substantially change sensitive organic forest soils which in turn affects the rate of decomposition of organic matter. This disturbs whole forest ecosystems which, especially in the period of global climatic changes, are among the most important reservoirs able for fixation of carbon dioxide from the atmosphere.