The Specific and Total CO2 Emission Activity of Wood-Decaying Fungi and their Response to Increase in Temperature

1. Introduction

Climate change, its causes and environmental consequences is one of the most discussed problems in ecology, also in works on the ecology of woody debris and its decomposition [1,2,3,4,5]. This is far from accidental, since woody debris is a globally significant natural reservoir of carbon; in Russian forests alone its reserves amount to about 5.5 Gt. This huge mass of woody debris is at various stages of its biological decomposition – a large-scale and specific to forest ecosystems process, during of which in Russian forest annually about 214 Mt C-CO₂ is emitted into the atmosphere. This makes debris the second natural source of carbon dioxide after soil [1,6].

The main role in the decomposition of debris in forests of temperate latitudes is played by xylotrophic fungi − Basidiomycota, Agaricomycetes [7,8,9,10]. This is, perhaps, the only group of organisms in the modern biosphere that has a unique set of interconnected ecological and physiological adaptations to the woody habitat and is capable of decomposing the lignocellulosic complex of wood [11]. They determine the main parameters of CO₂ emission activity of woody debris and this makes them one of the globally significant regulators of the gas composition of the atmosphere [12], factors of climate stability and change [5].

One of the key questions arising from climate change relates to future carbon dynamics, which largely depends on the temperature sensitivity of decomposition processes. They play an important role in the global carbon cycle and, through feedbacks, can potentially influence climate change [5,13,14]. All currently available data clearly indicate a close connection between the CO₂ emission activity of xylotrophic fungi, wood debris and temperature [3,4,5,15,16,17,18,19,20,21]. In particular, in one of our recent works [22], we showed that an increase in temperature from 20 to 30 °C has a non-additive, possibly synergistic effect on the CO₂ emission activity of xylotrophic fungi, causing its exponential growth.

Considering the role of xylotrophic fungi as factors of stability and climate change, this phenomenon undoubtedly requires its most careful study and, above all, this concerns the temperature response of CO₂ emission activity of xylotrophic fungi to an increase in temperature. This determined the purpose of this work – analysis of the relationships between the CO₂ emission activity of xylotrophic fungi and temperature, testing the hypothesis of an exponential increase in CO₂ emission with increasing temperature in the range of 10-30 °C, which is relevant for temperate latitudes.

2. Materials and Methods

The study of the temperature response of CO₂ emission activity dikaryotic mycelium of five species xylotrophic fungi growth on wort agar was carried out: Fomes fomentarius (L.) Fr., F. inzengae (Ces. & De Not.) Cooke, Fomitopsis betulina (Bull.) B.K. Cui, M. L. Han & Y.C. Dai, F. pinicola (Sw.) P. Karst., Phellinus igniarius (L.) Quél (Figure 1). Dikaryotic cultures were isolated from basidiocarps of the corresponding species of the fungi using traditional methods [23] and wort (4%) – agar (2%) as a nutrient medium (MA). Use of pure fungi cultures allow to solve of one most difficult problem in the study of xylotrophic fungi − the assessment of mycelium biomass in wood. When mycelium develops on artificial nutrient media, an indicator of its biomass can be such an easily assessed characteristic as the area it occupies in Petri dishes.

The species identification of basidiocarps was determined on anatomical and morphological characteristics [24], and their species names are given according to Index Fungorum [25]. F. fomentarius strains were typed using ITS region sequencing; according to phylogenetic analysis, strains collected on Populus L. belong to F. inzengae, and on Betula L. belong to F. fomentarius sensu stricto – to two cryptic taxa [26,27,28].

The analysis scheme was as follows. Petri dishes (9 cm in diameter, 3 for each strain) were inoculated with a piece of agar (about 5 mm) with the mycelium of the fungus being studied and kept for several days at +25 °C. When the mycelium began to grow around the inoculum, its border was marked by felt-tip pen on the underside of the Petri dishes and dishes were placed in open exposure chambers with a volume of 0.5 l and placed in a thermostat at +10 °C for 2 hours. Then the chambers were sealed, their CO₂ content was measured and placed in a thermostat at +10 °C for one hour. At the end of the exposure, CO₂ measurements in the chambers again were made, after which they were opened and left with closed Petri dishes inside for a day in a thermostat at +10 °C. After 24 hours, the size of the mycelium in Petri dishes was measured and its border was marked, the chambers were closed, CO₂ was measured in them and placed in a thermostat at +10 °C for an hour. At the end of the exposure, the CO₂ content in the chambers was measured again. According to the same scheme, growth of mycelium and its emission activity were assessed at +20 °C, +25 °C, +30 °C and +35 °C.

The CO₂ content in the chambers was measured using a Gasmet DX-4030 FTIR spectrometer (Gasmet Technologies Oy, Finland) with an accuracy of ±50 ppm. The emission activity of the mycelium was assessed by the difference in CO₂ concentration in the chambers at the beginning of the exposure and at it end and was calculated in μg of CO₂, taking into account the volume of the exposure chambers and Petri dishes, exposure duration.

Specific CO₂ emission was calculated in µg CO₂/cm²/h by equation (1):

SEA = ΔСО₂ × (V₁−V₂) / Vm × M / S × 0.27 × 273/T,

(1)

The total CO₂ emission, or the total amount of carbon dioxide emitted by the mycelium, was calculated in µg CO₂/h by equation (2):

TEA = ΔСО₂ × (V₁−V₂) / Vm × M × 0.27 × 273/T,

(2)

where SEA is the specific CO₂ emission, TEA is the total CO₂ emission, ΔCO₂ is the amount of CO₂ released by the mycelium during exposure (ppm/h), V₁ is the chamber volume (l), V₂ is the sample volume (l), Vm is the molar volume (22.4 l/mol), M – molar mass of CO₂ (44 g/mol), S – area occupied by mycelium (cm²), T – temperature in Kelvin (K).

The temperature coefficient (Q₁₀) of specific CO₂ emission, showing the multiplicity of its change with a temperature increase of 10 °C, was calculated by equation (3):

Q₁₀SEA = SEA₁/SEA₂,

(3)

where Q₁₀SEA is the temperature coefficient of specific emission, SEA₁ is the specific emission at 10 and 20 °C, and SEA₂ at 20 and 30 °C, respectively.

The temperature dependence of mycelium growth was assessed by the increase in the area it occupied on MA during the day (cm²/day) and by the temperature coefficient (Q₁₀) of growth calculated by similar equation (4):

Q₁₀SM = V₂/V₁,

(4)

where Q₁₀SM is the temperature coefficient of growth, V₁ is the intensity of mycelium growth (cm²/day) at 10 and 20 °C, and V₂ is the intensity of mycelium growth at 20 and 30 °C, respectively.

Statistical data processing was performed using the Statistica 10.0 program (StatSoft Inc., USA). Arithmetic means (m) are given with standard errors (SE). The Pearson correlation coefficient (r) was used to characterize the relationships between variables. Student's t-test was used for pairwise comparisons; one-way analysis of variance (ANOVA) was used for multiple comparisons of means. When describing the results of statistical evaluation, the values of the corresponding criteria and its significance are given.

3. Results

Figure 2 shows the dynamics of CO₂ emission activity of five species dikaryotic mycelium at MA in the range of 10-30 °C. It can be seen that the temperature dynamics of total (TEA) and specific (SEA) CO₂ emission activity are significantly different: SEA is linear (determination coefficient 0.91-0.98), TEA is exponential (determination coefficient 0.94-0.99).

The Table 1, Table 2 and Table 3 shows that the temperature rise from 10 to 20 °C increase the specific CO₂ emission activity mycelium from 1.3 (F. betulina) to 2.1 (F pinicola, collected on Picea), on average 1.8 times. An increase in temperature from 20 to 30 °C enhances SEA by 1.2 (F. fomentarius s. str.) – 1.9 (F. pinicola, collected on Picea), on average 1.6 times. In other words, the temperature coefficient of the specific CO₂ emission activity of the mycelium of the studied group of xylotrophic fungi ranges from 1.6 to 1.8. An increase in temperature from 10 to 30 °C (3 times) causes a corresponding increasing in SEA – 2.9 times. At 35 °C, SEA decreases in some of the analyzed fungi, while in F. betulina and F pinicola (collected on Betula) it remains at the same level as at 30 °C.

The specific CO₂ emission activity of the mycelium does not show any relationship connection with its size, in our case, with its area. Thus in F. betulina strain at 10 °C SEA of the same level (18˗19 µg CO₂/cm²/h) for mycelium with an area of 10 and 13 cm², and at 20 °C it is equal 24-25 µg CO₂/cm²/h for mycelium with an area of 13 and 21 cm² (Table 1). In F. pinicola (strain collected on Picea) at 10 °C, SEA equal to 19 μg CO₂/cm²/h is recorded in mycelium with an area of 11 and 14 cm², and in the strain collected on Betula 23 μg CO₂/cm²/h in mycelium of 13 and 18 cm². The same is observed at 30 °C: in the strain collected on Betula, the mycelium of 29 cm² and 38 cm² has SEA equal to 73 µg CO₂/cm²/h, and in the strain collected on Picea 73˗74 µg CO₂/cm²/h in the mycelium with an area of 27 cm² and 35 cm² (Table 2). There is also no relationship between SEA and mycelium area in the F. fomentarius s. str. and F. inzengae strains (Table 3).

The specific CO₂ emission activity depends on the species of fungus and in the range 10-20 °C, its average value varies from 27.5±1.95 (F. betulina) to 118.8±9.53 μg CO₂/cm²/h (F. inzengae). In F. betulina, F. pinicola it is significantly – F_{(1, 142)} = 90.160, p = 0.001 – lower (varies from 27.5±1.95 to 47.4±4.19, on average 40.3±2.32 μg CO₂/cm²/h) than in F. fomentarius s. str. and Ph. igniarius (varies from 60.1±4.59 to 118.8±9.53, on average 95.4±5.31 µg CO₂/cm²/h). At the same time, F. pinicola strains isolated from basidiocarps collected on Betula and Picea have SEA of the same level (Table 2).

The response of total CO₂ emission activity to an increase in temperature is more pronounced than in the case of specific activity. If, with an increase in temperature from 10 to 20 °C, SEA, as noted, increases by 1.3-2.1 times, on average 1.8 times only, then TEA increases by 3 (F. betulina) – 6 (F. fomentarius s. str.) times, on average 4 times. When the temperature increases from 20 to 30 °C, TEA increases by 2 (F. betulina) – 4 (F. fomentarius s. str., F. pinicola), on average 3 times, while SEA 1.6 times. An increase in temperature from 10 to 30 °C enhances the TEA of the mycelium of F. betulina and Ph. igniarius by 6, F. pinicola by 10, F. inzengae by 13 and F. fomentarius s. str. by 21 times – on average 11 times. At the same time, as noted above, SEA will increase by 2.9 times. Like SEA, TEA reaches its maximum at 30 °C; at 35 °C it decreases or remains at the same level as at 30 °C. TEA varies depending on the fungus species: the highest (2000-3000 μg CO₂/h) in the mycelium of F. fomentarius s. str., F. inzengae, F. pinicola and 2-3 times lower (does not exceed 1000 μg CO₂/h) in the mycelium F. betulina and Ph. igniarius (Table 1, 2, 3).

The total emission activity of the mycelium depends not only on temperature, but also on its size. For example, at 20 °C, an increase in the mycelium area of F. betulina by 1.6 times (from 13 to 21 cm²) is accompanied by a similar 1.6-fold increase in TEA (from 303 to 490 μg CO₂/h). Increase in mycelium size Ph. igniarius by 1.5 times (from 6 to 9 cm²) at 20 °C leads to an increase in its TEA by 1.6 times (Table 1). In F. pinicola at 20 °C, an increase in the size of the mycelium by 1.2˗1.3 times is accompanied by an increase in TEA by 1.2˗1.5 times (Table 2). An increase in TEA proportional to the increase in mycelium area is also observed in the F. fomentarius s. str. and F. inzengae strains (Table 3).

The size of the mycelium reflects the intensity of its growth, which is positively related to temperature. The correlation coefficient of the daily increase in mycelium area with temperature for F. betulina is 0.61, F. pinicola 0.57 (strain collected on Picea) – 0.76 (strain collected on Betula), F. inzengae and Ph. igniarius 0.85, F. fomentarius s. str. 0.97. The temperature coefficient (Q₁₀) of mycelium growth with an increase in temperature from 10 to 20 °C varies from 1.3 (F. inzengae) to 2.0 (F. fomentarius s. str.), and on average is 1.5; it has the same average value when the temperature increases from 20 to 30 °C. At 30 °C growth rate of F. betulina and Ph. igniarius mycelium reaches its maximum, and F. pinicola, F. inzengae, F. fomentarius s. str. – at 35 °C (Table 1, Table 2 and Table 3).

4. Discussion

There is an opinion that for the majority of representatives of the boreal microbiota, adapted to an average summer temperature of about +15 °C, an increase in temperature to 30 °C will be tantamount to temperature shock [6]. However, as the results of this study show, in the range of 10-30 °C, xylotrophic fungi respond positively to increased temperature. Thus, in this range, the specific CO₂ emission activity of the mycelium, which is an indicator of its respiratory activity, increases on average 1.7 times with an increase in temperature by 10 °C and 3 times with its increase from 10 to 30 °C: 1.7 x 1.7 = 2.9. In other words, the specific CO₂ emission activity of the mycelium of xylotrophic fungi obeys the Van't Hoff rule and this determines the linear nature of its temperature dynamics. The specific emission activity of the mycelium does not depend on the size of the mycelium and its relationship with temperature is described by the following equation (5):

SEA_T2 = SEA_T1 x Q₁₀SEA ^(T2-T1)/10,

(5)

where SEA_T1 and SEA_T2 are specific CO₂ emission at temperature T₁ and T₂, respectively; Q_10SEA – temperature coefficient of specific CO₂ emission activity.

Thus, the only driver of the specific CO₂ emission activity of the mycelium of xylotrophic fungi is temperature, or rather its temperature sensitivity, the indicator of which is the temperature coefficient. The latter, in the range of 10-30 °C, varies depending on the species from 1.2 to 2.1, with an average of 1.7. We also recorded similar Q₁₀ values of specific CO₂ emission activity when analyzing the gas exchange of wood residues destroyed by xylotrophic fungi: 2.0-2.1 [12,29]. The close positive relationship between specific emission activity and temperature determines its unstable nature; one of the results of this is a fluctuation in the intensity of CO₂ gas exchange of wood residues during the day: an increase in the daytime and a decrease in the night [30].

The total CO₂ emission activity of the mycelium is determined by its specific emission activity and size. Accordingly, its temperature dynamics has two drivers: temperature sensitivity of a) specific emission and b) mycelium growth, indicators of which are the temperature coefficients of specific emission and mycelium growth. The relationship between total emission and temperature is described by the following equation (6):

TEA_T2 = TEA_T1 x Q₁₀SEA ^(T2-T1)/10 x Q₁₀SM ^(T2-T1)/10,

(6)

where TEA_T2 and TEA_T1 are the total CO₂ emission at temperature T₁ and T₂, respectively; Q₁₀SEA and Q₁₀SM are the temperature coefficient of specific CO₂ emission and mycelium growth, respectively.

Depending on the species of fungi, the Q₁₀ of mycelium growth ranges from 1.3 to 2.0, and on average it is 1.5 and the almost identical Q₁₀ of specific emission − 1.7. Therefore, an increase in temperature in the range of 10-30 °C causes an almost equal increase in two unidirectional processes – specific CO₂ emission activity and mycelium growth. Their joint action causes an exponential increase in total CO₂ emissions. The dependence on the mycelium size determines another very important feature of the total CO₂ emission activity of xylotrophic fungi. Due to the fact that the growth of mycelium is an irreversible increase in its size and mass, the temperature dynamics of the total emission also has the character of a directed, irreversible process. The total emission reaches its maximum at 30-35 °C, a temperature at which both the maximum specific CO₂ emission activity and the size of the mycelium are observed.

5. Conclusions

In the range of summer temperatures (10-30 °C) that are relevant for temperate latitudes, the CO₂ emission activity of xylotrophic fungi is closely and positively related to temperature. Their specific CO₂ emission activity determined by the respiratory activity of the mycelium and does not depend on its size. The only driver of specific emissions is temperature, an increase in which causes its proportional (linear) increase. The total CO₂ emission activity, which is an indicator of the amount of CO₂ emitted, depends on the size and specific emission activity of the mycelium. It has the character of an irreversible, directional process that increases exponentially with increasing temperature to 30˗35 °C. This gives fairly strong grounds to assume that climate warming will lead to an exponential increase in the CO₂ emission activity of woody debris, which, in turn, could potentially contribute to the acceleration of climate change.