Fungal Methane Production Controlled by Oxygen Levels and Temperature

1. Introduction

Methane (CH₄) is a potent climate gas, with a greenhouse gas (GHG) potential approximately 100 times greater than carbon dioxide (CO₂) over a 10-year period [1]. A significant portion of global CH₄ emissions originates from biotic sources, exceeding contributions from abiotic sources like fossil fuel and biomass burning, as well as geogenic processes [2,3]

Contrary to the earlier belief that biotic CH₄ production occurs exclusively under anaerobic conditions by methanogenic archaea in environments such as wetlands, landfills and rice paddies, and in the digestion system of termites and ruminants, recent research has revealed that biotic CH₄ can also be produced in the presence of oxygen (O₂). The first evidence of CH₄ formation by plants under aerobic conditions was presented by [4], and subsequent research expanded this finding to a range of eukaryotic CH₄ and prokaryotic sources, including mosses and lichens [5], marine algae [6,7], terrestrial and marine cyanobacteria [8], plant cell cultures [9,10], non-methanogenic archaea [10], animals [11,12], human cell cultures and humans [10,13,14,15,16], as well as fungi [10,17,18].

For some years, the mechanisms behind CH₄ formation in these organisms remained elusive. However, a recent breakthrough came with the discovery by [10] of a universal non-enzymatic CH₄ formation mechanism potentially occurring in all organisms that produce reactive oxygen species ROS. This mechanism, based on Fenton chemistry, involves the reaction of ROS with free iron (II) ions and methylated precursor compounds within cells, encompassing all three domains of life.

Despite these advancements, little is known about the detailed mechanism and the physical and chemical factors that drive CH₄ formation in many newly discovered sources and organisms. This is especially true for (saprotrophic) fungi that play an essential role in decomposing organic matter such as wood lignocellulose, thus playing a crucial role in the global carbon recycling [19]. The challenge lies in determining fungal biomass and correlating it with CH₄ emissions. This is further complicated by the species and medium dependency of these emissions (e.g., [18]), as well as the potential for yet unknown CH₄ formation pathways, which complicates our understanding of the global impact of these emissions.

Before the studies by [17] and [18], which found that saprotrophic fungi directly produce CH₄ under aerobic conditions without the presence of methanogenic archaea, it was assumed that fungi initiated the decomposition process by breaking down macromolecules, like those in wood, thereby providing the precursor compounds for CH₄ production by methanogenic archaea in anoxic microsites (e.g., [20,21,22,23]). Another CH₄ producing pathway, involving facultative anaerobic fungi and a halomethane dependent pathway, was identified by [24], where CH₄ formation correlated with the formation of chloromethane (CH₃Cl), as previously reported by [25]. These authors further highlight, that the function of the enzymes involved in the halomethane dependent CH₄ formation pathway are independent of O₂ and thus might also be involved in observed fungal CH₄ production by [17] and [18].

A common consensus in the field is that fungal CH₄ emissions are strongly dependent on the fungal species and the wood substrates [17,18,26,27,28]. However, the influence of key parameters like O₂ availability and temperature on fungal CH₄ formation has not been investigated, even though these factors strongly influence the physiological activity and growth of fungi. Studies have shown that the activity and growth of xylothrophic fungi depend on prevailing O₂ mixing ratios [29,30,31]. For instance, xylothrophic fungi, which include the two investigated fungi in this study, can consume all available O₂ in their woody habitat and still grow under anoxic conditions [31]. On the other hand, studies have indicated that below a concentration of 0.2% O₂, fungal growth is completely inhibited [29,30], while the decay of wood debris by saprotrophic fungi decreased with decreasing O₂ and increasing CO₂ mixing ratios, and vice versa [29,32]. This suggests that prevailing O₂ concentrations significantly influence fungal activity and metabolism, potentially controlling fungal CH₄ emissions.

Temperature is another critical driver for fungal metabolism. Numerous studies have investigated the relationship between wood decay and temperature [27,28,33,34,35], finding that increased temperatures led to higher CH₄ emissions due to wood decay. This observation is likely linked to the role of saprotrophic fungi as significant producers of extracellular enzymes needed for wood decomposition, which is predicted to increase due to higher temperatures [33]. Fungal growth even quadrupled with a 10 °C increase in temperature across a tropical elevation gradient [34], indicating a substantial impact of temperature on the amount of prevailing fungal biomass and, consequently, on CH₄ emissions by xylothrophic fungi.

In our study, we investigated the effects of different O₂ levels and temperatures on fungal CH₄ production by two saprotrophic fungi, Pleurotus sapidus and Laetiporus sulphureus. Both fungal species were grown on various substrates including beech wood, pine wood, and grass, and incubated under sterile conditions. Oxygen consumption rates were measured under different temperatures and patterns of CH₄ production as well as CH₄ production normalized to O₂ consumption (CH₄__norm) were examined.

2. Results

In order to evaluate the dependency of fungal CH₄ formation on prevailing O₂ concentrations starting at ambient levels (20.9% O₂, see section 2.1) and at elevated levels (starting at > 90% O₂, see section 2.2) as well as different temperature (17 to 40°C, section 2.3) two different saprotrophic fungal species were incubated with different growth media (beech, pine, grass). For methodological details, we refer to section 4. Materials and Methods. Please note that CH₄ formation and O₂ consumption rates were based on a per flask basis and not related to fungal dried biomass because it was not possible to calculate the amount of fungal biomass after each measurement step. Thus, in all conducted incubation experiments CH₄ production rates were normalized to the O₂ consumption rates (CH₄__norm) to directly link CH₄ production to the metabolic activity of the fungi, inferred from O₂ consumption (Table 1). In addition, changes in CO₂ concentrations in the flask were also measured and their formation rates estimated and is also an indicator for the metabolic activity of the fungi. As the focus of the manuscript is on the role of O₂ the accompanied CO₂ data is shown in the supplement (Text S1 and Figure S1). Please also note that all presented CH₄ formation, CH₄__norm rates were corrected by subtracting the observed CH₄ rates in the medium controls.

2.1. Dependance of fungal CH₄ production on ambient O₂ concentrations

All incubation experiments in which the two fungal species P. sapidus or L. sulphureus were grown on different substrates at various O₂ levels showed measurable CH₄ formation rates compared to the respective substrate control (Table 1). Calculated CH₄ formation and O₂ consumption rates were in the range of 0 to 5.34 ± 0.64 nmol h^-1 and 0.06 ± 0.01 to -0.88 ± 0.06 mmol h^-1, respectively.

No measurable CH₄ formation was observed when O₂ levels were below ~0.5%. As soon as O₂ was reintroduced, following a O₂ induced dilution of CH₄ levels in the vials, this led to an immediate increase in CH₄ formation, indicating a rapid response to the availability of O₂ for fungal metabolism. This is exemplarily shown in Figure 1 (arrows indicate the addition of O₂) for L. sulphureus (Figure 1A) and P. sapidus (Figure 1B) grown on beech wood.

For both fungi, the amount of CH₄ gradually increased within the flasks when O₂ was present. Notably, CH₄ formation rates substantially decreased in both fungi when O₂ mixing ratios fell, eventually ceasing completely, indicating no further CH₄ formation. However, upon reintroduction of O₂ to the flasks (Figure 1, as indicated by arrows, e.g., at an incubation time of 370 h for L. sulphureus grown on beech wood), the amount of O₂ initially increased (from 0.05 ± 0.03 to 16.8 ± 0.7 mmol) while that of CH₄ decreased (from 240.0 ± 7.8 to 190.3 ± 7 nmol), a result of the dilution effect from the supplemented gas volume. Subsequently, CH₄ formation resumed immediately leading to a consequent increase in CH₄ (~ 60 nmol). This pattern was repeatedly observed during the incubation of the two fungi grown on beech wood (Figure 1). It is important to note that during the incubation of P. sapidus grown on beech wood (Figure 1B), the CH₄ yield in the flask gradually decreased with successive O₂ additions from 167 nmol at the start of the incubation to 94.7 ± 6.0 nmol at the end of the incubation. This reduction is attributed to the dilution effect of the O₂ additions, which surpassed the fungal CH₄ formation rate. Nonetheless, a distinct increase in CH₄ was noted following each O₂ addition. Controls containing pine wood and grass (excluding beech due to unavailability, however [17] showed that beech controls show negligible CO₂ emissions) exhibited much lower but still measurable CH₄ as well as CO₂ emission rates regardless of prevailing O₂ levels (Figure S1). A more detailed description and discussion of these data can be found in the supplement (Text S1).

Beyond the dependency of fungal CH₄ formation on the presence of O₂, we further found that the prevailing mixing ratios of O₂ in the incubation flasks influenced the CH₄ formation rates by P. sapidus and L. sulphureus. While in all experiments O₂ consumption rates generally were higher when higher O₂ mixing ratios prevailed ranging from -0.23 ± 0.002 to 0.02 mmol h^-1 and -0.88 ± 0.06 to 0.06 ± 0.01 mmol h^-1 for O₂ mixing ratios from 0 to 5% and 5 to 21%, respectively (Table 1), this observation was more obvious for CH₄ production (0.001 ± 0.03 to 2.16 nmol h^-1 and 0.09 ± 0.02 to 5.34 to 0.64 nmol h^-1, respectively) and CH₄__norm rates (-0.02 ± 0.70 to 11.6 ± 0.2 (*10^-6) and 0.70 ± 0.12 to 13.7 ± 1.0 (*10-⁶), respectively). Generally, we observed differences between CH₄ production rates and CH₄__norm rates of P. sapidus and L. sulphureus.

While CH₄ production rates were similar when both fungi were grown on beech wood (0.51 ± 0.44 nmol h^-1 for L. sulphureus and 0.48 ± 0.31 nmol h^-1 for P. sapidus), L. sulphureus showed higher rates when grown on pine wood (4.55 ± 1.49 nmol h^-1 for L. sulphureus and 1.70 ± 0.65 nmol h^-1 for P. sapidus; p = 0.005). Similarly, we found that CH_{4_norm} rates were generally higher for L. sulphureus compared to P. sapidus (e.g., Figure 2; beech wood: 3.00 ± 1.92 (*10^-6) and 0.95 ± 0.76 (*10-⁶), respectively, p < 0.001; pine wood: 12.9 ± 1.65 (*10-⁶) and 4.38 ± 0.58 (*10-⁶), respectively; p = 0.003). For P. sapidus (except for when grown on grass at 17 °C) higher CH₄__norm rates were observed when O₂ levels ranged between 0 to 5% compared to higher levels between 5% to ambient levels (pine wood: 4.18 ± 1.26 vs. 2.08 ± 1.23 (*10-⁶), respectively, p = 0.057; grass: 0.19 ± 0.05 vs 0.86 ± 0.62 (*10-⁶) at 17 °C, p = 0.063 and 11.6 ± 0.17 vs. 3.41 ± 1.73 (*10-⁶) at 40 °C, p < 0.001; Table 1). For L. sulphureus, we observed an opposite trend where higher CH₄__norm rates prevailed in a range between 5 to 21% (beech wood: -0.02 ± 0.70 vs. 3.76 ± 1.28 (*10-⁶), respectively, p = 0.002; pine wood at 27°C: 10.4 vs. 13.7 ± 1.00 (*10-⁶); e.g., Figure 2, Table 1).

In all incubations CO₂ mixing ratios demonstrated an opposite trend to O₂ levels, serving as a clear indicator of the fungi’s metabolic activities. Please note, that that CO₂ measurements were conducted less frequently than those for CH₄ and O₂, due to logistical reasons. During experiments where O₂ was added, CO₂ concentrations sometimes exceeded 20% in mixing ratios, a level expected when all O₂ was consumed and converted to CO₂ (supplement Text S1 and Figure S1).

2.2. Fungal CH₄ production starting at elevated O₂ mixing ratios of near 100%

In another approach investigating the O₂ dependency of fungal CH₄ formation, both L. sulphureus and P. sapidus were grown on beech at a temperature of 27 °C and exposed to a starting O₂ mixing ratio of approximately 95%, flushing the flasks with pure O₂ (Figure 3). At the beginning of the experiment the CH₄ yield in the incubation flasks was 27.3 ± 5.2 nmol for L. sulphureus and 16.9 ± 1.1 nmol for P. sapidus (reflecting prevailing CH₄ mixing ratios of around 0.2 and 0.4 ppmv in the flasks, respectively). These values increased to 161.3 ± 23.7 nmol (2.2 ± 0.3 ppmv) and 61.6 ± 7.1 nmol (0.8 ± 0.1 ppmv) over 1055 h and 70 h, respectively, whilst O₂ levels decreased to 16.6 ± 5.3% and 13.9 ± 3.5%. This corresponds to a CH₄ formation rate of 0.6 ± 0.1 nmol h^-1 for P. sapidus and a lower rate of 0.09 ± 0.02 nmol h^-1 for L. sulphureus (p = 0.002). Interestingly, the O₂ consumption rate varied substantially between the two fungal species. P. sapidus had a much higher rate accounting for -0.88 ± 0.06 mmol h^-1, in contrast to L. sulphureus, which reached a considerably lower O₂ consumption rate of -0.06 ± 0.001 mmol h^-1 (p < 0.001). This disparity in O₂ consumption directly influenced the CH₄__norm rates, which were substantially higher for L. sulphureus compared to P. sapidus, accounting for 1.56 ± 0.25 (*10-⁶) and 0.70 ± 0.12 (*10-⁶), respectively (p = 0.02).

When comparing these CH₄__norm rates to those starting at ambient O₂ levels of 21%, we found that CH₄__norm rates were in a similar range for P. sapidus (0.95 ± 0.76 (*10-⁶)) and lower compared to L. sulphureus with 3.76 ± 1.28 (*10-⁶).

2.3. Temperature dependency of fungal CH₄ formation, O₂ consumption and CH₄__norm rates

P. sapidus and L. sulphureus, grown on pine wood and grass (only P. sapidus), were chosen to investigate whether CH₄ formation and O₂ consumption, as well as CH₄__norm rates, differed based on different temperatures: 17 °C, 27 °C, and 40 °C. Similar to what is described in sections 2.1 and 2.2, the above-mentioned rates of CH₄ formation, O₂ consumption, and CH₄__norm rates showed a clear influence of prevailing O₂ mixing ratios. This influence, along with that of different temperatures, is additionally presented in this section.

Figure 4A shows the trend of the amount of CH₄ and O₂ levels of the incubation experiments of L. sulphureus grown on pine wood at three different temperatures (17, 27, 40 °C), while Figure 4B,C show the course of these parameters of P. sapidus grown on pine wood (17 °C and 40 °C) and grass (17 °C and 27 °C), respectively, along with the corresponding controls for pine wood and grass. Please note that for P. sapidus grown on pine wood and grass, only two temperature steps were available due to logistical reasons. All three incubation experiments clearly showed that CH₄ production rates were highest at a temperature of 27 °C (p = 0.02 for L. sulphureus and p = 0.001 for P. sapidus grown on grass) or at 40 °C for P. sapidus grown on pine wood compared to the rates at a temperature of 17°C (p = 0.002; Figure 5, Table 1). Similar observations were made for O₂ consumption rates which were highest at a temperature of 27 °C compared to the other temperature steps. Moreover, calculated CH₄__norm rates at each O₂ level (see Table 1 or Figure 5) this pattern. However, we observed a more complex distribution of CH₄__norm rates when we considered the different O₂ levels within each incubation experiment (L. sulphureus grown on pine and P. sapidus grown on grass and pine) depending on the fungal species. For incubations with L. sulphureus grown on pine wood at 27 °C, CH₄__norm rates were higher for O₂ mixing ratios between 5 and 21% (13.69 ± 0.97 (*10-⁶)) compared to rates below 5% O₂ (10.4 (*10-⁶)). However, for P. sapidus grown on pine wood, CH₄__norm rates were higher when O₂ mixing ratios were < 5% compared to O₂ levels between 5°C and 21 °C with rates accounting for 4.18 ± 1.26 (*10-⁶) vs. 2.08 ± 1.23 (*10-⁶) and 4.94 ± 0.25 (*10-⁶) vs. 3.97 ± 0.38 (*10-⁶) for 17 °C and 27 °C, respectively. The same observation was made for P. sapidus grown on grass at a temperature of 40 °C with values of 11.6 ± 0.17 (*10-⁶) vs. 3.41 ± 1.73 (*10-⁶) for O₂ mixing ratios below 5% and above 5%, respectively. Contrastingly at a temperature of 17 °C, P. sapidus grown on grass exhibited higher CH₄__norm rates when O₂ levels ranged between 5 and 21% (0.86 ± 0.62 (*10-⁶)) opposed to O₂ mixing ratios below 5% (0.19 ± 0.05 (*10-⁶)). Pine wood and grass controls showed a comparatively small increase in CH₄ levels over time depending on temperature with CH₄ formation rates ranging from 0.3 ± 0.05 to 3.36 ± 0.53 nmol h^-1 for pine wood and 0.01 ± 0.001 to 0.31 ± 0.01 nmol h^-1 for grass (Table 1, Text S2 and Figure S2).

3. Discussion

3.1. Dependence of fungal CH₄ formation on O₂ levels

Despite existing studies on the growth, wood decay, and eco-physiological adaptations of xylothrophic fungi, the impact of O₂ levels on CH₄ production rates and CH₄__norm rates have not been explored to date (e.g., [36]). Our results clearly demonstrate that fungal metabolism due to the availability of O₂ is a crucial factor driving fungal CH₄ production. In our experiments, when O₂ was completely consumed by the fungi (meaning below ~0.5%; as indicated by the sensitivity of the deployed O₂ sensors), CH₄ formation ceased. This finding contradicts earlier beliefs that linked CH₄ formation in wood debris to anoxic microsites and the activity of methanogenic archaea (Figure 1 and Figure 4; [20,22,23,37,38], since if this was the case for our experiments, we would expect a strong increase in CH₄ levels once O₂ was depleted. However, our fungal incubations were performed under sterile conditions and excluding the activity of bacteria and archaea (see [17]). This is further in line with previous findings of [18] that showed that δ¹³C-CH₄ values of fungal CH₄ covers a wide range from -42 to -70 ‰, which is not exclusively indicative of methanogenic CH₄ but overlapping with many other CH₄ sources such as thermogenic degradation of organic matter and other eukaryotes such as algae and cyanobacteria [8,39].

Intriguingly, upon reintroduction of O₂ to the incubation flasks, inducing aerobic metabolism, O₂ consumption and CO₂ production, fungal CH₄ formation promptly resumed (e.g., Figure 1B). Thus, our study distinctly establishes for the first time that aerobic CH₄ formation by the investigated fungal species, P. sapidus and L. sulphureus, and likely saprotrophic fungi in general, occurs exclusively for metabolically active cells in the presence of O₂. This is in line with previous results by [10], showing that bacteria such as Bacillus subtilis produce CH₄ when they are active and O₂ is present. Conversely, CH₄ production by this organism halts when either the cells are in a dormant state or O₂ is absent. Thus, the pattern of CH₄ formation, dependent on O₂ levels and the metabolic activity of B. subtilis, appears to be a clear analogy to the two fungal species investigated in this study. Therefore, it seems likely that fungal CH₄ was produced via a mechanism similar to that described by [10], via the generation of a methyl radicals by oxidative demethylation of a methylated nitrogen, sulfur or oxygen moiety in the presence of ROS and iron (II).

Further exploring this dependency, we discovered that the level of O₂ present in the incubation flasks significantly influenced CH₄ formation by P. sapidus and L. sulphureus. In all experiments, O₂ consumption rates were generally higher when higher O₂ mixing ratios were present. This observation aligns with prior studies indicating that the growth and wood decay activities of saprotrophic fungi diminish when O₂ levels fall below 1.5% and 10% or 1%, respectively, and CO₂ levels rise accordingly [29,32]. Contrarily, [31] found that xylothrophic basidiomycetes could completely exhaust O₂ in their environment and withstand high CO₂ levels, even up to 100%. These fungi are, therefore, facultative anaerobes that produce CO₂ in O₂-deprived environments. Our observations support this, as we noticed a significant increase in CO₂ levels, even when O₂ was entirely consumed in the incubations containing medium and fungi (Figure S1). It is noteworthy, however, that CO₂ mixing ratios rose more rapidly when O₂ was available to the fungi.

Our study further found that O₂ consumption, CH₄ production and CH₄__norm rates differed substantially between two fungal species P. sapidus and L. sulphureus. These were most prominent in the O₂ consumption rates, where P. sapidus exhibited much higher rates compared to L. sulphureus under similar incubation conditions. This could be due to differences in biomass; however, this parameter could not be determined within the scope of this study. Another reason could be differences in their metabolic activity as they are different types of white rot and brown rot fungi, respectively. And finally, the growth substrates might have impacted the observed rates. Several studies have found that fungal CH₄ production, also in relation to CO₂ emissions as an indicator of metabolic activity, is dependent on the growth substrate and substrate quality [17,18,27]. In our experiments, the most likely explanation for the observed differences is their prevalence of being decomposed by the different enzyme sets of L. sulphureus, a brown rot fungus, and P. sapidus, a white rot fungus.

Generally, there were notable differences in CH₄ production rates and CH₄__norm rates between these two fungal species. In line with a previous study by [17], and [18], CH₄ production rates were typically up to 2.5 times higher in incubations containing L. sulphureus (grown on pine) compared to those with P. sapidus grown on pine (Table 1) and similar in magnitude when both fungi were grown on beech wood. However, we now demonstrate for the first time that CH₄__norm rates, normalized to O₂ consumption, also exhibited a similar trend with L. sulphureus showing up to 2.5 times higher values than P. sapidus (Figure 2 and Figure 5).

Moreover, we observed distinct differences between P. sapidus and L. sulphureus concerning CH₄__norm rates under varying O₂ levels. For P. sapidus, CH₄__norm rates were often higher when O₂ levels ranged from 0 to 5%, compared to higher levels between 5% and ambient mixing ratios (Figure B,D,F; P. sapidus grown on pine and grass). Conversely, for L. sulphureus, higher CH₄__norm rates were observed in a range between 5 to 20%. The underlying reason for this disparity remains unclear, but a potential explanation might lie in the differences in their metabolic pathways. P. sapidus, a white rot fungus, predominantly uses oxidative enzymes to decompose wood compounds such as lignin, cellulose, and hemicellulose. In contrast, L. sulphureus, a brown rot fungus, relies on non-enzymatic oxidative systems to primarily depolymerize cellulose and, to a lesser extent, lignin, by generating ROS. Brown rot fungi deploy a mechanism dependent on Fenton-type reactions with ROS for wood decomposition [40,41,42]. However, the specific O₂ requirements for both mechanisms are not well understood. While the O₂ requirement for brown rot fungi is relatively known due to the direct production of ROS, it is more complex for white rot fungi, where O₂ is utilized as a substrate for the enzymes associated with wood decay [41,43,44]. This aspect requires further evaluation and could be linked to the observed differences in CH₄ production and CH₄__norm rates between P. sapidus and L. sulphureus.

In general, the growth and wood decay of these fungi at different O₂ levels indicate that metabolic activity is closely connected to fungal CH₄ formation, given that no CH₄ is produced by either fungus when O₂ is absent. While the exact mechanism of CH₄ formation by these fungi remains elusive, initial evidence by [17], which identified methionine as a precursor of fungal CH₄, suggests that the universal CH₄ formation mechanism proposed by [10] involving Fenton chemistry with methylated compounds and ROS likely represents a significant contributor to the observed CH₄ formation. Nonetheless, the potential involvement of other mechanisms, such as the halomethane-dependent pathway reported by [24], cannot be ruled out. Although demonstrated under anaerobic conditions, the activity of the relevant enzymes for this mechanism also persists under aerobic conditions, presenting another possible CH₄ formation mechanism that warrants future investigation, particularly under aerobic conditions.

3.2. Temperature influence on fungal CH₄ formation dynamics

To date, there is a noticeable gap in research regarding the influence of temperature on fungal CH₄ formation. Previous studies have primarily focused on the growth or decomposition rates of wood by basidiomycetes, which are also critical factors for fungal CH₄ emissions. Previous studies have consistently shown that both fungal growth (Meier 2010 and others) and wood decomposition rates [27,28,33] increase with rising temperature. However, it should be noted that the fungal biomass and changes during incubation experiments could not be determined during this study and thus CH₄ formation rates per unit biomass of fungi could not be calculated. Instead, we use consumption of O₂ and CO₂ production rates in relation to CH₄ formation as an indicator for fungal metabolic activity (see supplement Text S1 and Figure S1). We observed that the highest CH₄ formation as well as CH₄__norm rates occurred at 27 °C for both studied fungi. This peak in activity was likely attributed to the temperature being close to the optimal metabolism. At temperatures both lower (17 °C) and higher (40 °C) than this, a decrease in the CH₄__norm rates was noted, suggesting a decline in metabolic activity, as furthermore indicated by lower O₂ consumption and CO₂ production rates.

It is important to acknowledge that higher fungal biomass, resulting from elevated growth rates, most likely leads to increased CH₄ formation. This is due to CH₄ being produced by the fungus itself, as reported by [10], who found that two fungal species produced CH₄ and that elevated levels of ROS, which can increase in organisms as a stress response, even amplified the observed CH₄ formation. This aligns with findings from [17] and [18], which showed fungal CH₄ formation independent of the presence of methanogenic archaea. Therefore, it appears that CH₄ formation may be a function of not only the fungal biomass and experienced stress (ROS) levels but also the metabolic activity of the fungal species.

At 17°C, P. sapidus grown on grass exhibited smaller CH₄ production rates compared to when it was grown on pine wood, despite similar O₂ consumption rates across different O₂ regimes. Consequently, CH₄__norm rates were much higher for P. sapidus grown on pine wood. This indicates a substrate-specific component regulating CH₄ formation rates, supporting previous studies that highlighted the strong effect of fungal substrate on CH₄ formation and decomposition rates [18,27]. Additionally, CH₄ production rates for P. sapidus grown on grass and pine wood increased at elevated temperatures of 40 °C and 27 °C, respectively, likely due to higher metabolic activity, as indicated by increased O₂ consumption rates, particularly when O₂ mixing ratios exceeded 5%. This could also be influenced by increased stress levels, especially for P. sapidus grown on grass at 40 °C, similar to the previously discussed higher CH₄ formation in fungi due to increased ROS [10].

A similar pattern was noted for L. sulphureus grown on pine wood at 17 °C, 27 °C, and 40 °C. The highest CH₄ production rates were found at 27 °C, which is presumably closest to the optimal fungal growth temperature, whereas the lowest CH₄ formation rates at 17 °C coincided with the lowest O₂ consumption rates, indicating reduced metabolic activity at this temperature compared to 27°C and 40°C. However, contrastingly, CH₄__norm rates were in a similar range for both 17 °C and 40 °C, suggesting that while metabolic activity (inferred from O₂ consumption) was higher at 40°C, the ratio of CH₄ formation to O₂ consumption remained in the same range of around 3.5 (10^-6). [28], also observed that CH₄ and CO₂ levels clearly increased with temperature, ranging from 5 °C to 25 °C, during the decomposition of woody debris in a northern boreal forest, indicating an aerobic mechanism for CH₄ formation as reported by [10,17,18], which opposes the notion of an exclusive anaerobic origin of CH₄ by methanogenic archaea in anoxic microsites of these woody debris.

At various temperatures, we found that without O₂, no or substantially less CH₄ was emitted during our incubation experiments. Notably, CH₄ formation observed in the controls was also temperature-dependent, suggesting an additional abiotic mechanism as previously suggested by [17,18]. For results of control and explanations of abiotic CH₄ formation we refer to the Supplement Text S2 and Figure S2. Regarding abiotic formation of CH₄ and CO₂ from the studied substrates much more studies are required to fully comprehend this phenomenon.

4. Materials and Methods

4.1. Selected fungi

In this study, we selected P. sapidus (Pleurotaceae, DSMZ 8266) and L. sulphureus (Polyporacaeae, DSMZ 1014) as our focus due to their documented CH₄ emission properties, as reported in previous studies by [17,18]. These organisms were specifically chosen not only for their CH₄ emission capabilities but also for their distinct ecological and physiological traits. P. sapidus, a white rot fungus, and L. sulphureus, a brown rot fungus, are both notable for their ease of cultivation and management in laboratory settings, making them ideal species for our experimental analysis.

4.2. Incubation experiments

We investigated the O₂ and temperature dependency of fungal CH₄ emissions, focusing on two types of fungi: P. sapidus and L. sulphureus. P. sapidus was cultivated on a variety of media including beech, pine, and a grass mixture, whereas L. sulphureus was grown on beech and pine. The cultivation process involved using autoclaved wood chips from these trees and grass mixture, sterilized at 121°C and 2 bar pressure for 20 minutes.

These sterilized media were then placed into 2.7 L glass flasks (Weck, Germany) and inoculated with pure fungal submerged cultures, maintaining sterile conditions as described by [17]. In parallel, controls of the medium were prepared in the similar way, except for the inoculation with fungal cultures. To facilitate gas exchange, each flask was sealed with a rubber band and a glass lid, which incorporated a hole plugged with a cotton stopper. Before the beginning of the incubation period, the flasks were aerated under sterile conditions to establish the initial atmospheric ratios of CH₄, O₂, and CO₂. Subsequently, the cotton stoppers were replaced with sterile silicone stoppers (Saint-Gobain Performance Plastics, France) to limit uncontrolled gas exchange while allowing for precise gas sampling.

In order to investigate the temperature dependency of fungal CH₄ emissions, the fungi were placed in a climate chamber set at three different temperatures: 17°C, 27°C, and 40°C. The duration of incubation varied, extending up to ~1100 h (ca. 46 days).

Gas samplings were performed as follows. First the pressure within the glass flasks was measured using an EX- portable pressure measuring instrument (GHM Messtechnik GmbH, Germany) with a precision of ± 1%. Then a plastic syringe (Plastikpak, USA) was used to extract 10 ml of gas for CH₄ measurements and 6 ml for CO₂. Measurement of CH₄ and CO₂ mixing ratios was performed according to the procedures detailed in section 4.3, with atmospheric air replacing the extracted gas. Alongside gas sampling, we continuously monitored O₂ concentrations within the flasks using calibrated O₂ spots (section 4.3).

Furthermore, to maintain specific O₂ mixing ratios and testing the hypothesis of O₂ dependency of fungal CH₄ emissions, especially in cases where O₂ was depleted or fell below certain concentrations, we regulated the O₂ levels in the flasks by adding pure O₂ until the desired concentration (usually to atmospheric O₂ levels), which was immediately controlled using the calibrated O₂ spots. For the pine wood control incubated at 40 °C CH₄ formation was, additionally to ambient O₂ mixing ratios, investigated without the presence of O₂, by exchanging the whole headspace volume with pure helium.

4.3. Measurement of CH₄, CO₂ and O₂ concentrations

Mixing ratios of CH₄ in this study were measured using a gas chromatograph coupled with a flame ionization detector (GC-FID; Shimadzu 14b, Japan). To facilitate this, sample gas was injected into the GC-FID through a six-port valve (Valco Instruments, USA), which was linked to a chemical trap filled with Drierite®. This setup was crucial for drying the gas before entering the analytical system via a sample loop with a volume of 2 ml that was employed using a 20 ml plastic syringe (Plastipak BD, USA). The GC itself was equipped with a stainless-steel column (3.175 mm in inner diameter) packed with a 60-80 mesh molecular sieve 5A (Supelco, USA), effectively separating CH₄ from other gas components in the samples. The oven temperature was maintained constantly at 125 °C. To quantify CH₄ within the samples, daily measurements (ranging from 3 to 4) of two reference standards with CH₄ mixing ratios of 2.192 and 9.655 ppmv were conducted.

For measuring CO₂ mixing ratios, a GC coupled with a barrier discharge ionization detector (BID; Shimadzu, Japan) was employed. Here, 50 µl of sample gas was injected into the GC-BID via an autosampler AOC-20-i (Shimadzu, Japan) using a split injection method (5:1). This GC was equipped with an 8; Shimadzu, Japan). The quantification of CO₂ was achieved by measuring various reference standards with mixing ratios (400 ppm, 0.5%, 10%, and 40% by volume), each conducted in triplicate. As part of quality control, one reference standard was measured after every 6 to 9 single measurements.

Oxygen (O₂) concentrations were determined using non-invasive optical sensors (O₂ spots; PSt3 sensor type) and a Fibox 4 portable measuring instrument (both from PreSens Precision Sensing GmbH, Germany). These O₂ spots were installed in the glass flasks before the start of the incubation experiments and were calibrated using a two-point calibration with ambient air (20.9% O₂) and helium (0% O₂). The precision of the O₂ spots is 0.4% at 20.9% O₂ and 0.05% at 0.2% O₂.

4.4. Calculations and statistical methods

In this study, all CH₄ emission rates were carefully normalized against the respective O₂ consumption rates to ensure the investigation of CH₄ formation based on the fungal metabolism. The CH₄ formation rates, based on O₂ levels, were categorized into three distinct groups: 0% O₂, 0-5% O₂, and >5 to 95% O₂. This categorization was pivotal as it was observed that below an O₂ mixing ratio of 5%, there was a noticeable reduction in CH₄ production.

To systematically categorize the normalized CH₄ rates within these defined O₂ levels for each flask, sections corresponding to the respective O₂ mixing ratios were manually defined. Within these sections, CH₄ emission and O₂ consumption rates were calculated using linear regression analysis for all incubation experiments. Importantly, respective CH₄ formation rates of the controls were subtracted from the calculated rates in the control of the respective medium.

For each incubation experiment, arithmetic means and standard deviations were calculated to discern differences between the treatments. Furthermore, t-tests were conducted to provide statistical backing for the observations made. However, it is important to note that the study adhered to the recommendations of the American Statistical Association. Consequently, p-values and other statistical parameters were not solely relied upon as the criteria for drawing conclusions [45]. Thus, the term “statistically significant” was consciously avoided in the interpretation of the results.

5. Conclusions

This study is the first, to our knowledge, to investigate how different O₂ mixing ratios and temperatures control CH₄ emissions in two saprotrophic fungi, L. sulphureus and P. sapidus. We discovered that CH₄ formation rates are highly dependent on the prevailing O₂ mixing ratios. Notably, in all our incubation experiments, we observed that fungal CH₄ formation rates diminished when O₂ mixing ratios fell below approximately 0.5%. Conversely, CH₄ formation increased immediately after O₂ was reintroduced or when O₂ levels remained above this threshold, unambiguously highlighting the role of aerobic metabolism for fungal CH₄ formation. Furthermore, we found that CH_{4_norm} rates varied based on the fungal species and their substrates, including beech wood, pine wood, and grass. These findings suggest that the investigated fungal species, and likely all fungi, produce CH₄ per se, and that aerobic metabolism controlled by O₂ levels is a critical factor for this process. This further challenges the previous assumption that in the fungal realm CH₄ is only formed under anoxic conditions and that saprotrophic fungi simply provide methanogenic archaea with precursor compounds via the decomposition of woody components. Additionally, our study shows that temperature has a substantial effect on fungal CH₄ formation. We observed lower CH₄ formation and CH₄_norm rates at temperatures of 17 °C and 40 °C, while the highest rates of CH₄ formation occurred at 27 °C, indicating that this temperature is comparatively closer to the optimum metabolic activity for the investigated fungi.

Thus, our research clearly demonstrates that both the availability of O₂ and temperature are key in controlling fungal CH₄ emissions. From an environmental perspective, temperature increases due to climate change could significantly increase fungal CH₄ emissions through enhanced fungal growth and biomass. Therefore, understanding CH₄ formation and its controlling factors, especially temperature and O₂ levels, is crucial for assessing the role of fungal CH₄ emissions in global CH₄ fluxes. Additionally, exploring these fungal CH₄ dynamics in various environments such as forests, soils and aquatic systems [46] under both aerobic and anaerobic conditions is necessary to better understand their impact on carbon and greenhouse gas dynamics.