Abstract

   Fungi inhabiting deep subseafloor sediments have been shown to possess
   anaerobic methane (CH[4]) production capabilities under atmospheric
   conditions. However, their ability to produce CH[4] under in situ
   conditions with high hydrostatic pressure (HHP) remains unclear. Here,
   Schizophyllum commune 20R-7-F01, isolated from ~2 km below the
   seafloor, was cultured in Seawater Medium (SM) in culture bottles
   fitted with sterile syringes for pressure equilibration. Subsequently,
   these culture bottles were transferred into 1 L stainless steel
   pressure vessels at 30 °C for 5 days to simulate in situ HHP and
   anaerobic environments. Our comprehensive analysis of bioactivity,
   biomass, and transcriptomics revealed that the S. commune not only
   survived but significantly enhanced CH[4] production, reaching
   approximately 2.5 times higher levels under 35 MPa HHP compared to 0.1
   MPa standard atmospheric pressure. Pathways associated with
   carbohydrate metabolism, methylation, hydrolase activity, cysteine and
   methionine metabolism, and oxidoreductase activity were notably
   activated under HHP. Specifically, key genes involved in fungal
   anaerobic CH[4] synthesis, including methyltransferase mct1 and
   dehalogenase dh3, were upregulated 7.9- and 12.5-fold, respectively,
   under HHP. Enhanced CH[4] production under HHP was primarily attributed
   to oxidative stress induced by pressure, supported by intracellular
   reactive oxygen species (ROS) levels and comparative treatments with
   cadmium chloride and hydrogen peroxide. These results may provide a
   strong theoretical basis and practical guidance for future studies on
   the contribution of fungi to global CH[4] flux.

   Keywords: anaerobic, HHP, Schizophyllum commune 20R-7-F01, CH[4],
   transcriptomics, ROS

1. Introduction

   Methane (CH[4]) is a potent greenhouse gas pivotal to global climate
   dynamics, primarily sourced from biogenic emissions driven by microbial
   activity, which constitute approximately 90% of the global CH[4]
   budget, estimated at 380–755 Tg annually. Significant contributors
   include coal beds, seafloor sediments, and subsurface reservoirs, with
   deep-sea sediments alone contributing around 20% of these emissions
   [[32]1,[33]2,[34]3].

   Archaea are well documented as major producers of CH[4], utilizing
   various biochemical pathways, such as CO[2] reduction with H[2],
   acetate reduction, and methylotrophic pathways involving methanol and
   methoxy-group-containing substrates [[35]4]. Recent research has also
   identified aerobic bacterial pathways contributing to CH[4] production,
   including methylthio-alkane reductase’s involvement in methionine
   biosynthesis and C-P lyase in phosphonate ester degradation
   [[36]5,[37]6]. Furthermore, both plant and animal cells have been
   observed releasing CH[4] under aerobic conditions independently of
   endosymbionts, although the precise mechanisms remain elusive
   [[38]7,[39]8]. In contrast, fungi, a vital group of eukaryotic
   organisms, have received less attention concerning CH[4] production.
   Lenhart et al. (2012) provided initial evidence that wood-decaying
   fungi such as Pleurotus sapidus, Trametes versicolor, Lentinula edodes,
   Laetiporus sulphureus, and Hypholoma fasciculare produce CH[4] under
   aerobic conditions, identifying serine as a precursor for methane
   synthesis [[40]9]. Subsequent studies by Ernst et al. (2022) presented
   contradictory findings, showing that fungi like Saccharomyces
   cerevisiae S288C and Aspergillus niger DSM 821 primarily generate CH[4]
   via Fenton chemistry rather than enzymatic reactions [[41]10].

   Recent research by Huang et al. (2022), employing biochemical, genetic,
   and stable isotopic tracer analyses, revealed that strains of
   Schizophyllum commune 20R-7-F01, isolated from coal-bearing sediments
   ~2 km below the seafloor (under 35 MPa hydrostatic pressure), utilized
   a novel halomethane-dependent pathway for CH[4] production during
   anaerobic degradation of phenol, benzoic acid mono- and polymers, and
   cyclic sugars [[42]11]. The taxonomic classification of S. commune is
   complicated by its widespread distribution and genetic diversity.
   Traditionally viewed as a single species, recent genomic analyses
   reveal significant variation among strains. Notably, the subseafloor S.
   commune 20R-7-F01 shows genetic divergence from terrestrial strains
   like S. commune H4-8, with many genes lacking orthologs [[43]12].
   Similar anaerobic methanogenic pathways have been confirmed in other
   wood-rot fungi, such as Agaricus bisporus, Hypsizygus marmoreus, and
   Pleurotus ostreatus [[44]13,[45]14,[46]15]. Nonetheless, uncertainties
   persist regarding the ability of these fungi to produce CH[4] under in
   situ high hydrostatic pressure (HHP) conditions, as well as specific
   mechanism governing CH[4] production in response to HHP.

   In this study, we cultured the fungal strain S. commune 20R-7-F01
   anaerobically in 1 L stainless steel vessels at 30 °C to mimic in situ
   subseafloor environments. Our findings revealed a significant increase
   in CH[4] production by this subseafloor fungus under elevated
   hydrostatic pressure (HP), primarily attributed to the induction of
   reactive oxygen species (ROS) by HHP. These results likely highlight
   the potential role of fungi as CH[4] producers in the deep biosphere,
   an aspect that may have been previously underestimated in global CH[4]
   budgets.

2. Materials and Methods

2.1. High Hydrostatic Pressure Cultivation Experiments

   Schizophyllum commune 20R-7-F01 (CGMCC 11604) was isolated from a
   sediment core collected at a depth of 1966 m below seafloor (mbsf) from
   the Western Pacific Ocean [[47]16]. Mycelial inocula were prepared
   following the method described by Zain Ul Arifeen et al. (2021). For
   the high hydrostatic pressure (HHP) cultivation experiments, fresh
   mycelial inocula (7 g) were introduced into 170 mL sterile culture
   bottles containing Seawater Medium (SM). The SM composition included
   CaCl[2] (2.99 g/L), MgCl[2] (4.17 g/L), KBr (0.10 g/L), NH[4]Cl (0.16
   g/L), KCl (5.05 g/L), NaCl (33.43 g/L), H[3]BO[3](0.02 g/L), Na[2]SO[4]
   (0.21 g/L), and C[6]H[12]O[6] (20 g/L). The bottles were then purged
   with 99.99% N[2] for 15 min to remove oxygen from the culture bottles
   [[48]17]. Culture bottles, fitted with sterile syringes for pressure
   equilibration, were incubated at 30 °C under HP of 15 MPa and 35 MPa,
   achieved by manual pumping of water into the vessel(TOP INDUSTRIE,
   Paris, France). A control culture under standard atmospheric pressure
   (0.1 MPa) was maintained under identical conditions. Fungal mycelia
   were harvested after 1, 3, and 5 days of culture, and one vial of
   mycelia was filtered through sterile gauze, rinsed three times with
   deionized water, immediately treated with liquid nitrogen, and stored
   at −80 °C for transcriptome analysis. Additionally, three replicates of
   harvested mycelia underwent the same gauze filtration and rinsing steps
   before being immediately utilized for biomass and CH[4] quantification.
   Simultaneously, one vial of mycelia was also employed for assessing
   cellular activity following the aforementioned procedures.

   For the ROS testing experiments, fresh mycelial inocula (7 g) were
   inoculated into 170 mL culture bottles containing SM supplemented with
   0.75 mM, 1.5 mM, and 3 mM concentrations of cadmium chloride
   (Sigma-Aldrich, Shanghai, China) or hydrogen peroxide (Sigma-Aldrich,
   Shanghai, China) [[49]18,[50]19]. Culture bottles were incubated at 30
   °C under standard atmospheric pressure. Fungal mycelia were harvested
   after 1, 3, and 5 days of incubation. Subsequently, three replicates of
   harvested mycelia underwent the same gauze filtration and rinsing steps
   before being immediately utilized for biomass and CH[4] quantification.
   Simultaneously, one vial of mycelia was also employed for assessing
   cellular activity following the aforementioned procedures.

2.2. Assessment of Fungal Hyphal Vitality and Biomass Determination

   To assess fungal mycelial viability, a 0.4% trypan blue (Sigma-Aldrich,
   Shanghai, China) staining technique was employed [[51]20]. Viable
   mycelial cells were identified as colorless under optical microscopy
   (XIUILAB, Shanghai, China), whereas non-viable cells were stained blue.
   Mycelia harvested by filtration were dried in a 65 °C oven for one day
   to determine biomass. Dry weights were measured to quantify biomass
   production [[52]21].

2.3. Assessment of ROS, O^2−, OH·, H[2]O[2], and CH[4] Levels in Fungal
Mycelia

   Intracellular levels of ROS in fungal mycelia were assessed following
   established methodologies [[53]22,[54]23]. The
   2′,7′-dichlorofluorescein diacetate (DCFH-DA, Beyotime, Shanghai,
   China) probe was introduced into cells, and ROS concentrations were
   determined through fluorescence microscopy examination and subsequent
   quantification using ImageJ software (version 1.55i). The intracellular
   content of O^2− in fungal mycelia was determined as per established
   protocols [[55]24,[56]25]. The superoxide anion reacts with
   hydroxylamine hydrochloride to form NO^2−, which, upon reaction with
   p-aminobenzenesulfonami-de and naphthalene ethylenediamine
   hydrochloride, produces a red azo compound with a characteristic
   absorption peak at 530 nm. The content of O^2− can be determined based
   on the absorbance at 530 nm. The intracellular content of OH· in fungal
   mycelia was determined as per established protocols [[57]26]. The
   hydroxyphenyl fluorescein (HPF,) probe was introduced into cells, and
   OH· concentrations were determined through fluorescence microscopy
   examination and subsequent quantification using ImageJ software
   (version 1.55i). The intracellular content of H[2]O[2] in fungal
   mycelia was determined as per established protocols [[58]27,[59]28].
   Absorbance at 415 nm, resulting from the formation of a titanium
   peroxide complex (Ti^4+ and H[2]O[2]), was measured to quantify
   H[2]O[2]concentrations. Intracellular CH[4] levels in fungal mycelia
   were evaluated following detailed procedures, utilizing gas
   chromatography (GC, HP-Agilent, Shanghai, China) for accurate
   quantification of CH[4].

2.4. Transcriptomic Analysis

   Mycelial samples for transcriptome sequencing were labeled as follows:
   “d1_01M”, “d3_01M”, “d5_01M”, “d1_15M”, “d3_15M”, “d5_15M”, “d1_35M”,
   “d3_35M”, and “d5_35M”. Here, “dn” denotes sampling days (1, 3, and 5
   days), and “nM” indicates pressure levels (0.1, 15, and 35 MPa) applied
   to the strain. Total RNA extraction utilized TRIzol reagent (TIANGEN,
   Beijing, China) per the manufacturer’s instructions, followed by cDNA
   library construction. Sequencing employed the Illumina HiSeq platform
   with default RNA protocols (Meiji, Shanghai, China). Clean reads were
   obtained by removing adapters, sequences with >10% N bases, and
   low-quality sequences (Phred score Q ≤ 5, >50% of reads) ([60]Table
   S1). Clean reads were mapped to the strain 20R-7-F01 assembled genome
   (BioProject ID: PRJNA544166) using TopHat2 [[61]29]. Gene expression
   levels were quantified in Transcripts Per Million (TPM) using Cufflinks
   software (version 2.2.1) [[62]30]. Raw RNA-seq data were deposited in
   the NCBI Sequence Read Archive under BioProject ID PRJNA1101667.

   Differential gene expression analyses utilized the DESeq method DESeq2
   [[63]31], applying a threshold of p-value < 0.05 and |Log2
   (fold-change)| ≥ 1 to identify significant DEGs [[64]32]. Functional
   analysis of DEGs included gene ontology (GO) and Kyoto Encyclopedia of
   Genes and Genomes (KEGG) enrichment analyses using clusterProfiler in R
   (v4.3.0). Enriched pathways were visualized with the Pathview package,
   setting the threshold for enriched gene annotations at p-value < 0.05.
   DEGs related to key pathways underwent hierarchical clustering, with
   correlation analysis performed using psych and reshape 2 packages in R
   (v4.3.0). A correlation network of pathway genes was constructed using
   Cytoscape software version 3.9.1
   ([65]https://cytoscape.org/releasenotes.html, accessed on 12 May 2024).

2.5. Quantitative Real-Time PCR Analysis

   Quantitative real-time PCR (qRT-PCR) followed the protocol outlined by
   Zain Ul Arifeen et al. Fungal cultures were maintained under identical
   conditions and durations to those for RNA-seq samples. SYBR qPCR Master
   Mix (Vazyme, Nanjing, China) and specific primer pairs for each gene
   ([66]Table S2) were used for qRT-PCR analysis. Thermal cycling
   conditions included initial denaturation at 95 °C for 30 s, followed by
   43 cycles of 95 °C for 10 s, 58.5 °C for 30 s, and 72 °C for 30 s.
   Relative gene expression was calculated using the 2^−ΔΔCT method
   [[67]33].

2.6. Statistical Analysis

   The data were presented as mean ± standard deviation. One-way analysis
   of variance (ANOVA) or Student’s t-test, performed using GraphPad Prism
   version 8.0.2, was used to analyze significant differences between
   treatments (p < 0.05).

3. Results and Discussion

3.1. Impact of High Hydrostatic Pressure on Fungal Methane Productivity

   To assess the effect of HHP on fungal CH[4] productivity, strain
   20R-7-F01 was cultured in bottles under varying HHP conditions, and the
   CH[4] yield in the headspace was quantified using GC. The results
   demonstrated a significant enhancement in the CH[4] production of
   strain 20R-7-F01 with increasing HP([68]Figure 1). Specifically, CH[4]
   production at 15 MPa was approximately 1.3, 1.7, and 1.9 times higher
   on days 1, 3, and 5 of culture, respectively, compared to atmospheric
   conditions. Furthermore, at 35 MPa (equivalent to in situ pressure),
   CH[4] production increased to approximately 2.0, 2.4, and 2.5 times
   higher than at atmospheric pressure. This substantial increase suggests
   a strong influence of HHP in enhancing CH[4] production by strain
   20-7-1. The effect of hydrostatic pressure on biological methanogenesis
   may be a universal phenomenon that affects all methanogens, but similar
   observations have only been noted in archaea, such as Thermophilic
   marburgensis, which exhibited CH[4] production levels approximately 3
   times higher than atmospheric levels when cultured at 50 MPa [[69]34].
   The mechanism behind enhanced CH[4] production under HHP in archaea is
   hypothesized to involve oxidative stress induced by HP. However, it
   remains unclear whether analogous mechanisms govern the impact of HHP
   on fungal CH[4] production.

Figure 1.

   [70]Figure 1
   [71]Open in a new tab

   Methane production by strain 20R-7-F01 under varying hydrostatic
   pressures. Note: * represents p < 0.05; ** represents p < 0.01; ***
   represents p < 0.001.

3.2. Transcriptomic Analysis of Methane Synthesis Genes Under High
Hydrostatic Pressure

   The transcriptomic analysis revealed significant upregulation of key
   genes involved in methane synthesis under high hydrostatic pressure
   conditions in S. commune 20R-7-F01. Following cultivation at 15 MPa for
   3 days, the expression levels of mct1, dh3, and ms increased by
   5.9-fold, 2.8-fold, and 2.7-fold, respectively, compared to ambient
   pressure (0.1 MPa) ([72]Table 1). Similarly, at 35 MPa, these genes
   showed increases of 7.9-fold, 4.5-fold, and 3.1-fold, respectively
   ([73]Table 1). The quantitative PCR validated these findings,
   demonstrating significant upregulation of mct1, dh3, and ms under HHP
   conditions ([74]Table 1). This enhanced methane production is primarily
   attributed to the upregulation of genes encoding key enzymes involved
   in methane synthesis.

Table 1.

   Relative expression of genes associated with methane synthesis in S.
   commune 20R-7-F01.
   1 d 3 d 5 d
   RNA-Seq qPCR RNA-Seq qPCR RNA-Seq qPCR
   15 MPa 35 MPa 15 MPa 35 MPa 15 MPa 35 MPa 15 MPa 35 MPa 15 MPa 35 MPa
   15 MPa 35 MPa
   mct1 1.16 1.69 1.21 ± 0.03 1.85 ± 0.11 2.56 2.99 2.82 ± 0.17 3.25 ±
   0.21 3.49 5.15 3.17 ± 0.15 5.36 ± 0.19
   mct2 0.42 0.73 0.37 ± 0.02 0.61 ± 0.05 0.52 0.85 0.44 ± 0.05 0.65 ±
   0.07 −0.09 −0.08 −0.1 ± 0.03 −1.7 ± 0.03
   dh1 0.91 0.62 0.85 ± 0.04 0.88 ± 0.06 0.82 −0.31 0.73 ± 0.02 −0.43 ±
   0.04 0.81 −0.73 0.66 ± 0.05 −0.88 ± 0.05
   dh2 0.21 0.89 0.27 ± 0.03 0.73 ± 0.09 0.85 0.55 0.91 ± 0.06 0.77 ± 0.08
   1.48 0.87 1.12 ± 0.11 0.69 ± 0.07
   dh3 0.68 1.07 0.93 ± 0.06 1.21 ± 0.16 1.48 2.16 1.69 ± 0.11 2.49 ± 0.19
   2.11 3.21 1.95 ± 0.13 3.11 ± 0.11
   dh4 0.56 2.05 0.71 ± 0.03 2.31 ± 0.14 −0.26 −0.24 −0.43 ± 0.03 −0.22 ±
   0.01 0.65 −0.23 0.51 ± 0.04 −0.28 ± 0.01
   dh5 −1.72 −2.04 −1.66 ± 0.04 −2.15 ± 0.15 −1.39 −1.36 −1.31 ± 0.12
   −1.46 ± 0.06 −0.13 −0.18 −0.22 ± 0.01 −0.32 ± 0.03
   ms 1.04 1.12 1.12 ± 0.02 1.25 ± 0.08 1.42 1.63 1.66 ± 0.07 1.77 ± 0.02
   1.48 1.51 1.31 ± 0.08 1.63 ± 0.07
   [75]Open in a new tab

   The transcriptional correlation analysis identified 2316 differentially
   expressed genes (DEGs) highly correlated (|p| ≥ 0.9) with mct1 (689
   DEGs), dh3 (817 DEGs), and ms (810 DEGs) ([76]Table S1). The gene
   ontology (GO) enrichment analysis revealed that these DEGs were
   enriched in activities associated with oxidoreductase functions,
   carbohydrate metabolism, methylation, ATP binding, hydrolase activity,
   metal ion binding, and transmembrane transport. Notably, oxidoreductase
   activity exhibited the highest enrichment, comprising 8.5% of the total
   2316 DEGs (p = 6.95 × 10^^−11) ([77]Figure 2A, [78]Table S1). The KEGG
   pathway enrichment analysis further indicated significant enrichment of
   these DEGs in pathways such as peroxisomes, glycolysis/gluconeogenesis,
   the tricarboxylic acid cycle, and the pentose phosphate pathway under
   HHP conditions ([79]Figure 2B).

Figure 2.

   [80]Figure 2
   [81]Open in a new tab

   Enrichment analysis of significantly differentially expressed genes
   (DEGs). Panel (A) depicts GO enrichment analyses of the 2316 DEGs.
   Panel (B) shows KEGG enrichment analyses of the 2316 DEGs. Description
   of the top 30 enriched GO and KEGG pathways.

   The hierarchical clustering analysis illustrated the upregulation of
   genes involved in oxidoreductase activities, particularly those
   implicated in oxidative stress response (e.g., SOD, CAT, and BCP), in
   S. commune 20R-7-F01 under HHP conditions ([82]Figure 3, [83]Table S2).
   Collectively, these findings suggest that the enhancement of methane
   synthesis metabolism under HHP conditions may be linked to alterations
   in oxidoreductase activities. Similar observations in other piezophilic
   organisms, such as Sporosarcina psychrophila DSM 6497 and Shewanella
   piezotolerans WP3, underscore that S. commune 20R-7-F01, like other
   piezophiles, counters oxidative stress induced by HHP through the
   activation of oxidative–reductive pathways [[84]35,[85]36].

Figure 3.

   [86]Figure 3
   [87]Open in a new tab

   Hierarchical clustering heat map analysis of DEGs in the oxidoreductase
   activity pathway.

   Furthermore, the investigation of DEGs related to antioxidant genes
   revealed significant correlations with key genes involved in methane
   biosynthesis in strain 20R-7-F01 ([88]Figure 4). A total of 197 DEGs of
   antioxidant genes were identified, with 54 (27.4%) showing notable
   correlations with mct1, 79 (40.1%) with dh3, and 64 (32.5%) with metE.
   These findings indicate that these antioxidant genes were significantly
   involved in methane production by S. commune under HHP conditions.
   Enhanced methane release in response to oxidative stress induced by HHP
   may serve as a protective mechanism against biological membrane damage
   caused by reactive oxygen species (ROS) [[89]37,[90]38]. In summary,
   our study highlights that the increased methane production observed in
   S. commune 20R-7-F01 under HHP conditions is a response to oxidative
   stress induced by HHP, mediated through the upregulation of genes
   associated with both methane synthesis and antioxidant defense
   mechanisms. These findings contribute to our understanding of microbial
   adaptation to extreme environmental conditions, emphasizing the role of
   methane production in stress response strategies.

Figure 4.

   [91]Figure 4
   [92]Open in a new tab

   A network diagram illustrating the interactions between DEGs associated
   with methane production and oxidative stress. Each node represents a
   gene, while edges represent interactions.

3.3. ROS and H[2]O[2] Induced in Fungal Cells by High Hydrostatic Pressure

   To explore the enhanced methane production of S. commune 20R-7-F01
   under high hydrostatic pressure (HHP) as a potential response to
   elevated fungal cell ROS and H[2]O[2] levels induced by HHP, we
   assessed intracellular ROS and H[2]O[2] levels, as well as the
   activities of antioxidative enzymes (SOD, CAT, POD) at 0.1 (control),
   15, and 35 MPa pressures. Our results revealed significant increases in
   ROS and H[2]O[2] levels in fungal cells under HHP compared to 0.1 MPa,
   accompanied by reduced fungal cell viability ([93]Table 2, [94]Figure
   S1). For instance, after 5 days of cultivation, the ROS and H[2]O[2]
   levels were 7.04-fold and 6.12-fold higher at 15 MPa, and 10.33-fold
   and 8.51-fold higher at 35 MPa, respectively, compared to at 0.1 MPa,
   yet the cells were clearly labeled blue, with trypan blue. Further
   analysis demonstrated that the increase in ROS levels induced by HHP
   was primarily due to changes in H[2]O[2] levels, while the
   contributions of superoxide anion (O^2−) and hydroxyl radical (OH·)
   were relatively insignificant ([95]Table S3). This is similar to
   findings by Zhe et al. (2018), who reported oxidative damage in the
   deep-sea bacterium Shewanella piezotolerans WP3 due to elevated
   intracellular H[2]O[2] levels under 20 MPa conditions [[96]39].
   Additionally, the antioxidative enzyme activities within the fungal
   hyphae increased with pressure, notably with peroxidase (POD) activity
   at 35 MPa after 5 days of cultivation, which was 5.95 times higher than
   at 0.1 MPa over the same period. This is consistent with the results
   shown in [97]Figure 3. The heightened activities of SOD, CAT, and POD
   in scavenging ROS- and H[2]O[2]-induced cellular damage under HHP
   suggest that S. commune, in response to HHP stress, employed metabolic
   mechanisms akin to those observed in the deep-sea bacterium Shewanella
   piezotolerans WP3 and yeast Saccharomyces cerevisiae [[98]40,[99]41].

Table 2.

   Oxidative levels of S. commune 20R-7-F01 under high hydrostatic
   pressure.
   ROS (AU/g) H[2]O[2] (μmol/g)
   0.1 MPa 15 MPa 35 MPa 0.1 MPa 15 MPa 35 MPa
   1 d 0.37 ± 0.09 1.14 ± 0.22 2.84 ± 0.23 0.31 ± 0.03 0.99 ± 0.12 2.33 ±
   0.21
   3 d 0.85 ± 0.13 4.46 ± 0.31 6.37 ± 0.21 0.76 ± 0.09 3.66 ± 0.35 4.34 ±
   0.37
   5 d 0.89 ± 0.11 6.27 ± 0.58 9.19 ± 0.77 0.77 ± 0.08 4.71 ± 0.29 6.55 ±
   0.49
   SOD (U/g) CAT (U/g) POD (U/g)
   0.1 MPa 15 MPa 35 MPa 0.1 MPa 15 MPa 35MPa 0.1 MPa 15 MPa 35 MPa
   1 d 7.65 ± 0.63 20.94 ± 1.66 26.89 ± 1.53 9.40 ± 0.55 14.87 ± 1.13
   23.28 ± 1.51 5.95 ± 0.62 9.18 ± 0.92 35.25 ± 2.53
   3 d 14.27 ± 1.37 26.57 ± 1.38 37.78 ± 1.22 12.73 ± 1.06 23.21 ± 1.47
   37.25 ± 1.73 15.95 ± 1.33 24.62 ± 1.31 94.56 ± 3.22
   5 d 15.92 ± 1.03 28.45 ± 1.54 43.22 ± 1.71 14.54 ± 1.25 25.51 ± 1.64
   40.03 ± 2.21 16.35 ± 1.03 25.50 ± 1.52 97.29 ± 3.71
   [100]Open in a new tab

   After establishing the occurrence of oxidative damage in S. commune
   20R-7-F01 under HHP conditions, we conducted a correlation analysis
   between intracellular ROS and H[2]O[2] levels, antioxidant enzyme
   activities (SOD, CAT, and POD), and methane content across different
   pressures. The results depicted in [101]Figure 5 reveal that at 0.1
   MPa, there existed a moderate positive correlation between methane
   production in the strain and intracellular ROS and H[2]O[2] levels, as
   well as antioxidant enzyme activities, although this was not
   statistically significant. In contrast, at 15 MPa and 35 MPa,
   significant positive correlations were observed between methane
   production and intracellular ROS and H[2]O[2] levels, as well as
   antioxidant enzyme activities. Particularly notable was the pronounced
   positive correlation between methane production and H[2]O[2] levels.
   For instance, at 15 MPa, the correlation coefficient between methane
   production and H[2]O[2] levels in the strain reached 0.9334. Similarly,
   at 35 MPa, the correlation coefficient between methane production and
   H[2]O[2] levels also reached 0.9686. These results indicate that
   alongside the increase in intracellular H[2]O[2] levels in S. commune
   20R-7-F01, there is a corresponding increase in methane release. This
   further corroborates the conclusion from our transcriptome analysis
   showing that enhancing methane metabolism in S. commune 20R-7-F01 is a
   fungal response mechanism to HHP-induced ROS. While Ernst et al. (2022)
   [[102]10] have demonstrated the existence of an ROS-driven methane
   production mechanism in general organisms, this is based on Fenton
   chemistry rather than a direct ROS-driven pathway specific to methane
   production in organisms. Similarly, although Mauerhofer et al. (2021)
   [[103]34] found an increase in methane production by methanogenic
   archaea with increasing HHP, they did not identify the fundamental
   reasons behind the HHP-induced enhancement of methane production in
   archaea [[104]33]. Here, we provide the first evidence that HHP can
   promote methane production in S. commune 20R-7-F01 for adapting to
   oxidative stress.

Figure 5.

   [105]Figure 5
   [106]Open in a new tab

   The correlation between methane production and the activities of ROS,
   H[2]O[2], SOD, CAT, and POD in S. commune 20R-7-F01.

3.4. Experimental Evidence of ROS Contribution to Increased Methane
Production in S. commune 20R-7-F01

   To investigate the influence of oxidative stress induced by H[2]O[2] or
   CdCl[2] on methane production by S. commune 20R-7-F01, we cultured the
   fungus in a liquid mPD medium supplemented with varying concentrations
   (0.75 mM, 1.5 mM, and 3 mM) of these stressors. Control cultures
   lacking H[2]O[2] or CdCl[2] served as a baseline. Methane production
   capacity was assessed under atmospheric pressure conditions after 1, 3,
   and 5 days of cultivation. As detailed in [107]Table 3, the methane
   production markedly increased with increasing concentrations of
   H[2]O[2]or CdCl[2]. For instance, exposure to 0.75 mM CdCl[2] resulted
   in methane production 1.45 times higher than the control after five
   days, which further increased to 2.44 times higher with 3 mM CdCl[2].
   Similarly, exposure to 0.75 mM H[2]O[2] led to a 1.74-fold enhancement
   in methane production relative to the control condition, while
   increasing the H[2]O[2] concentration to 3 mM further augmented methane
   production by a factor of 3.06. It is noteworthy that supplementation
   of the mPD culture medium with BHT to scavenge intracellular ROS under
   different oxidative stress conditions led to a corresponding decrease
   in methane production by the strain. For example, the addition of
   antioxidant BHT (5 mM) to the fungal mycelium exposed to 3 mM H[2]O[2]
   for five days resulted in a significant reduction in methane production
   from 2.63 mmol/g to 2.23 mmol/g. Concurrently, increasing
   concentrations of H[2]O[2] or CdCl[2] were correlated with elevated
   levels of intracellular ROS ([108]Table S4), accompanied by reduced
   fungal cell viability and biomass ([109]Figures S2 and S3). For
   instance, exposure to 3 mM H[2]O[2] led to a decrease in biomass by
   45.27 mg relative to the control, accompanied by significant trypan
   blue staining of the cells. These findings collectively underscore that
   oxidative stress induced by H[2]O[2] or CdCl[2] promotes methane
   production by S. commune 20R-7-F01. Thus, oxidative damage induced by
   HHP likely contributes to the increased methane production by this
   fungus. Similar observations by Gu et al. and Samma et al. in alfalfa
   roots indicate that elevated ROS levels from metal stressors enhance
   methane emission [[110]17,[111]18].

Table 3.

   Effects of varying oxidative stress conditions on methane production in
   S. commune 20R-7-F01.
   CK CdCl[2] CdCl[2] + BHT H[2]O[2] H[2]O[2] + BHT
   0.75 1.5 3 0.75 1.5 3 0.75 1.5 3 0.75 1.5 3
   1 d 0.56 ± 0.04 0.64 ± 0.03 0.91 ± 0.05 1.07 ± 0.02 0.60 ± 0.01 0.81 ±
   0.03 0.97 ± 0.01 0.76 ± 0.02 1.03 ± 0.04 1.26 ± 0.06 0.68 ± 0.01 0.87 ±
   0.03 1.08 ± 0.01
   3 d 0.81 ± 0.03 0.90 ± 0.06 1.54 ± 0.07 1.82 ± 0.05 0.83 ± 0.04 1.40 ±
   0.06 1.65 ± 0.07 1.05 ± 0.05 1.74 ± 0.05 2.48 ± 0.09 0.89 ± 0.02 1.52 ±
   0.04 2.07 ± 0.04
   5 d 0.86 ± 0.06 1.25 ± 0.08 1.69 ± 0.06 2.10 ± 0.09 1.15 ± 0.05 1.53 ±
   0.07 1.87 ± 0.03 1.50 ± 0.03 2.09 ± 0.11 2.63 ± 0.12 1.26 ± 0.06 1.85 ±
   0.08 2.23 ± 0.05
   [112]Open in a new tab

4. Conclusions

   In conclusion, our study demonstrates that Schizophyllum commune
   20R-7-F01, isolated from the subseafloor sediment approximately 2 km
   below the seabed, exhibited enhanced methane (CH[4]) production
   capabilities under in situ temperature, high hydrostatic pressure, and
   anaerobic conditions. Through comprehensive analyses encompassing
   biological activity assays, biomass quantification, transcriptomics,
   and metabolomics, we found that S. commune not only survived but
   significantly increased CH[4] production under HHP conditions. Pathways
   related to carbohydrate metabolism, methylation, hydrolase activity,
   and the metabolism of cysteine and methionine, as well as activities of
   redox enzymes, were notably activated under HHP. Specifically, critical
   genes involved in fungal anaerobic CH[4] synthesis, such as
   methyltransferase mct1 and dehalogenase dh3, were markedly upregulated.
   The observed enhancement in CH[4] production under HHP was primarily
   attributed to pressure-induced oxidative stress, supported by
   comparative analyses of intracellular ROS levels and treatments
   involving cadmium chloride and hydrogen peroxide. These findings may
   indicate a potentially significant role for deep subseafloor sediment
   fungi in global methane generation, which has not been unaccounted for
   in previous estimations. Further elucidation of the mechanisms
   governing methane production by sediment fungi in the deep biosphere
   promises to advance our understanding of the Earth’s additional sources
   of methane that contribute to global climate change.

Supplementary Materials

   The following supporting information can be downloaded at:
   [113]https://www.mdpi.com/article/10.3390/microorganisms12112160/s1.
   [114]microorganisms-12-02160-s001.zip^ (564.3KB, zip)

Author Contributions

   M.Z.: designed the project, conducted the research work, analyzed and
   interpreted the data, drafted the manuscript. D.L. and J.L.:
   participated in data analysis. J.F.: edited the manuscript. C.L.:
   project administration, supervision, conceptualization, funding
   acquisition, manuscript writing—review and editing. All authors have
   read and agreed to the published version of the manuscript.

Institutional Review Board Statement

   The study protocol was approved by the Institutional Review Board at
   the University of Minnesota (9 August 2024), and all participants
   provided written informed consent.

Data Availability Statement

   No data were used for the research described in the article.

Conflicts of Interest

   The authors declare no conflicts of interest.

Funding Statement

   This work was supported by the National Natural Science Foundation of
   China (nos. 92251303, 41973073 and 41773083), and the Science and
   Technology Innovation Program of Jiangsu Province (no. BK20220036).

Footnotes

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References