ABSTRACT
Deep-sea sediments harbor abundant microbial communities extending vertically up to ~2.5 km below the seafloor. Despite their prevalence, the reasons for their large community sizes and low energy fluxes remain unclear. Particularly, the reliance of fungi, the predominant eukaryotic group, on amino acids in these energy-limited, anaerobic conditions is poorly understood. We investigated the role of amino acids in the growth and development of the fungus Schizophyllum commune 20R-7-F01, isolated from anaerobic sub-seafloor sediments. The fungus efficiently used all amino acids as carbon sources, and some as nitrogen sources, with specific amino acids influencing sexual reproduction and fruit-body formation. Notably, amino acids with hydrocarbon chains or methyl groups appeared crucial for fruit-body production. The upregulation of genes, metabolites, and pathways related to amino acid metabolism in the fungus under anaerobic conditions underscores the significance of amino acids as energy and nutrient sources in such environments. Amino acids not only served as carbon/nitrogen sources but also contributed to fungal fruit-body formation under low oxygen conditions, vital for long-term fungal survival in the energy-limited deep biosphere. This study sheds light on the crucial role of amino acids in fungal growth and reproduction in energy-limited anaerobic conditions.
IMPORTANCE
In the depths beneath the ocean floor, where darkness, anaerobic conditions, and energy scarcity prevail, life persists against all odds. This study illuminates the pivotal role of amino acids, the fundamental building blocks of life, as a vital energy for deep subseafloor fungi. Our research uncovers how these fungi not only rely on amino acids for survival but also utilize them to reproduce, forming fruit bodies in environments deprived of oxygen and energy. This revelation not only elucidates the mechanisms enabling fungal survival in extreme conditions but also hints at the essentiality of amino acids as nutrients for other deep-sea microbes. By unraveling these mysteries of the hidden biosphere, our study opens new frontiers in understanding the resilience and adaptation of life in the most inhospitable environments on our planet.
KEYWORDS: deep biosphere, necromass, amino acids, subseafloor, fungi, anaerobic
INTRODUCTION
Deep-seafloor sediments harbor an extensive microbial community, estimated to reach a staggering 1029 cells (1), creating what scientists refer to as the “deep biosphere” (2). This biosphere extends vertically up to approximately 2.5 km below the seafloor (kmbsf) (3). Despite our awareness of the abundant life in the deep biosphere, understanding how such vast microbial communities sustain themselves with limited energy sources remains a puzzle. The mechanisms enabling this phenomenon are still largely unknown.
All three domains of life, Archaea (4), Bacteria (5), and Eukarya (6), have been discovered in these sediments, with fungi particularly prevalent as the dominant eukaryotic organisms (7, 8). However, our knowledge of how fungi thrive in the energy-limited, anoxic conditions of submarine environments is sparse. Specifically, we lack understanding regarding how subseafloor fungi withstand the energy-limited anaerobic conditions of the deep biosphere and whether they have adapted to extremely low energy fluxes or exist in a dormant, metabolically inactive state.
Microbial necromass, primarily composed of amino acids, is hypothesized to serve as the primary organic matter in subseafloor sediments. This necromass provides a potential energy source for microorganisms, which may have evolved specific adaptations to utilize amino acids for energy (9, 10). Several observations support the ongoing fermentation of amino acids in deep subseafloor sediments. (i) Amino acid concentrations decrease from micromolar levels at the sediment surface to nanomolar concentrations in the fermentation zone in sediments older than one million years (9). (ii) Genes associated with amino acid metabolism exhibit relatively high expression levels in the fermentation zone across various anoxic subseafloor settings (7, 11). (iii) Accumulation of ammonium, short-chain fatty acids, and the amino acid fermentation product γ-amino butyric acid is observed in the fermentation zones of anoxic sediments (12–14). Amino acid fermentation typically occurs through the Stickland reactions, where one amino acid is oxidized while another is reduced (15). For example, Clostridium sticklandii, a bacterial generalist amino acid fermenter, can metabolize all amino acids through the Stickland reactions except alanine and glutamine (16). Glycine reductase, a key enzyme in the Stickland reactions, generates acetyl phosphate for substrate-level phosphorylation from glycine reduction (17). However, no expression of glycine reductase has been detected in subseafloor sediments (7), suggesting alternative mechanisms for amino acid degradation by microbes in these environments.
The fermentation of amino acids in subseafloor sediments produces typical fermentation by-products, including CO2, NH4+, acetate, butyrate, and some H2 (18). These by-products not only support the microorganisms involved in the fermentation process but also contribute to the survival of non-fermenting microbes. Consequently, it is believed that microorganisms in the deep subseafloor biosphere have evolved mechanisms to utilize amino acids as a carbon and energy source, enabling them to survive in extreme energy-limited conditions over millions of years.
Our laboratory previously isolated several fungal species, including Schizophyllum commune 20R-7-F01, from anoxic subseafloor sediments retrieved at a depth of approximately ~2,457 m below the seafloor off the Shimokita Peninsula, Japan. This isolation process adhered to stringent anaerobic protocols and contamination control measures (19). Phylogenetic analysis revealed that these subseafloor fungi share ancestry with terrestrial species, likely introduced through sinking organic matter deposition (19–21). The presence of plant fossils in the sediment cores further bolsters this hypothesis (3, 22).
In contrast to the majority of marine fungi, these subseafloor isolates displayed remarkable adaptability, thriving in both anoxic and aerobic environments (19, 23). This adaptability is underpinned by unique metabolic strategies, such as ethanol fermentation, augmented amino acid production, alleviation of mitochondrial numbers, and activation of phagocytosis (23–26). Interestingly, sexual reproduction seems to be constrained under anoxic conditions, suggesting a potential shift toward asexual modes of reproduction (24).
Furthermore, our previous research has elucidated the role and significance of amino acids in the anaerobic survival of subseafloor fungi. S. commune 20R-7-F01 has been shown to synthesize amino acids, particularly branched-chain amino acids (BCAAs), in response to anoxic conditions to adapt to low oxygen levels (23). Moreover, amino acids contribute to growth promotion and fruit-body formation under hypoxic conditions (24). Several studies also suggest that amino acids may serve as primary organic substrates for microbial metabolism in subseafloor sedimentary environments (10, 27–29). However, there is still a lack of knowledge regarding which specific amino acids act as carbon and/or nitrogen sources, the adequacy of energy derived from amino acid metabolism to support microbial growth, and the impact of amino acids on sexual reproduction, i.e., the life cycle of fungi. To address these inquiries comprehensively, we conducted an extensive experimental study utilizing a subseafloor fungus to investigate its ability to use amino acids as an energy source under anaerobic conditions. Specifically, we grew Schizophyllum commune 20R-7-F01 in media containing amino acids as the sole carbon source under anaerobic conditions (Fig. 1).
Fig 1.
Subseafloor sediments harbor a diverse community of fungi, yet how they survive in these energy-limited and anaerobic conditions remains unclear. This study investigates the role of extracellular amino acids in sustaining fungi in subseafloor sediments. We isolated Schizophyllum commune 20R-7-F01 from ~2 kmbsf and tested its ability to grow on amino acids using a combination of transcriptomic, metabolomic, and culture-based experiments. [Far-left image JAMSTEC (30), reprinted with permission; other images adapted from references (19) and (24).]
RESULTS
Transcriptomic and metabolomic response of the fungus to anaerobic conditions
To investigate how subseafloor fungi respond to oxygen-deprived conditions, we cultured S. commune 20R-7-F01 under both anaerobic and aerobic conditions in artificial seawater medium (ASW) supplemented with glucose as the sole carbon source for 7 days. Following this, we conducted comprehensive metabolomic and transcriptomic analyses to elucidate the molecular mechanisms underlying fungal adaptation to anaerobiosis, comparing them to the responses observed under aerobic conditions (Fig. 1). The transcriptomic analysis revealed a total of 299 upregulated genes (P-value ≤ 0.05; log2FC ≥ 1.5) (Table S1; Fig. 2), with 99 of these genes directly associated with amino acid metabolism, particularly emphasizing branched-chain amino acid biosynthesis (Table S2). Concurrently, the metabolomic analysis identified 111 metabolites (Table S3; Fig. 2), among which 56 were upregulated (P-value ≤ 0.05; VIP ≥ 1.0; log2FC ≥ 1.5) (Table S3), with 47 of these being amino acids or their derivatives (Table S4). Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis further confirmed the link between the upregulated genes and metabolites and amino acid biosynthesis, highlighting pathways such as phenylalanine, tyrosine, cystine, methionine, glycine, serine, threonine, glutathione, beta-alanine, valine, leucine, and isoleucine metabolism (Fig. 2E). Moreover, the transcriptomic analysis specifically revealed the upregulation of amino acid-related pathways, including valine, leucine, and isoleucine biosynthesis, and phenylalanine metabolism (Fig. 2F). Notably, 25% of the pathways (63 pathways) were concurrently upregulated in both the transcriptomic and metabolomic analyses, with most of these pathways being related to either amino acid metabolism or the biosynthesis and degradation of organic acids (Fig. 2G). These comprehensive analyses strongly support the vital role of amino acids in the survival of fungi under anaerobic conditions. Based on these findings, we hypothesized that amino acids might be critical for the survival of the fungal community in the absence of oxygen.
Fig 2.
Profile of transcriptomic and metabolomic data and comparison of upregulated pathways in S. commune 20R-7-F01 during anaerobic growth. (A) Volcano plot represents a global comparison of transcriptional profiles between anaerobically and aerobically grown fungus. Red and green color dots represent the up and downregulated genes with a threshold of log2FC ≥ 1 and adjusted P-value < 0.05, respectively. (B) Hierarchical clustering in heatmap format of all differentially expressed genes (DEGs) in S. commune 20R-7-F01, grown under anaerobic and aerobic conditions. Each column represents experimental conditions, whereas each row represents a differentially expressed gene. Red color shows upregulation, while green color represents downregulation of DEGs. The terms WO1, WO2, and WO3 represent the three replicates of the fungus grown under anaerobic conditions, while the terms O1, O2, and O3 represent three replicates of the fungus grown under aerobic conditions. (C) Metabolomic analysis of the culture broth of S. commune 20R-7-F01, grown under anaerobic and aerobic conditions for 7 days. (D) The KEGG enrichment pathways of differentially expressed metabolites. (E and F) The results of KEGG enrichment analysis for upregulated genes and metabolites, respectively. (G) Comparison of pathways upregulated in both transcriptomics and metabolomics, highlighting the genes and metabolites that are commonly upregulated during anaerobic growth compared to aerobic conditions.
Effect of amino acids on fungal growth and development under anaerobic conditions
To explore the effect of amino acids on fungal growth and development under anaerobic conditions, S. commune 20R-7-F01 was cultivated on solid minimal medium (MM) supplemented with or without individual amino acids as carbon or nitrogen sources under aerobic and anaerobic conditions. Growth and development, excluding fruit-body formation, were monitored over a 7-day incubation period. Since this fungus cannot produce fruit bodies anaerobically (24), cultures were then exposed to hypoxic conditions (1% pO2) to induce fruit-body formation. The emergence and progression of fruit bodies were subsequently monitored. Results indicated a positive impact of all amino acids on fungal growth and development under anaerobic conditions (Table 1). Notably, certain amino acids, including Ala, Val, Leu, Ile, Ser, Thr, and Asp, were associated with primordia or fruit-body formation. However, the size of these primordia was significantly smaller than that produced on control (glucose-only) medium. Interestingly, amino acids featuring simple hydrocarbon or methyl group side chains played a pivotal role in fruit body development. While no significant effect of amino acids on fungal growth was noted under aerobic conditions. These findings underscore the multifaceted importance of amino acids beyond their roles as carbon or nitrogen sources, particularly in facilitating fungal growth and fruit-body formation under low-oxygen environments.
TABLE 1.
Impact of amino acids on the growth and development of subseafloor fungus S. commune 20R-7-F01 under anaerobic conditionsa
| Amino acids | Carbon source | Nitrogen source | Primordia formation | Fruit-body formation |
|---|---|---|---|---|
| Gly | + | − | − | − |
| Ala | + | + | + | + |
| Val | + | + | + | + |
| Leu | + | + | + | + |
| Ile | + | + | + | + |
| Met | + | + | − | − |
| Phe | + | + | − | − |
| Trp | + | + | − | − |
| Pro | + | + | + | − |
| Ser | + | + | + | + |
| Thr | + | + | + | + |
| Cys | + | − | − | − |
| Tyr | + | − | − | − |
| Asn | + | + | + | − |
| Gln | + | + | + | − |
| Asp | + | + | + | + |
| Glu | + | − | − | − |
| Lys | + | + | − | − |
| Arg | + | + | − | − |
| His | + | + | + | − |
The fungus was grown on MM medium containing individual amino acids as a sole carbon/nitrogen source or added as a supplement to check its effect on growth and development. “+” and “−” indicate the positive and negative effects of amino acids, respectively, on fungal growth and development.
Quantitative assessment of the impact of amino acids as a carbon source on fungal anaerobic growth
To evaluate the utilization of amino acids as the exclusive carbon source under anaerobic conditions, the fungus was cultured in a liquid MM formulated in an artificial marine water medium (AMW) with individual amino acids serving as the sole carbon source. After a 7-day incubation period, the growth yield was quantified. Control experiments involved culturing the fungus in media either lacking amino acids or containing glucose. The results revealed the fungus’s capability to metabolize all amino acids as a carbon source, albeit with varying efficiencies. Notably, Ala, Val, Leu, Ile, and Gln yielded biomass levels ranging from 0.60 to 0.71 g/L, comparable to those obtained with glucose (0.76 g/L). This suggests the favorability of these amino acids as carbon sources for the anaerobic growth of subseafloor fungi. In contrast, significantly lower biomass production (0.24–0.33 g/L) was observed with Gly, Trp, Cys, Asp, Lys, and Arg (Fig. 3), indicating their limited suitability for the growth of subseafloor fungi under anaerobic conditions.
Fig 3.
Impact of amino acids, as a carbon source, on the anaerobic growth of subseafloor S. commune 20R-7-F01. The biomass of the strain cultured on individual amino acid or glucose (Glc) as a carbon source under anaerobic conditions is shown. Fungal growth in the culture was determined after 7 days, and relative growth was quantified by normalizing the values to the control culture with no addition of amino acids. The horizontal line indicates the relative growth of the control culture containing glucose as the carbon source. An independent sample t-test (P value ≤ 0.05) was used to identify amino acids forming equal (**) or less (*) biomass compared to the control.
Quantitative assessment of the impact of amino acids as a nitrogen source on fungal anaerobic growth
The fungus was cultured in liquid MM prepared in an AMW medium with glucose as the carbon source and amino acids as the nitrogen source. After 7 days of anaerobic incubation, biomass was measured. As a control, the fungus was grown under the same conditions without amino acids but with (NH4)2SO4 as the sole nitrogen source. The results showed that certain amino acids, including Asn, Gln, Asp, Lys, and Arg, were effectively utilized by the fungus, generating biomass levels ranging from 0.51 to 0.61 g/L, comparable to those produced with (NH4)2SO4, indicating their potential suitability as nitrogen sources under anaerobic conditions (Fig. 4). Conversely, Ile, Met, Pro, Ser, and Thr resulted in lower biomass production (0.22–0.43 g/L) compared to the (NH4)2SO4 control, suggesting their lesser efficiency as nitrogen sources for fungal growth under anaerobic conditions. Amino acids Gly, Cys, Tyr, and Glu led to extremely low biomass production, indicating their unsuitability as nitrogen sources for the fungus.
Fig 4.
Impact of amino acids as nitrogen sources on the anaerobic growth of subseafloor S. commune 20R-7-F01. The biomass of the strain cultured on individual amino acid or ammonium sulfate as a nitrogen source under anaerobic conditions is presented. Fungal growth in the culture was determined after 7 days, and relative growth was quantified by normalizing the values to the control culture with no addition of amino acids. The horizontal line indicates the relative growth of the control culture containing (NH4)2SO4 as the nitrogen source. An independent sample t-test (P value ≤ 0.05) was used to identify amino acids forming equal (**) or less (*) biomass compared to the control.
Role of amino acids in primordia formation
To determine the role of amino acids in primordia formation, the fungus S. commune 20R-7-F01 was grown on solid MM containing individual amino acids or glucose (control) as the carbon source, with (NH4)2SO4 as the nitrogen source. The experiment was conducted under anaerobic conditions, and the development of primordia was monitored closely. Intriguingly, the fungus could initiate primordia on Ala, Val, Leu, Ile, Pro, Ser, Thr, Asp, and Lys, but the quantity and size of these primordia were significantly lower compared to those grown on glucose (Table 1; Fig. 5). This observation implies that, following glucose, amino acids could serve as a viable carbon source for the fungus, providing adequate energy through fermentation to support fungal growth and initiate early development under anaerobic conditions.
Fig 5.
Influence of amino acids on primordia formation. Primordia formation was observed in the fungus cultured under anaerobic conditions on solid MM supplemented with (A) glucose, (B) Ala, (C) Val, (D) Leu, (E) Ile, (F) Pro, (G) Ser, (H) Asp, (I) Thr, and (J) Lys as sole carbon sources. Yellow arrow indicates the presence of primordium, highlighting the impact of amino acids on this developmental process.
Role of amino acids in fruit-body formation
To investigate the impact of amino acids on fruit-body formation, the cultures were exposed to low oxygen (1% pO2) and light (10 hours day⁻¹) to stimulate fruit-body formation. Interestingly, fruit bodies were only formed on cultures grown on Asp, Ile, Ser, Val, Leu, Thr, and Ala (Table 1; Fig. 6). However, these fruit bodies were smaller, less numerous, and lacked spores compared to the large, mature fruit bodies with spores grown on glucose (Fig. 6). This observation suggests that the fungus derived sufficient energy from amino acid to initiate fruit-body formation. Nevertheless, this energy might not be adequate to complete the entire life cycle, as evidenced by the smaller size, less number, and immaturity of fruit bodies lacking spores. It is noteworthy that in the complete absence of oxygen, no fruit body formation was observed, regardless of the carbon source used.
Fig 6.
Effect of amino acids on fruit-body formation. The fungus was grown under hypoxic conditions (1% pO2) on MM solid medium supplemented with (A) glucose, (B) Asp, (C) Ile, (D) Ser, (E) Val, (F) Leu, (G) Thr, and (H) Ala as a sole carbon source. Yellow arrow shows the fruit body.
DISCUSSION
Microbes residing in subseafloor sediments exhibit robustness in energy-limited, oxygen-deprived settings, relying on unconventional organic compounds such as amino acids for sustenance. However, it remains unknown whether amino acid fermentation provides sufficient energy for the growth and development of subseafloor fungi. Here, we delve into the metabolic capabilities of S. commune 20R-7-F01, isolated from ~2.0 kmbsf subseafloor sediments, to harness amino acids as a primary energy reservoir under anaerobic conditions. Our investigations unveil a remarkable revelation that not only does the fungus flourish on amino acids but it also orchestrates its entire life cycle in the absence of oxygen, solely reliant on these organic compounds. This profound discovery illuminates the mechanisms underpinning microbial viability in the profound depths of the biosphere, underscoring the pivotal role of amino acids in sustaining life within energy-scarce environments.
Amino acids as energy source in subseafloor sediments
The heightened activity observed in genes and pathways associated with amino acid degradation during fungal growth under anaerobic conditions underscores the pivotal role of amino acids as a primary energy source for subseafloor organisms. Our comprehensive analysis revealed that 25% (63 pathways), mainly predominantly linked to amino acid and organic acid biosynthesis and degradation, exhibited consistent upregulation in both transcriptomic and metabolomic assessments, thus emphasizing the intrinsic relationship between amino acids and energy dynamics in anaerobic settings (Fig. 2). These findings are consistent with prior research demonstrating the upregulation of amino acid biosynthesis and degradation pathways in subseafloor sediments, highlighting the specialized adaptation of microbial communities to utilize amino acids as a fundamental energy source (9, 10).
Moreover, the identification of transcripts related to amino acid transport and metabolism across various deep sediments, particularly their higher expression in anoxic subseafloor environments (7, 11), further underscores the critical role of amino acids in sustaining microbial life within these habitats. Furthermore, amino acids contribute approximately 4%–5% to the total organic carbon content in the youngest sediment, a proportion that diminishes to less than 0.1% in sediment aged over several million years, indicating the preferential degradation of amino acids by microbial communities over geological time (10, 27, 28, 31). Notably, a substantial portion of the transcript derived from anaerobic Peru Margin sediments, attributed to amino acid metabolism, was associated with fungi (4), highlighting an overlooked role of fungi in amino acid degradation within subseafloor sediments. Our prior investigations have emphasized the significance of amino acids in the anaerobic survival of the subseafloor fungus, S. commune 20R-7-F01. In these studies, the fungus exhibited heightened metabolism of BCAAs, indicative of its capacity to cope with the energy constraints imposed by anaerobic conditions (23). Collectively, these lines of evidence substantiate the notion that amino acids serve as a viable energy source and may play a crucial role in the survival of subseafloor microorganisms within the energy-limited anaerobic environments.
Amino acids’ metabolism under anaerobic conditions
Despite the recognized significance of amino acids in deep sediments, the precise mechanisms microbes employ for their degradation and the adequacy of energy derived from amino acid fermentation for microbial growth remain elusive. The fungus’s ability to produce biomass comparable to that of glucose when cultivated on various amino acids (e.g., Ala, Val, Leu, Ile, and Gln) (Fig. 3) suggests the potential of amino acids as favorable carbon sources under anaerobic conditions.
Microbes utilize amino acids to support their anaerobic growth. For instance, newly isolated Archaea from the fermentation zone of anoxic deep-sea sediments (28) and two novel Archaea obtained from marine sediment (32, 33) were found to show amino acid-based metabolism. Prokaryotes typically convert amino acids to 2-oxoacids, which are then oxidized to generate CO2 and acyl-CoA. Acyl-CoA is used for ATP generation through substrate-level phosphorylation (34). Meanwhile, the electrons generated from the oxidative steps of amino acid fermentation, such as deaminations and decarboxylations, are excreted, typically in the form of reduced molecules such as H2, formate, acetate, and butyrate (35). CO2 and H2 resulting from this process can be used by methanogens for ATP synthesis and carbon assimilation, while other end products of amino acid fermentation, namely acetate, short-chain fatty acids, and NH4+, accumulate transiently in the environment (13).
In contrast, our findings show that fungi can grow and complete their life cycle (Fig. 5 and 6) by utilizing amino acids as carbon and energy sources under oxygen-deficient conditions, albeit with significantly lower biomass production compared to aerobic conditions (24). Our findings suggest a potentially novel fermentation pathway in deep-sea fungi, where amino acids could serve as both electron donors and acceptors during energy metabolism. This distinctive process, possibly evolved as an adaptation to the limited energy sources of the deep biosphere, may facilitate the sustained growth and even the formation of fruiting bodies under anaerobic conditions solely through amino acid utilization. However, it is imperative to conduct further investigation and validation across a broader range of fungal species and geographical locations to comprehensively assess the extent of this phenomenon.
Roles of amino acids on fungal anaerobic growth and development
Amino acids play vital roles in fungal growth, development, and reproduction by serving as essential nutrients and signaling molecules (36). Notably, specific amino acids, such as Ile and Leu, have been demonstrated to influence appressorium formation, hyphae growth, conidia formation, and morphogenesis in pathogenic fungi (37–39). Our investigation unveiled the fungus’s capacity to utilize all amino acids as carbon sources under anaerobic conditions, with Ala, Val, Leu, Ile, and Gln exhibiting comparable efficacy to glucose in biomass production (Fig. 3). Additionally, amino acids Asn, Gln, Asp, Lys, and Arg efficiently served as nitrogen sources, supporting similar biomass production as observed with standard nitrogen-rich medium (Fig. 4).
Interestingly, we observed that when grown on amino acids containing side chains consisting of either a simple hydrocarbon (Val, Leu, Ile, Ser, Thr, and Asp) or a methyl group (Ala), the fungus produced primordia and immature fruit bodies. This suggests that the hydrocarbon or methyl group of amino acids may have unique functions in fruit-body development under low oxygen conditions.
Our findings indicate that subseafloor fungus S. commune not only utilizes amino acids for ATP regeneration (23) but also relies on them to support growth and development in anoxic conditions (Fig. 2 and 5). Other fungal strains isolated from subseafloor sediments also demonstrate the ability to use amino acids as carbon and/or nitrogen sources under anaerobic conditions, suggesting that amino acids may serve as primary organic substrates for a broader range of fungi in the deep biosphere (Table S3). This phenomenon may contribute to the diversity and richness of fungi in the submarine environment, which is characterized by extreme deficiencies in energy resources (10).
Conclusion
Our study reveals the crucial role of amino acids in supporting the anaerobic growth and development of subseafloor fungi. The fungus S. commune 20R-7-F01 demonstrates remarkable metabolic versatility by utilizing various amino acids as carbon and nitrogen sources under anaerobic conditions, resulting in biomass production comparable to that of glucose and standard nitrogen-rich medium. Moreover, our results indicate a potential novel fermentation process in subseafloor fungi, where amino acids may serve as both electron acceptors and energy sources, enabling the fungus to sustain normal growth and even produce fruit bodies in hypoxic conditions. Importantly, specific side chains of amino acids appear to play a role in fruit body development under low oxygen conditions. Overall, this research yields valuable insights into the energy dynamics and adaptive strategies of subseafloor fungi, contributing to our understanding of microbial life in extreme environments and its ecological significance within marine ecosystems.
MATERIALS AND METHODS
Fungal strain, media, and culture conditions
The fungus utilized in this study was Schizophyllum commune 20R-7-F01 (CGMCC 11604), isolated from the ~2.0 kmbsf deep sediments during IODP Expedition 337 (19). To prepare the inoculum for liquid medium experiments, the fungus was initially activated three times on mPDA (potato dextrose agar dissolved in artificial marine water). Artificial marine water composition included CaCl2 (2.99 g L−1), MgCl2 (4.17 g L−1), KBr (0.10 g L−1), NH4Cl (0.16 g L−1), KCl (5.05 g L−1), NaCl (33.43 g L−1), H3BO3 (0.02 g L−1), and Na2SO4 (4.26 g L−1).
For inoculum preparation, mycelia were obtained from the outer edge of the fungal colony on mPDA and homogenized. The resulting homogenized fungal mycelia were transferred to a 500-mL conical flask with 200 mL of sterilized mPD (mPDA without agar) and incubated at 30°C and 150 rpm until small mycelial colonies formed. Colonies were separated from the medium, washed three times with double distilled water, and used as inocula for subsequent experiments.
The minimal medium used for amino acids’ study consisted of KH2PO4 (0.46 g L−1), K2HPO4·3H2O (1.25 g L−1), MgSO4·7H2O (0.5 g L−1), resazurin (0.01 g L−1) as an oxygen indicator, FeCl3 (5 g L−1), and a trace element solution (1 mL L−1). Solid MM was prepared by adding 20 g of agar. The trace element solution contained H3BO3 (60 mg L−1), ammonium molybdate tetrahydrate (40 mg L−1), CuSO4 (0.2 g L−1), ZnSO4 (2.0 g L−1), CoCl (0.4 g L−1), and Ca(NO3)2 (1.2 g L−1).
Preparation of low-oxygen conditions
To establish anaerobic conditions, a 30-mL liquid medium in a 50-mL serum glass bottle was subjected to a 10-minute purge with high-purity helium gas (99.99%). Subsequently, the bottles were immediately sealed with rubber stoppers and aluminum caps to maintain the anaerobic environment. To sustain anoxic conditions, the bottles underwent a 5-minute flush with the same gas every 24 hours throughout the entire incubation period. For solid medium plates, an anaerobic jar was employed to create anaerobic conditions by flushing it with a mixed gas (He, H2, and CO2) for 20 minutes. The presence of anaerobic conditions was monitored using resazurin paper strips as an anaerobic indicator.
To create a hypoxic environment (1% pO2), the anaerobic jar was purged for 20 minutes initially and then for 5 minutes every 24 hours thereafter using a pre-mixed gas mixture comprising 1% oxygen balanced with helium obtained from a commercial gas supplier. For aerobic conditions, standard environmental conditions were maintained throughout the experiment.
Examining the influence of amino acids on fungal growth, development, and fruit-body formation
The fungus was cultivated on solid MM plates with or without individual amino acids at a concentration of 20 mM, as described by Zain Ul Arifeen et al. (24). The cultivation process involved exposing the fungus to both aerobic and anaerobic conditions. The growth and development, including fruit-body formation, were closely monitored during this experiment. Under anaerobic conditions, the fungus was allowed to grow until primordia developed. At this stage, the fungus was transitioned to hypoxic conditions (1% pO2) and provided with light for 10 hours per day to facilitate the development of fruit bodies.
It is important to highlight that S. commune 20R-7-F01 lacks the ability to produce fruit bodies in the absence of oxygen, as noted by Zain Ul Arifeen et al. (24). Therefore, the impact of amino acids on fruit-body formation was specifically investigated under hypoxic conditions (1% pO2) in this study. This approach aimed to comprehensively examine the influence of amino acids on fungal growth, development, and the formation of fruit bodies, shedding light on the role of oxygen availability in this process. For control, the fungus was also grown aerobically on the same medium with or without amino acids. However, no significant difference in growth was observed under aerobic conditions. Therefore, we focused subsequent experiments on evaluating the impact of amino acids on fungal growth and development under anaerobic conditions.
Investigating the function of amino acids as carbon or nitrogen sources
To explore the fungus’s capacity to utilize individual amino acids as a carbon source, the fungus cultures were grown in liquid MM under anaerobic conditions for a duration of 7 days. Within the MM, individual L-amino acids (20 mM) were incorporated as the sole carbon source, and glucose was included as a positive control. Furthermore, a control culture lacking any amino acids was included for comparative purposes.
In order to assess the role of individual amino acids as a nitrogen source, the fungus was cultured in MM with glucose as the exclusive carbon source. Individual L-amino acids (5 mM) were introduced as the sole nitrogen source, and the outcomes were compared to a control culture cultivated with ammonium sulfate [(NH4)2SO4] as the standard nitrogen source.
Following the incubation, the fungal cultures were subjected to filtration using Miracloth filters to isolate the mycelium. The collected mycelium was then dried at 70°C for 24 hours. Relative growth was quantified by normalizing the dry weight of the fungal biomass from each treatment to the control culture lacking amino acid addition (for the carbon source experiment) or the control culture with ammonium sulfate (for the nitrogen source experiment). This methodological approach aimed to comprehensively elucidate the role of individual amino acids as either carbon or nitrogen sources for fungal growth and development.
Un-targeted LC–MS/MS-based metabolomics analysis
To investigate the metabolites and metabolic pathways upregulated under anaerobic conditions, we performed a comprehensive untargeted LC–MS/MS-based metabolite profiling of the culture broth of S. commune 20R-7-F01. The fungus was grown in liquid MM prepared in ASW with glucose as the sole carbon source under both aerobic and anaerobic conditions in triplicates. Samples were collected after 7 days of inoculation and subjected to metabolite extraction using established methods (24). In brief, metabolites were extracted using an 80% methanol solution containing 0.1% formic acid and then centrifuged at 4°C and 15,000 g for 10 minutes. The resulting supernatant was further processed by replacing it with 60% methanol and filtered prior to LC–MS/MS analysis. For the analysis, a UHPLC system coupled with an Orbitrap Q Exactive HF-X mass spectrometer (Thermo Fisher) was used, along with a Thermo Hyperil Gold column (C18). Compound Discoverer 3.0 (CD 3.0, Thermo Fisher) was employed for processing the raw data.
To obtain accurate qualitative and relative quantitative results, the mzCloud (https://www.mzcloud.org/) and ChemSpider (https://www.chemspider.com/) databases were utilized (40). Significantly different metabolites were identified by setting the threshold value to VIP > 1.0, FC > 1.5, or FC < 0.667 and P value < 0.05. The pathways of the differential metabolites were identified using the Kyoto Encyclopedia of Genes and Genomes (https://www.genome.jp/kegg/) database (41). Principal component analyses were performed using metaX (42). This rigorous approach allowed us to comprehensively elucidate the metabolites and metabolic pathways that are upregulated under anaerobic conditions, providing valuable insights into the metabolic adaptations of S. commune 20R-7-F01 in response to anaerobic conditions.
Transcriptome analysis
RNA was isolated from three replicates of S. commune 20R-7-F01, cultivated for 1 week under both anaerobic (treatment) and aerobic (control) conditions in liquid MM prepared in artificial seawater containing glucose as the sole carbon source, following TRIzol reagent (TIANGEN, China) manufacturer’s instructions. To eliminate any genomic DNA contamination, the RNA samples were treated with RNase-Free DNase set (NEB, Ipswich, MA, CA, USA). The integrity and purity of the RNA were assessed using an RNA Lab-On-A-Chip on Agilent Bioanalyzer 2100 (Agilent Technologies, Palo Alto, CA, USA). The extracted RNA was stored in a liquid nitrogen tank and then sent to Personal Biotechnology Company (Shanghai, China) for subsequent processing, including cDNA construction, library preparation, and sequencing. Sequencing was performed using the Illumina HiSeq 2000 platform, and the resulting reads were subjected to cleaning and quality assessment using the fastQC program. Only high-quality clean reads were retained for further analysis. A reference genome index was generated using the Bowtie2 algorithm, and the filtered reads were aligned to the reference genome using Tophat2 (43). Transcript assembly and abundance estimation were carried out using Cufflinks (44).
The expression levels of genes were normalized and calculated using the RPKM value. To identify differentially expressed genes (DEGs) between the two samples, we utilized the “DESeq” package in the R programming language (45). DEGs were determined based on the criteria of an adjusted P-value of ≤0.05 and an absolute value of log2 (expression fold change) ≥ 1 (46). Subsequently, GO annotation and KEGG enrichment analysis were performed on the DEGs to identify the major GO categories and pathways influenced by hypoxic incubation. This comprehensive transcriptome analysis provides insights into the gene expression changes in S. commune 20R-7-F01 in response to anaerobic conditions, shedding light on the molecular mechanisms underlying its metabolic adaptations to low-oxygen environments.
ACKNOWLEDGMENTS
This work was supported by the Science and Technology Innovation Program of Jiangsu Province (no. BK20220036), the National Natural Science Foundation of China (nos. 42273077, 41973073, and 41773083), and the Jiangsu Agricultural Science and Technology Innovation Fund, China [no. CX (22)2021].
M.Z.U.A. designed the project, conducted the research work, analyzed and interpreted the data, and drafted the manuscript. S.A., X.W., and S.H. helped in revising the manuscript, significantly enhancing the overall quality and substantially improving the English language. C.H. obtained funding, participated in project designing, interpretation and analysis of the data, and drafting of the manuscript. The final manuscript was read and approved by all authors.
Contributor Information
Changhong Liu, Email: chliu@nju.edu.cn.
Jennifer F. Biddle, University of Delaware, Lewes, Delaware, USA
DATA AVAILABILITY
The sequencing data have been deposited in the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA) under the BioProject ID PRJNA626396.
SUPPLEMENTAL MATERIAL
The following material is available online at https://doi.org/10.1128/aem.01279-24.
Tables S1 to S5.
ASM does not own the copyrights to Supplemental Material that may be linked to, or accessed through, an article. The authors have granted ASM a non-exclusive, world-wide license to publish the Supplemental Material files. Please contact the corresponding author directly for reuse.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Tables S1 to S5.
Data Availability Statement
The sequencing data have been deposited in the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA) under the BioProject ID PRJNA626396.