Respiration and Substrate Utilization by Marine Fungi: Their Ecological Role in an Upwelling Ecosystem

Abstract

Marine fungi are relevant ecological components of microbial communities and play crucial roles in the degradation of organic matter and the cycling of nutrients in oceanic ecosystems. However, their metabolic adaptations to dynamic, nutrient-rich coastal upwelling environments remain scarcely known. The aim of our research is to contribute to the understanding of the ecological and biogeochemical roles of marine fungi in upwelling systems. To this end, we investigated the metabolic responses of three fungal strains (Sarocladium sp., Penicillium sp., and Fusarium sp.) isolated from the coastal upwelling off central-south Chile. The growth and respiration rates of these marine fungi were comprehensively evaluated using a diverse array of substrates, including glucose, cellobiose, sucrose, glycogen, asparagine, alanine, and glycine. The highest respiration rates were consistently recorded with glycogen, with no significant differences observed among species. Glucose yielded higher respiration rates in Sarocladium sp., and sucrose stimulated greater respiration in Penicillium sp. Phenotype microarrays were used to evaluate mycelium growth on various organic and inorganic nitrogen, phosphorus, and sulfur substrates. All three species exhibited high utilization indices for all substrate types, with organic components promoting the greatest growth. l-Amino acids were the preferred nitrogen sources. Adenosine and guanosine were the primary sources of phosphorus, while cysteine and methionine were the main sources of sulfur necessary for growth. Based on these species’ preferred substrates for growth and respiration, we offer new insights into the previously underexplored ecological roles of these fungi in the degradation of marine organic matter and biogeochemical cycling in upwelling ecosystems.

1. Introduction

Fungi are ubiquitous in marine ecosystems and inhabit diverse environments, including coastal waters, estuaries, mangroves, sediments, deep-sea hydrothermal vents, and the open ocean [1–4]. Compared to their terrestrial counterparts and other marine microbes, such as bacteria and archaea, marine fungi have historically been an understudied group [3,5–7]. They encompass a wide array of species across Ascomycota, Basidiomycota, Blastocladiomycota, Chytridiomycota, and yeasts [6,8,9]. The ecological role of marine fungi is primarily associated with the decomposition of organic matter from plants and animals [7,10,11]. Marine fungi can also function as pathogens, or engage in mutualistic or parasitic relationships with animals, plants, algae, cyanobacteria, phytoplankton, zooplankton, sponges, and seaweeds [10,12–15].

Marine fungi play an essential role in biogeochemical cycles and in the transformation of organic compounds and biomass into elements such as carbon (using a wide variety of carbohydrates, from simple sugars to complex carbohydrates), nitrogen (e.g., denitrification, production of N₂O) [16,17], phosphorus (e.g., recycling proteins) [18], sulfur (e.g., dimethylsulfoniopropionate hydrolysis) [19], and metals (Mn(II) mineralization) [20]. They also produce siderophores for Fe³⁺ solubilization [21]. Additionally, marine fungi degrade fossil fuels [22,23] and xenobiotics, such as polycyclic aromatic hydrocarbons and pesticides [22,24–26].

An active community of marine fungi inhabits the water column and sediments of the upwelling system off the coast of south-central Chile (36.5° S) [27–29], which has one of the highest rates of primary production in the world’s oceans [30–32]. This system is also characterized by a high abundance of organic material [27,28,33–35]. However, the role of fungi in degrading organic matter and nutrient cycling in this upwelling ecosystem remains largely unexplored.

In this coastal upwelling ecosystem, fungal biomass can be comparable to that of prokaryotes, particularly during periods of high autotrophic biomass when fungi process a significant amount of photosynthetic carbon [27]. Despite their remarkable physiological versatility and extensive enzymatic capabilities, there is a critical lack of detailed physiological and metabolic data on marine fungi from upwelling regions. Additionally, their specific roles in nutrient cycling, particularly with regard to nitrogen, phosphorus, and sulfur, remain unclear.

The aim of our research is to contribute to the understanding of the ecological and biogeochemical roles of marine fungi in upwelling systems. To this end, we investigated the metabolic responses of three fungal strains (Sarocladium sp., Penicillium sp., and Fusarium sp.) isolated from the highly productive coastal upwelling system off central-south Chile. Specifically, we aimed to quantify their respiration rates across a wide range of carbohydrate and amino acid substrates of varying complexity (including glucose, cellobiose, sucrose, glycogen, asparagine, alanine, and glycine) and to assess their capacity for utilizing key nitrogen, phosphorus, and sulfur substrates. This approach is crucial for elucidating the specific metabolic adaptations of these fungi to the variable nutrient conditions of the upwelling ecosystem, thereby offering novel and critical insights into their previously unexplored ecological and biogeochemical roles in marine organic matter degradation and nutrient cycling, driven by their remarkable physiological versatility and extensive enzymatic capabilities.

2. Materials and methods

2.1. Collection, isolation, and identification

Seawater (450 mL) samples were collected from the Coastal Time Series Oceanographic Station (St. 18 at 36.5°S, 73.1°W) of the Center for Oceanographic Research in the eastern South Pacific, aboard R/V Kay-Kay II (Department of Oceanography, University of Concepción).

The water samples were stored in sterile containers at the in situ temperature, and protected from direct sunlight until processing in the laboratory. Water samples (450 mL) were filtered through sterile Millipore membrane filters (Burlington, MA), with a pore size of 0.45 µm and a diameter of 47 mm (Millipore, Burlington, MA, Catalog No.: HAWP04700), and then frozen in liquid nitrogen. Filters were placed on Petri dishes containing a sterile, solid glucose-yeast extract agar medium (Sigma Aldrich, St. Louis, MO, Catalog No. Y1625), with streptomycin sulfate (0.15 g L⁻¹; Catalog No. S9137) and penicillin (0.5 g L⁻¹; Sigma Aldrich, St. Louis, MO, Catalog No. P7794), to prevent bacterial growth. The Petri dishes were then cultivated in a Purifier Biological Safety Cabinet (LabConco, Logic class II, Kansas City, MO). After about one week, once the colonies had fully developed, they were isolated through successive subcultures on a sterile solid medium (Emerson’s YpSs agar [36]), which was prepared in sterile seawater containing yeast extract, soluble starch, dipotassium phosphate, and magnesium sulfate. The colonies were then maintained in darkness at 4 °C. The most frequent colonies were isolated and cultured in Emerson’s YpSs agar. The isolates were stored in the fungal species bank of the Laboratory of Marine Organic Geochemistry (MOG) at the University of Concepción.

The DNA of the cultured strains was extracted using a Power Soil DNA Isolation Kit (MO BIO Laboratories, Carlsbad, CA, Catalog No. 12888-50) and used as a template to amplify the internal transcribed spacer region. The specific fungal primer ITS1-F [37] and the universal primer ITS4 [38] reverse were used. PCR cycles were run in duplicate on a TC-PRO thermal cycler (BOECO, Hamburg, Germany) and consisted of an initial 2-min step at 95 °C, followed by 35 cycles of 30 s at 95 °C, 30 s at 55 °C, and 1 min at 72 °C. A final 5-min step at 72 °C was performed to complete any partial polymerizations. A PCR mix without template DNA was used as a control [29]. The PCR products were stained with GelRed (Biotium, Inc., Fremont, CA) and run on a 1.2% agarose gel. They were then visualized under UV transillumination [29]. The PCR products were sequenced for identification analysis at the Automated DNA Sequencing Service of MACROGEN (Seoul, South Korea; http://dna.macrogen.com/eng). Genera and species were identified by matching sequences with those stored in GenBank based on previous ITS rDNA definitions in fungi using BLAST (National Center for Biotechnology Information (NCBI)). Sequences larger than 600 bp that were 99% identical to GenBank sequences were considered to be of the same species, and sequences that were 97% identical were considered to be of the same genus [39]. A single isolate from each strain was used in the experiments.

Three strains, representative of the most common cultivable species found in the study area, were selected from the MOG Laboratory’s fungal species bank. Strain MOG ID: CH58, Penicillium sp. (GenBank: MH231242.1); strain MOG ID: CH114, Fusarium sp. (GenBank: MH231240.1); and strain MOG ID 84; Sarocladium sp. (Sarocladium strictum; GenBank: KY401135.1).

2.2. Fungal respiration

The respiration rates of the three studied species were determined in the presence of seven substrates (carbohydrates and amino acids) of different structural complexities. The respiration experiments with different substrates used a modified liquid medium base [40,41], with three concentrations (10⁻⁶, 10⁻³, 1 g L⁻¹) for each of the following substrates: glucose, cellobiose, glycogen, sucrose, asparagine, alanine, and glycine. Each culture also contained 2.4 g of MgSO₄·7H₂O, 1.21 g of Tris buffer (pH 7.5), in 1 L of aged, autoclaved, filtered seawater. All of these substrates and reagents were acquired from Sigma-Aldrich (St. Louis, MO). Oxygen consumption was recorded using a respirometer with Optode sensors (FIBOX 3, PreSens, Regensburg, Germany), based on the principle that molecular oxygen quenches luminescence. Experiments were carried out in triplicate in 12 mL bottles with integrated spot sensors inside each bottle, which allowed oxygen consumption to be recorded every hour without opening the bottles. The experiments were performed in the dark at 20 °C with sample stabilization completed in about 30 min (until reaching 20 °C); measurements were carried out for 1 h or for up to 4 h when the respiration rate was low. The calculation of oxygen consumption rates was based on the first linear part of the curve. We omitted the first 5–10 min while awaiting stabilization of the instrument, during which time there was a clear variation in oxygen.

2.3. Growth based on substrate use

The Biolog phenotype microarray (PM) test panels were used as substrate sources. PM3 and PM4 media formulations are the property of Biolog Inc. (U.S. Patent No. 6,727,076; Hayward, CA) and are based on modifications of the minimal media [42]. The Biolog PM3 microplate test panel has 96 wells containing various nitrogen sources (N/N: nucleotides and nucleosides; IN: inorganic nitrogen; An: amines; Ad: amides; d-AA: d-amino acids; l-AA: l-amino acids; Di-AA: dipeptides; FA: fatty acids; M: miscellaneous; and A-1 control well). The PM4 microplate test panel includes 60 wells with phosphorus sources (RN: ribonucleotide; PAA: phosphoamino acids; OP: organic phosphorus; Ch: cholines; CH: carbohydrates; MI: metabolic intermediates; IC: inorganic compounds; PA: phosphonic acids; and A-1 control well) and 36 wells with sulfur sources (AA: amino acids; AA-S: amino acids derivatives; AASnp: non-protein amino acids; OS: organic sulfur; IS: inorganic sulfur; CH-der: carbohydrate derivatives; Chol: cholic acid; acid: sulfinic acid; pep: di- and tripeptides; and F-1 control well) [43,44].

For the analysis of the PMs, the following steps were taken for the nitrogen, phosphorus, and sulfur growth profiles. A pure culture of a fungal species was grown on a 2% malt extract agar (Sigma-Aldrich, St. Louis, MO) plate with aged, autoclaved filtered seawater until enough conidia were present to prepare a suspension. All culture media contained streptomycin sulfate and penicillin (0.15 and 0.5 g L⁻¹, respectively) to prevent bacterial growth and were cultivated in a Purifier Biological Safety Cabinet (LabConco, Logic class II). The conidia were swabbed from the surface of the agar plate and suspended at a specified density in an inoculating fluid. The inoculating fluid used for the PMs contained no nitrogen, phosphorus, or sulfur sources (IF-0 Inoculating Fluid 72211; Biolog, Hayward, CA). The initial density was the same for all species, and was measured as the initial optical density (O.D.) at 750 nm [45,46]. Each spore suspension was poured into a sterile filling reservoir and PMs were inoculated by pipetting 100 μL per well, using glucose as the carbon source. PM3 and PM4 were incubated in the dark at 20 °C for 120 h and read every 24 h using a Elx800 spectrophotometer (BioTek Instruments, Winooski, VT) at 750 nm. Each isolate was replicated three times.

2.4. Metabolic pathways

The PM primarily provides information on growth rates based on different nutrients. Integrating these data into a biochemical map can suggest potential enzymatic pathways or steps unique to a particular strain, species, or genus [46]. It is important to consider that in the absence of tetrazolium oxidation measurements, these estimates are considered indirect, and the exponential growth phase must be carefully determined at the time, to ensure that the measured growth occurred during an active growth period.

2.5. Data analysis

The assumption of normality was tested for each data set using the Shapiro–Wilk and Kolmogorov–Smirnov normality tests. All statistical analyses were performed using Statistica v.13 (Tibco Software, Inc., Palo Alto, CA). A two-way ANOVA was performed to assess differences in respiration rates between species in relation to different substrate concentrations (factors = species, substrate concentration). Additionally, a two-way ANOVA was used to evaluate the differences in respiration rates attained by each species when using different substrates (factors = substrate type, concentration).

In relation to substrate utilization by the strains, a cluster analysis was performed using O.D. values (as a percentage of the total) at 96 h, which corresponds to the linear growth phase for most substrate sources. This was applied to identify the groups of nitrogen, phosphorus, and sulfur sources (from the experimental data set), which produced similar growth. A two-way ANOVA (factors = growth, species, and days) was performed to detect differences in species growth rates when using different substrates. Reaction kinetics were measured using growth rates between days 0 and 2, as well as between days 2 and 4. These rates were then compared to the value of the integrated area under the curve over the five days of culture.

An operational application of the Shannon index (H*) was used as a measure of the number of substrates utilized by fungi (substrate richness), as well as the diversity and the extent of utilization of particular substrates (substrate evenness). H* is defined as follows:

$$H\ast =-{\sum }_{i=1}^N=\mathit{\text{pi}}\left(\mathrm{\text{ln}}\hspace{0.17em}pi\right),$$

where pi is the proportion of mycelial growth on substrate i (O.D. at 96 h) of total of microplate, and N is the number of substrates on a plate (95 for nitrogen, 59 for phosphorus, and 35 for sulfur) [47,48]. Here, H* describes the ability of the fungal species to use a greater or lesser number of nitrogen, phosphorus or sulfur sources, thus acting as an index of physiological diversity. Filamentous fungi that degrade more substrates and/or to degrade them with similar efficiency have higher H* values.

3. Results

3.1. Respiration of carbohydrates and amino acids

Exposure to different concentrations of monosaccharides, disaccharides, and polysaccharides resulted in an increase in respiration rates. All of these compounds produced higher respiration rates at higher substrate concentrations. The highest respiration rate was produced by glycogen (140 µmol O₂ L⁻¹ h⁻¹; Figure 1(D)), followed by sucrose (Figure 1(C)), glucose (Figure 1(A)), l-asparagine (Figure 1(E)), l-alanine (Figure 1(F)), cellobiose, and l-glycine (>40 µmol O₂ L⁻¹ h⁻¹; Figure 1(B,G)). Cellobiose was the only substrate for which there were no significant differences in respiration rates at different concentrations (F = 12.35, p = 0.13).

Figure 1.

Respiration rate (µmol O₂ L⁻¹ h⁻¹) of Sarocladium sp. (blue dots), Penicillium sp. (dark gray dots), and Fusarium sp. (light gray dots) exposed to: A: glucose; B: cellobiose; C: sucrose; D: glycogen; E: l-asparagine; F: l-alanine; G: l-glycine (concentration in g L⁻¹). Fungal biomass (OD₇₅₀) measured when exposed to: H: glucose; I: cellobiose; J: sucrose; K: glycogen; L: l-asparagine; M: l-alanine; N: l-glycine. The results of the statistical analyses are shown in each figure.

The three analyzed species exhibited statistically significant differences in respiration rates across substrates and concentrations (p < 0.005). These responses are species-specific, indicating significant differences in respiration rates for each substrate. Sarocladium sp. exhibited the highest respiration rates with glucose (Figure 1(A)), cellobiose (Figure 1(B)), asparagine (Figure 1(E)), and alanine (Figure 1(F)). Penicillium sp. showed the highest respiration rates with sucrose (Figure 1(C)), and Fusarium sp. showed the highest rates with glycogen (Figure 1(D)) and glycine (Figure 1(G)). Statistically significant differences were observed in the respiration rates of Sarocladium sp., Penicillium sp., and Fusarium sp. across different substrate types (p < 0.001) and substrate concentrations (p < 0.001).

3.2. Utilization of nitrogen substrates

The analyzed strains showed high utilization of particular substrates, with H*-values of 4.2, 4.3, and 4.2 for the species Sarocladium sp., Penicillium sp., and Fusarium sp., respectively. These results show that these species can use a high number of nitrogen sources, which indicates a high level of physiological diversity. All three analyzed species were capable of growing in all tested nitrogen substrates (Figure 2); however, significant differences in substrate use were observed among the 41 substrates studied, with species-specific responses in substrate use (χ² = 18.7 p = 0.0009; Figure 2). Sarocladium sp. exhibited greater growth than the other two species with these substrates (Suppl. Fig. 1).

Figure 2.

Cluster analysis and heatmap showing the utilization of nitrogen substrates by the marine fungi Sarocladium sp., Penicillium sp., and Fusarium sp. at 96 h. N/N: nucleotides and nucleosides; IN: inorganic nitrogen; An: amines; Ad: amides; d-AA: d-amino acids; l-AA: l-amino acids; Di-AA: dipeptides; FA: fatty acids; M: miscellaneous. The symbol “+” indicates a significant difference among the three species.

We identified the potential metabolic pathways of substrates that produce higher growth (cluster I) using the Kyoto Encyclopedia of Genes and Genomes (KEGG; www.genome.jp/kegg), which provides pathway maps for carbohydrate, amino acid, and nucleotide metabolism, among others. The substrates that produced the greatest increase in mycelium growth were amino acid metabolism compounds from the arginine and proline pathway: l-arginine, l-proline, glutamine, l-ornithine, putrescine (amine), and urea (organic nitrogen) (Suppl. Fig. 1).

The use of these substrates suggests the utilization of the aspartate and glutamate pathways. Significant substrate utilization differences were observed among species using nine of the 10 substrates in cluster I (Figure 2), which includes most of the l-AA used. Cluster I represents the use of the first 10 nitrogen substrates and accounts for 26.7%, 19.2%, and 22.3% of the total plate absorbance by Sarocladium sp., Penicillium sp., and Fusarium sp., respectively. The biomass began to differ from the control on day 2, exhibiting a sigmoid growth curve, and increased considerably by day 4 (data not shown). The areas under the calculated curves were closely related to the biomass on day 4 (Sarocladium sp. r² = 0.94, F = 81.9, p < 0.001; Penicillium sp. r² = 0.94, F = 91.9, p < 0.001; and Fusarium sp. r² = 0.89, F = 36.7, p < 0.001) (Suppl. Fig. 1).

The nitrogen substrates of cluster IIa promoted moderate growth of the studied strains. This cluster included 16 substrates, including: l-citrulline, γ-aminobutyric acid, nitrate, l-glutamic acid, l-aspartic acid, l-lysine, uric acid, adenine, glycine, and allantoin (sum of O.D.: 28.0 ± 1.9). This group primarily contained l-AA, followed by nucleotides/nucleosides (N/N), which are involved in amino acid and purine metabolism (Figure 2). Nitrate (cluster IIa) promoted moderate mycelial growth in all three studied strains (see Figure 2). Substrates in cluster IIa produced noticeable differences in biomass formation from days 2 to 4 (Suppl. Fig. 1). Significant differences in substrate use (p < 0.05) were observed among the species in 13 of the 26 cluster IIb substrates. The second part of cluster II is made up mainly of l-AA and dipeptides (di-AA), as well as ammonia and inorganic N (Figure 2). Substrates in group IIb include ammonia, l-valine, xanthine, l-methionine, l-leucine, and nitrite (see Figure 2). Significant differences in substrate use were observed for all three species in 50% of cluster II substrates (p < 0.05; Figure 2).

3.3. Utilization of phosphorus substrates

The number of utilized substrates was high, with values of 58, 59, and 54 (out of 59) for Sarocladium sp., Penicillium sp., and Fusarium sp., respectively (see Figure 3). Note that the Fusarium sp. strain does not utilize five of the phosphorus compounds (Figure 3). The utilization of particular substrates showed high H*-values of 3.98 (Sarocladium sp.), 4.0 (Penicillium sp.), and 3.98 (Fusarium sp.), indicating species-specific responses to substrate use (Figure 3). These results suggest high physiological diversity, meaning the species can use a large number of phosphorus substrates and degrade them efficiently.

Figure 3.

Cluster analysis and heatmap showing the utilization of phosphorus substrates by the marine fungi Sarocladium sp., Penicillium sp., and Fusarium sp. at 96 h. RN: ribonucleotide; PAA: phosphoamino acid; OP: organic phosphorus; Ch: cholines; CH: carbohydrates; MI: metabolic intermediates; IC: inorganic compounds; PA: phosphonic acids. The symbol “+” indicates a significant difference among the three species. White values indicate that the substrate was not used.

Cluster I consisted of only adenosine (ade) and guanosine (gua) monophosphate (MP) ribonucleotides (e.g., gua-2′-MP, ade-5′-MP, gua-2′,3′-cyclic-MP, gua-5′-MP, ade-2′-MP, and gua-3′,5′-cyclic MP) (Figure 3). Cluster I included substrates that produced high biomass in the studied species, with notable increases after two days and reaching maximum values by day 4 (Suppl. Fig. 2). There are significant differences in growth among the three species (χ² = 75.12, p < 0.0001). Penicillium sp. exhibited the greatest utilization of phosphorus compounds, followed by Fusarium sp. and Sarocladium sp. The use of gua and ade (e.g., O-phospho-l-threonine, carbamoyl phosphate, cytidine, phospho-l-arginine, and phospho-l-serine) (Figure 3) suggests these compounds are utilized in the biosynthesis of amino acids and purine metabolism. Cluster I (Figure 3) accounts for 26.6 ± 3.2% of the utilization of phosphorus substrates by the studied species. On day 2, growth was strongly related to the area under the O.D. curve over time (Sarocladium sp. r² = 0.84, F = 34.0, p < 0.001; Penicillium sp. r² = 0.97, F = 140.0, p < 0.001; and Fusarium sp. r² = 0.95, F = 20.0, p < 0.001).

Cluster II compounds (Figure 3) include PAAs, which may be involved in the metabolism of phosphonates and phosphinates, glycine metabolism, and amino acid metabolism; cytidine and uridine ribonucleotides, which are involved in pyrimidine metabolism; and carbohydrates, which are involved in the metabolism of amino sugars and fructose and mannose metabolism, among others. None of the phosphorus substrates in this second group produced statistically significant differences (p > 0.05) in growth among the three species (Suppl. Fig. 2).

3.4. Utilization of sulfur substrates

The species studied exhibited a high utilization of sulfur substrates, with H*-values of 3.22, 3.46, and 3.17 for Sarocladium sp., Penicillium sp., and Fusarium sp., respectively. All of these species used all sulfur substrates and exhibited species-specific responses in substrate use (χ² = 41.3, p < 0.0001) (Figure 4). Cysteine and methionine derivatives produced the greatest growth among all the studied species (Suppl. Fig. 3). This suggests the involvement of amino acid metabolism (using the KEGG), particularly that of cysteine and methionine. The cluster I substrates included l-cysteine sulfinic acid, l-methionine sulfone, hypotaurine, tetramethylene sulfone, and l-methionine sulfoxide, among others (Figure 4). Fusarium sp. exhibited greater growth with cluster I substrates than Penicillium sp. and Sarocladium sp. (Suppl. Fig. 3C). Sarocladium sp. uses sulfate, an inorganic compound that can be used in purine and cysteine/methionine metabolism, as one of its main substrates (Figure 4). A close relationship was observed between biomass on day 4 and the area under the curve (Sarocladium sp. r² = 0.49, F = 32.49, p < 0.001; Penicillium sp. r² = 0.44, F = 11.0, p = 0.002; and Fusarium sp. r² = 0.91, F = 230.0, p < 0.001). The mycelia in this cluster exhibited considerable growth by day 2, reaching its maximum value by day 4 (Suppl. Fig. 3).

Figure 4.

Cluster analysis and heatmap showing the utilization of sulfur substrates by the marine fungi Sarocladium sp., Penicillium sp., and Fusarium sp. at 96 h. AA: amino acids; AA-S: amino acid derivatives; AASnp: non-protein amino acids; OS: organic sulfur; IS: inorganic sulfur; CH-der: carbohydrate derivatives; Chol: cholic acids; acid: sulfinic acids; pep: di and tripeptides. The symbol “+” indicates a significant difference among the three species.

Cluster II (14 substrates) consists of a variety of substrates, including amino acids and inorganic compounds (Figure 4). This combination produced minimal growth with only slight differences in biomass on days 2 and 4 (Suppl. Fig. 3). This cluster included the inorganic substrates tetrathionate, thiophosphate, dithiophosphate, and sulfate, which are involved in the metabolism of cysteine, methionine, taurine, and hypotaurine, as well as organic compounds and amino acids (Figure 4).

4. Discussion

4.1. Marine fungi in the coastal upwelling zone

Marine fungi are an ecologically relevant component of microbial communities and play crucial roles in the degradation of organic matter and the cycling of nutrients in oceanic ecosystems [18]. However, their metabolic adaptations to dynamic, nutrient-rich coastal upwelling environments remain scarcely characterized [3,5–7]. The genus Penicillium is a dominant culturable genus among the filamentous fungal species found in the coastal zone off Central Chile, representing up to 82% of identified strains in some surveys, and it is known for its euryhaline and eurythermal capabilities [29]. On the other hand, the genera Fusarium and Sarocladium, despite being ubiquitous in numerous types of ecosystems, including marine environments, where they act as saprotrophs and pathogens, are less prevalent in samples from the coastal zone off Chile [29,33,35]. The presence of fungal species that thrive in terrestrial environments in coastal areas highlights the important connectivity between marine, terrestrial, and freshwater ecosystems [9,27].

Our results suggest that Sarocladium sp., Penicillium sp., and Fusarium sp. can use a wide variety of organic and inorganic nitrogen, phosphorus, and sulfur substrates (Figure 5), indicating their opportunistic nature. The observed species-specific preferences, such as Sarocladium sp.’s superior growth on nitrogen substrates and Fusarium sp.’s growth on sulfur compounds, suggest niche differentiation. This allows them to coexist and exploit different resources within the same environment. This metabolic partitioning underscores their intricate involvement in the biogeochemical cycles of nitrogen, phosphorus, and sulfur, a role that has previously been underappreciated. A previous study characterized the growth of these species on various carbon sources, noting a preference for carbohydrates and amino acids [34]. Understanding species-specific responses associated with the differentiated use of substrates by marine fungi is important because it advances our understanding of the ecological and biogeochemical role of fungi in the coastal ocean [6,33,49]. The efficient degradation of organic polymers like cellobiose and glycogen, coupled with the assimilation of various N, P, and S compounds, suggests these fungi are key players in the remineralization of organic matter and nutrient cycling. This is particularly relevant in the context of the marine biological carbon pump, in which fungi transform particulate organic matter (POM) into dissolved organic matter (DOM) [5,18]. Sarocladium sp. stood out by demonstrating the highest overall metabolic activity for carbohydrate utilization compared to the other tested fungal strains. Sarocladium sp. also exhibited high sulfate utilization, indicating its ability to sustain important metabolic processes such as purine and cysteine/methionine metabolism using inorganic sulfur sources.

Figure 5.

Utilization of carbon, nitrogen, phosphorus, and sulfur substrates, ordered according to their capacity to promote fungal growth. In the center, a scheme of the growth of a hypha is displayed, showing the main ultrastructural features in the lower half and the main processes in the upper half. Adapted from Fuentes and Quiñones [34].

The enzymatic capabilities implied by our results align with recent discoveries of a wide variety of carbohydrate-active enzymes (CAZymes) expressed by pelagic fungi, some of which, such as cellobiohydrolases (GH7), are unique to fungi and have not been detected in marine prokaryotes, highlighting their unique role in cellulose degradation [1,5].

During certain periods, fungal enzymatic activity can surpass that of prokaryotes [27], particularly in highly productive ecosystems such as the coastal upwelling zone of central-south Chile [27,35]. For instance, during periods of high phytoplankton biomass, over 90% of extracellular enzymatic activity in the water column has been observed to occur in size fractions (>25 µm) dominated by filamentous fungi, with minimal prokaryotic biomass [27]. This seasonal dominance aligns with the metabolic versatility demonstrated by local fungal isolates, such as Penicillium sp., Sarocladium sp., and Fusarium sp. These isolates have the capacity to utilize a wide array of substrates, including complex polymers like cellobiose and various amino acids. This capacity is directly linked to the production of diverse extracellular enzymes [5,18]. Our results show that a wide range of substrates promote optimal fungal growth, including compounds from the arginine and proline, aspartate and glutamate, purine metabolism (essential for nucleic acid formation), and catabolic pathways linking xanthine to urea metabolism and ATP/GTP recycling. We suggest that the high physiological plasticity of the isolated strains could explain the high rates of extracellular hydrolysis of proteins and carbohydrates recorded in the region [27,35]. This suggests that the metabolic capabilities of marine fungi could be a key factor in processing organic matter and biogeochemical cycles in this dynamic upwelling marine environment [27].

The findings presented here underscore the remarkable metabolic plasticity of these fungi, including their preference for organic substrates and extensive enzymatic capabilities. These characteristics reinforce their integral role in the marine microbial loop [1–4]. The metabolic plasticity observed in this study emphasizes the necessity of incorporating marine fungi into biogeochemical models of upwelling ecosystems. Future research should quantify the in situ enzymatic activities of these fungi to validate laboratory findings and accurately model their contributions. Understanding the cooperative and competitive interactions between fungi and prokaryotes in organic matter degradation is essential for comprehending carbon and nutrient cycling in upwelling coastal systems [27].

4.2. Respiration rates of marine fungi

With the exception of two previous studies [28,50], no information exists regarding the aerobic respiration rates of fungi inhabiting marine ecosystems. The three species studied here exhibited increased respiration rates and growth in response to elevated substrate concentrations, particularly glucose, sucrose, and glycogen. Our previous work found similar results for glucose [28].

The present study highlights the notable metabolic and physiological versatility of marine fungi. Their extensive enzymatic machinery and remarkable physiological plasticity enable them to efficiently degrade a wide range of organic compounds, from simple sugars to complex polymers. Among the carbohydrates tested, cellobiose (a building block of cellulose) exhibited the lowest respiration rates, which may be associated with the difficulty of breaking down the β-(1,4′) glycosidic bond. Enzymes that act on this bond are primarily found in fungi and bacteria [51,52]. The Basidiomycetes, Ascomycetes, and Zygomycetes actively participate in the degradation of cellulose, xylan, and lignin [53–56]. In our study, the species with the highest respiration rate was found to be on glycogen, which is a larger branched polymer than glucose or sucrose.

4.3. The role of marine fungi in the N, P, and S cycles

Studies of coastal ecosystems have shown that remineralization of nitrogen mediated by heterotrophic activity plays an important role in supporting primary production [57–60]. The nitrogenous compounds that produced the greatest growth in the three fungal species studied included l-AAs, amines, and N/N. Previous experimental work with Fusarium sp., Penicillium sp., and Sarocladium sp. showed that the preferred carbon sources include the amino acids l-proline and l-alanine, which may be used as sources of carbon and nitrogen in the glycolysis/gluconeogenesis pathway [34]. The high preference for amino acid use (Figure 2) further supports the notion that mycoplankton actively participates in the coastal marine N-cycle [7,61]. Their intense engagement in protein degradation and amino acid uptake suggests that these fungi play a key role in the remineralization of organic nitrogen [7,18].

The components of the nitrogen–nitrate–nitrite–ammonia system produced moderate growth in the three strains studied (Figures 2 and 3). In fungi, the enzymes glutamate dehydrogenase, glutamate synthase, and glutamine synthetase are crucial for ammonia assimilation [61]. In marine environments, NO₃⁻ is the major form of inorganic nitrogen that supports primary production and fuels new production in the ocean [62]. Assimilatory nitrate reductase catalyzes the first step in NO₃⁻ assimilation in plants and fungi [63,64]. The nitrate assimilation pathway involved in nitrate uptake in Aspergillus nidulans includes three genes that code for enzymes required for the absorption of nitrate and the enzymes nitrite reductase and nitrate reductase [65,66].

Nucleic acids are rich in phosphorus and nitrogen, which, in the coastal ocean, primarily originate from the breakdown of phytoplankton cells [67]. Among the three studied species, the ribonucleotides of ade and gua (purine bases) produced the greatest mycelial growth. Fungi have specific transporters in their plasma membranes that facilitate the assimilation of purine bases [68,69]. Pyrimidine ribonucleotides (cytidine and uridine) produced moderate growth rates in the studied strains. However, fungi cannot use pyrimidines as a nitrogen source; they can only be used in nucleic acid and nucleotide synthesis [70]. The hydrolysis of AMP to ade and GMP to guanine, by the action of nucleotidases, forms the extracellular arm of purine and phosphate rescue; ade and guanine can cross the cell membrane and be used as substrates for the production of purine nucleotides in the cell [71]. We found that the three studied strains exhibited moderate hyphal growth in the presence of inorganic substrates such as thiophosphate and phosphate, in which inorganic polyphosphates play an important role in energy metabolism [72]. It has been suggested that, under unfavorable conditions and stress, the hydrolysis of polyphosphates provides additional energy to maintain fungal cell processes [73].

Sulfur is found in amino acids, such as cysteine and methionine, as well as in polysaccharides and proteoglycans, and in several marine polysaccharides, especially in the cell wall of macroalgae, which are highly sulfated [74]. Although sulfur utilization has not received as much attention from the scientific community as carbon (C) or nitrogen (N), it is an important element in microbial ecology, biogeochemistry, and isotope geochemistry in the ocean [75]. Cysteine and methionine and their derivatives promoted the highest growth rates in the studied species. Methionine and cysteine are more easily oxidized than the other amino acids [76], which also provide carbon and nitrogen. Methionine initiates the synthesis of nearly all proteins in all organisms [76]. Cysteine plays a critical role in the structural stability and function of many proteins; sulfur is crucial for the function of several biologically important molecules [75,77]. Baltar et al. (2021) [1] show that oceanic pelagic fungi of the genus Rhodotorula produce sulfatase activity, while Sakaguchia dacryoidea exhibits fast hydrolysis and low substrate affinity. Orsi et al. [5] describe how fungi in sulfidic sediments assimilate more carbon from chemosynthetic bacteria than from diatom extracellular polymeric substances. This is related to a unique seafloor fungal community and a different composition of DOM than that found in the water column [5]. Moderate utilization of sulfonic acids and sulfones was observed in our study. However, Linder [78,79] demonstrated the extensive use of alternative sulfur sources, such as sulfoxides, sulfones, sulfonates, sulfamates, and sulfate esters, by 13 ascomycete yeast strains. Most of these strains can utilize multiple alternative sulfur sources, while others can only use sulfonates and sulfate esters, or are limited to aromatic sulfate esters.

Some fungal species can also oxidize inorganic sulfur [80,81] and produce compounds such as dimethyl sulfide (DMS) [19,82]. The growth rates of Sarocladium sp., Penicillium sp., and Fusarium sp. were slightly affected by inorganic substrates, such as sulfate, thiophosphate, tetrathionate, and dithiophosphate. Interestingly, fungi possess lyase enzymes that cleave compounds such as dimethyl sulfopropionate (DMSP), which is the biological precursor of DMS, a key natural source of sulfur in the ocean [83,84].

Our results indicate that Sarocladium sp., Penicillium sp., and Fusarium sp. possess the enzymes necessary to utilize a wide range of organic and inorganic substrates. This metabolic versatility positions them as active and competitive heterotrophs alongside bacteria in the race for nutrient acquisition [85]. Differences in substrate utilization and respiration among fungal species suggest niche differentiation, allowing species to coexist and exploit different resources within the same environment. Our results support the notion that marine fungi play a crucial role in degrading organic matter in their natural environment and are an important part of the marine microbial community. Accordingly, our findings support the increasingly accepted notion that marine fungi should be incorporated into current biogeochemical cycling models of coastal upwelling ecosystems.

Funding Statement

This work was supported by the Interdisciplinary Center for Aquaculture Research (INCAR) under Grant Numbers FONDAP-ANID 15110027 and 1523A0007.

Disclosure statement

No potential conflict of interest was reported by the author(s).