Introduction

In order to escape oligotrophic conditions in oceans, marine fungi often tend to evolve association with surrounding biota including seaweeds (Abdel-Gawad et al. 2014). Algal-inhabiting fungi are known as algicolous, and they are composed of a taxonomically diverse group of mutualists, endosymbionts, parasites, epiphytes, and saprobes that are of evolutionary, ecological, and commercial interest (Zuccaro and Mitchell 2005). Their adaptation is ensured by enzyme systems markedly different from those of terrestrial fungi. The cell walls of seaweeds constitute wide varieties of polysaccharides and hence extend a unique micro-niche sustaining growth of diverse microbial communities (Ivanova et al. 2002). Algal polysaccharides are known as phycocolloids, and they represent a major industry (Ioannou and Roussis 2009; Bixler & Porse 2011). The carbohydrate content of brown seaweeds is 30–50 %, consisting mostly of alginate and fucoidans. Alginates are linear polysaccharides composed of β-d-mannuronic and α-l-guluronic acids that are bound with 1,4-O-glycoside bonds. Fucoidans designate a family of fucose-rich polysaccharides, including sulfated fucogalacturonans found for example in Sargassum spp. (Ale and Meyer 2013). Red seaweeds are a very heterogeneous group with respect to the chemical composition of the cell walls. Usov and Elashvili (1991) reported the structural features of sulfated polysaccharides from different Laurencia complex species (including Palisada spp.) and found to be composed of sulfated and/or methylated polymers having an agarose backbone.

The marine macroalgae in the Red Sea of Egypt are dominated by red and brown algae, among which the brown alga Sargassum sp. and the red alga Palisada perforata are found in high abundance (Issa et al. 2014). A diverse association of invertebrates, protozoa, bacteria, and fungi carries out degradation of seaweed biomass in the marine environment. Algicolous fungi including ascomycetes and zygomycetes have been recorded in high numbers living on or in healthy seaweeds (Flewelling et al. 2013; Godinho et al. 2013; Furbino et al. 2014; Abdel-Gawad et al. 2014) as well as cast seaweed litter (Sridhar et al. 2012).

The role of marine algicolous fungi in the decomposition of marine algal materials is not well documented. Knowledge of how macroalgal polysaccharides are utilized by associated fungi is essential for understanding the flux of carbon in the seaweed ecosystems. In addition, finding new sources of enzymes degrading red and brown algal polysaccharides and selecting enzyme complexes for macroalgal thalli are beneficial to the development of seaweed cell technologies such as thallus maceration, protoplast isolation, and conversion of biomass into fermentable sugars for biofuel. This investigation sought to find new sources of fungi that are capable of degrading macroalgal biomass through enzymatic activities of fucoidanase, alginate lyase, agarase, amylase, and protease and to characterize potential application for the reutilization of the seaweed biomass.

Materials and methods

Algicolous fungi

Fifteen fungal isolates representing three major communities of algicolous fungi were used in the study (Table 1). Epiphytic fungi were isolated by surface washing technique (Abdel-Gawad et al. 2014). Endophytic fungi were isolated by surface sterilization method using 70 % ethanol (Kjer et al. 2010; Suryanarayanan et al. 2010). While saprophytic fungi were isolated by direct incubation (3 months, 150 rpm, and 28 °C) of fresh macroalgal thalli in sterile natural seawater (2.5 % w/v) and supplemented with chloramphenicol (200 mg L−1) to prevent bacterial growth, subsequently, the emerging hyphae were isolated and purified. Fungal identification was carried out morphologically (Abdel-Gawad et al. 2014).

Table 1 Details on algicolous fungi used in the study and their type of association with host macroalgae
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Pre-treatment of macroalgal biomass

Two macroalgae species, namely Sargassum sp. (brown algae) and P. perforata (red algae), were collected during summer from the intertidal zone of Hurghada, Egypt (27° 12′ N, 33° 50′ E). The macroalgal biomass was air-dried and powdered, and 2 % (w/v) was prepared in natural seawater and autoclaved (105 °C and 5 min) to release macroalgal components and polysaccharides. The autoclaved biomass was filtered and 10 mL was dispensed into each 25-mL glass bottle.

Fungal inoculum and fermentation

Fungi were grown on PDA medium for 2 weeks. Fungal spores were collected in sterile natural seawater and counted using a hemocytometer to give a final concentration of 1–4 × 104 propagules mL−1. One hundred microliters of each fungal suspension was inoculated in triplicate into 25-mL glass bottles containing the seaweed extract. All the bottles were incubated at 28 °C with agitation (150 rpm) for 10 days.

Enzymatic bioassay

After 10 days of fermentation, the fungal filtrate was collected and separated from mycelia by centrifugation (6000 rpm, 10 min). The supernatant was used for measuring different enzymatic activities. Fungi grown on Sargassum sp. as a sole carbon source were assayed for fucoidanase, alginate lyase (alginase), amylase, and protease activities, while for P. perforata, fungi were assayed for agarase, amylase, and protease activities.

For determining fucoidanase, alginase, amylase, agarase, and protease activities, a 0.5 % (w/v) of fucoidan, sodium alginate, soluble starch, 0.1 % (w/v) agar, and 1 % (w/v) casein were prepared in 0.1 M phosphate buffer (PB; pH 7.0), respectively. Fucoidan was extracted from Sargassum sp. as described by Ale et al. (2011, 2012), while other substrates were obtained commercially.

Each substrate was mixed with the culture supernatant (9:1 v/v), and the mixture was maintained at 37 °C for 1 h. Fucoidanase, amylase, and agarase activities were determined by measuring the amount of reducing sugars produced in the mixture by dinitrosalicylic acid reagent (DNS method, Miller, 1959). Alginase activity was determined by measuring the increase in the absorbance at 232 nm, because 4-deoxy-l-erythro-hex-4-ene pyranosyl uronic acid at the nonreducing terminus of the resultant oligomers formed by β-elimination provides a UV chromophore (232 nm) (Song et al. 2003). Protease activity was determined by measuring the increase in the absorbance at 280 nm after precipitation of insoluble casein by trichloroacetic acid (10 %, 1 mL) and centrifugation (6000 rpm, 10 min) (Belov et al. 1993). For determining thallus decomposing activity, 120 μg of Palisada or Sargassum powder (under 500 μm in size) was dispensed into each tube with 1800 μL PB and 200 μL fungal filtrate. The reaction mixture was incubated at 37 °C for 1 h with agitation (100 rpm). After incubation, the supernatant was obtained by centrifugation, and their reducing sugars were quantified in the same way.

One unit (U) of fucoidanase, amylase, agarase, and thallus decomposing activity was defined as the amount of enzyme able to release 1 μmol of reducing sugars per hour under the assay conditions. One unit (U) of alginase activity was arbitrarily defined as the amount of enzyme required to cause an increase of 0.01 optical density at 232 nm per hour under the assay conditions. Similarly, one unit (U) of protease activity was defined as the amount of enzyme required to cause an increase of 0.01 optical density at 280 nm per hour under the assay conditions. Concentration of protein in the fungal filtrate was determined by Lowry method (Lowry et al. 1951), and subsequently, the specific activity was calculated as activity (units) per milligram of protein.

Data analysis

Multiple comparisons of means were performed by the Tukey‘s B test. P values < 0.05 were considered significant for all the tests. The enzyme activities were also subjected to principal component analysis (PCA). The enzymatic activities values were normalized prior to the analysis, and the first two principal components were plotted to visualize the correlation among different enzyme activities and behavior of different types of algicolous fungi.

Results

Extracts of the brown seaweed Sargassum sp. and the red seaweed P. perforata supported germination and growth of all the studied marine algicolous fungi. After 5 to 10 days of fermentation, it was possible to visualize the formation of mycelial fragments or pellets in the culture of the seaweeds. The fungi were capable of degrading seaweed polysaccharides and release of reducing sugars, but the amount of reducing sugars formed after 10 days of incubation varied considerably between the studied fungi (Fig. 1). The amount of increase or decrease in the seaweed saccharification by reducing sugars after fermentation was calculated as percentage in relation to control (without fungus). Most of the fungi were able to increase the amount of reducing sugars after fermentation for 10 days (Fig. 1). The highest increase in reducing sugars was found in cultures of Lindra thalassiae on Sargassum (77.76 %) and Chrysosporium pruinosum on P. perforata (71.17 %) (Fig. 1).

Fig. 1
figure 1

Percentage increase or decrease in the amount of reducing sugars after fermentation of the macroalgal extract with algicolous fungi. Percentage values were calculated in relation to uninoculated extract

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Most of the studied fungi showed the ability to depolymerize the complex polysaccharides of fucoidan that found in the extract of Sargassum when used as a sole carbon source. The fucoidanase activity varied considerably between studied algicolous fungi. For instance, Aspergillus flavus, Cladosporium salinae, Curvularia lunata, and Setospheria rostrata showed the highest activity of fucoidanase, while Torula graminins showed the lowest activity. Acremonium sp., C. pruinosum, Scopulariopsis brevicaulis were unable to produce fucoidanase (Fig. 2a). Similarly, fucoidanase specific activity was higher in C. salinae and lower in T. graminins (Fig. 2b). It seemed that fungi associated with brown algae might be more able to derive their nutrition through special enzymes such as fucoidanases. For instance, C. salinae was isolated from the brown alga Padina pavonica, while Torula graminis was isolated from the red alga P. perforata.

Fig. 2
figure 2

Fucoidanase activity (a) and specific activity (b) of different algicolous fungi grown on Sargassum as a sole carbon source. Different values are means ± SD (n = 3). Different letters within a column indicate significant differences between samples at the level of P < 0.05

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In addition to fucoidanase, algicolous fungi were able to utilize alginate from the extract of Sargassum through alginase that has high activity in L. thalassiae (Fig. 3a). High specific activity of alginase was found in Acrophialophora sp. and Setosphaeria rostrata. Scopulariopsis candida showed the lowest activity and specific activity of alginase (Fig. 3b). Fungi isolated from brown algae showed quite higher activity of alginase than those from red algae, for instance, L. thalassiae and Acremonium sp. (Fig. 3a).

Fig. 3
figure 3

Alginase activity (a) and specific activity (b) of different algicolous fungi grown on Sargassum as a sole carbon source. Different values are means ± SD (n = 3). Different letters within a column indicate significant differences between samples at the level of P < 0.05

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All the tested algicolous fungi grown on P. perforata were able to produce extracellular agarase. A. flavus and C. lunata showed the highest activity and specific activity of agarase (Fig. 4a). The two fungi were isolated from the red alga P. perforata as saprophytes, which may account for their high agarase activity. The lowest agarase activity and specific activity was found in Acremonium sp., C. pruinosum, L. thalassiae, S. brevicaulis, and T. graminis.

Fig. 4
figure 4

Agarase activity (a) and specific activity (b) of different algicolous fungi grown on Palisada as a sole carbon source. Different values are means ± SD (n = 3). Different letters within a column indicate significant differences between samples at the level of P < 0.05

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All fungi grown on Sargassum and Palisada biomass expressed a week amylase activity except for A. flavus, which expressed a significantly higher specific amylase activity (Table 2). The ability of the fungus to express amylase depends on the biomass, for instance, Aspergillus terreus showed no amylase activity for Sargassum biomass, while amylase activity for Palisada biomass was evident (Table 2).

Table 2 Amylase and protease activities (U) and specific activities (U mg-1 protein) of algicolous fungi grown on Sargassum and Palisada as a sole carbon source
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Similarly, the ability of the fungus to express extracellular proteases depends on the biomass used for cultivation. For instance, all fungi cultivated on Palisada biomass expressed protease activities (Table 2). While some of these fungi showed no protease activity for Sargassum biomass (A. terreus, L. thalassiae, S. candida, Syncephalastrum racemosum and T. graminis). In case of Sargassum, the highest amylase activity was exhibited by C. pruinosum and specific activity by C. salinae. While in the case of Palisada biomass, S. brevicaulis and T. graminis showed the highest activity while A. terreus showed the highest specific activity (Table 2).

The crude enzyme preparations from the algicolous fungi were able to degrade macroalgal thalli when prepared as particles (under 500 μm in size) and reducing sugars were released. Thallus decomposing activity was evident for all tested algicolous fungi, except S. rostrata and S. racemosum showed no thallus decomposing activity against Sargassum powder. The enzymatic activities of algicolous fungi against both Sargassum and Palisada thalli were comparatively low compared to either fucoidanase or agarase assayed using pure enzyme substrates. The highest thallus decomposing activity was exhibited by A. terreus and A. flavus for Sargassum and Palisada, respectively (Table 3).

Table 3 Enzymatic activities (U) and specific activities (U mg-1 protein) of algicolous fungi against Sargassum and Palisada thalli
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Figure 5a shows a PCA analysis of enzyme activities of different algicolous fungi grown on the brown seaweed Sargassum. The activities of fucoidanase and alginase as well as protease and amylase occur on the opposite sides; therefore, they are significantly and negatively correlated. The majority of the studied algicolous fungi showed positive correlation to alginase and protease and negative correlation to fucoidanase and amylase. Similarly, in case of Palisada (Fig. 5b), agarase and amylase activities occur on the same side on the first component axis; therefore, they are significantly and positively correlated. None of the endophytic fungi grown on Palisada showed higher scores to agarase or amylase while some of the saprophytic fungi showed higher correlations to either amylase and agarase or protease.

Fig. 5
figure 5

Principal component analysis (PCA) of different enzyme activities during growth of algicolous fungi on the brown alga Sargassum (a) or the red alga Palisada (b) as a sole carbon source. Enzyme correlation circle: (a) components 1 and 2 accounted for 47 % and 25.4 % of the total variation, respectively; (b) components 1 and 2 accounted for 51.2 % and 29.1 % of the total variation, respectively

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Discussion

The present study shows that the seaweeds can be a source of nutrients for algicolous fungi; therefore, the closely associated fungi possess the necessary enzymatic systems capable of specific cleavage of macroalgal cell-wall polysaccharides into mono- and oligosaccharides. Algicolous fungi were capable of simultaneous saccharification and fermentation of seaweed extracts as a sole carbon source in natural seawater through different cocktail of enzymatic activities.

Twelve of the 15 studied algicolous fungi were able to degrade the sulfated hetero-polysaccharides of fucoidan found in the extract of Sargassum. The fucoidan-degrading ability may be part of the adaptive response of microorganisms colonizing seaweeds to the environment, through which these microorganisms satisfy their requirements for carbon, sulfur, and energy. Fucoidanase was reported in marine fungi such as Dendryphiella arenaria (Wu et al. 2011), Aspergillus niger, Penicillium purpurogenum, and Mucor sp. (Rodríguez-Jasso et al. 2008); however, most of them exhibit weak titles of activity. In the current study, higher fucoidanase activity reached 0.048 U · L−1 in C. salinae and lower activity was 0.009 U · L−1 in T. graminis using Sargassum extract. Low fucoidanase activity may be due to the specific role of fucoidans and fucoidanases in the metabolism of marine organisms as well as active proteases (Bakunina et al. 2000). The absence of fucoidanase in some of the algicolous fungi indicated that they could not degrade fucoidan as a component of brown algal cell walls but rely on growth on other substrates during growth.

Alginate lyases catalyze the degradation of alginate by a β-elimination of the alginate polymer to create an unsaturated uronic acid at the new nonreducing end (Wong et al. 2000). Alginate lyases have been isolated from a wide range of organisms, including algae, marine invertebrates, and marine and terrestrial microorganisms (Wong et al. 2000). Some marine fungi, such as Corollospora intermedia, Asteromyces cruciatus, Dendryphiella salina, D. arenaria, even if in these fungi production of alginolytic enzymes, seems an isolate-specific rather than a species-specific trait (Wainwright and Sherbrock-Cox, 1981; Schaumann and Weide, 1990). Algicolous fungi including truly marine and marine-derived species were able to produce alginase; thus, closely associated fungi with marine macroalgae may be a good source for extracellular alginate lyases. Most alginate-degrading bacteria were originally found to be closely associated with marine algae (Tang et al. 2009). In our study, L. thalassiae, Acrophialophora sp., and S. rostrata showed higher activity and specific activity of alginase. L. thalassiae, an indiscriminate pathogen in the ocean, is known to cause “raisin disease” in the brown algae Sargassum spp. and “Thalassia disease” in the seagrass Thalassia testudinum (Andrews, 1976; Porter, 1986). Absence of any disease symptoms was the main reason of identifying this fungus as an endophyte. High alginase in the marine brown algal pathogen L. thalassiae can indicate that colonization by this pathogen may occur by degradation of alginate rather than fucoidan; thus, brown algae with high content of fucoidan are less susceptible to infection that could be a part of mechanical resistance of macroalgae to pathogens. This result was confirmed by growth inhibitory action of fucoidan against L. thalassiae (data not shown).

Algicolous fungi were able to grow on Palisada extract which contained sulfated galactans in the cell wall. Emergence of agarase activity in the fungal cultures was accompanied by efficient growth of algicolous fungi. Agarase specific activity ranged between 1.29 ± 0.22 U mg−1 protein in L. thalassiae to 20.14 ± 0.61 U mg−1 protein in C. lunata. These values are quite higher than reported previously in terrestrial fungi (Nikolaeva et al. 1999), suggesting that closely associated fungi with marine macroalgae are better adapted and produce higher activities of specific enzymes than terrestrial counterparts. Surprisingly, agarase was found in the marine pathogen of brown algae; L. thalassiae. The infection of red algae by this fungus is not reported yet, but this finding supports the saprophytic nature of this fungus on red algal biomass. The existence of agarase in the fungal endophytes of the red alga P. perforata may indicate the enzymatic colonization process. However, fungal infection on Porphyra perforata (Polne-Fuller and Gibor, 1987) revealed that the fungus did not dissolve cell walls and did not produce a complete set of wall-degrading enzymes, although the hyphae penetrated into the extracellular matrix and through the walls into the cells.

Algicolous fungi grown on Sargassum and Palisada also exhibited amylases and proteases. The ability to produce proteases and/or amylases may be responsible for complete destruction of cells and hydrolysis of different seaweed components. Nikolaeva et al. (1999) reported that terrestrial fungi grown on agarophytic red algae were able to express proteases. The ability of fungi to express high activity of either amylase or protease depended on the taxon as well as the type of biomass used for growth. For instance, A. flavus showed higher activity of amylase, which reached 638.33 and 90.03 U mL−1 for Sargassum and Palisada, respectively. Generally, activities and specific activities of both amylases and proteases for most of algicolous fungi were low.

PCA demonstrated the correlation of different enzymes from algicolous fungi. Funcoidanases were found to be independent of alginate lyases that suggest antagonistic properties between the enzyme activities in the culture of Sargassum. This result indicated that efficient degradation of brown algal cell walls into fermentable sugars is either through funcoidanases or alginate lyases. On the other hand, agarases were correlated to amylases that strongly suggest the two enzymes synergistically degrade Palisada biomass.

In conclusion, metabolic processes always affect the natural ecosystem. Our findings showed that marine algicolous fungi might have an important impact on seaweed-based ecosystems. These fungi participate in the degradation of brown algal and red algal polysaccharides. They are involved in such diverse ecological processes as the following: remineralization of organic substances, nutrient recycling, detritus degradation and enrichment, biomass production, and finally the carbon cycle in general (Schaumann and Weide, 1990). The crude enzyme preparations from algicolous mycobiota adapted to assimilate red and brown seaweed biomasses contained a wide spectrum of enzymes necessary for seaweed cell wall degradation. Consequently, they can be used potentially for thallus maceration and protoplast isolation of these seaweed groups. However, these enzyme preparations may contain proteases, which were able to damage and destroy cells, as described previously (Nikolaeva et al. 1999). Efficient saccharification of macroalgae by fermentation with algicolous fungi would be a valuable resource for bioethanol production. In addition, the obtained tailor-made mono- and oligosaccharides have many biotechnological applications such as enhancing the resistance of crops to their pathogens, antidiabetic, hepatoprotective, and antioxidant properties (Chen et al. 2006; Vera et al. 2011). Thus, this eco-friendly treatment may be a good method to turn seaweed wastes into valuable resources with low energy consumption. For industrial application, an optimized scaled up process of the degradation needs to be demonstrated.