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Article

From Screening to Laboratory Scale-Up: Bioremediation Potential of Mushroom Strains Grown on Olive Mill Wastewater

by
Ilias Diamantis
1,2,
Spyridon Stamatiadis
1,2,
Eirini-Maria Melanouri
1,2,
Seraphim Papanikolaou
2 and
Panagiota Diamantopoulou
1,*
1
Hellenic Agricultural Organization ELGO-Dimitra, Institute of Technology of Agricultural Products (ITAP), Laboratory of Edible Fungi, 1, Sof. Venizelou, 14123 Lykovryssi, Greece
2
Laboratory of Food Microbiology and Biotechnology, Department of Food Science and Human Nutrition, Agricultural University of Athens, 75 Iera Odos, 11855 Athens, Greece
*
Author to whom correspondence should be addressed.
Biomass 2025, 5(3), 50; https://doi.org/10.3390/biomass5030050
Submission received: 7 July 2025 / Revised: 31 July 2025 / Accepted: 25 August 2025 / Published: 27 August 2025
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Abstract

Olive mill wastewater (OMW) is a phenol-rich effluent with high organic load, posing significant environmental disposal challenges in the Mediterranean countries. This study evaluated the bioremediation and valorization potential of OMW by eleven edible and/or medicinal fungal strains (Agrocybe cylindracea, Lentinula edodes, Pleurotus sapidus, Pleurotus sajor-caju, Flammulina velutipes, Ganoderma adspersum, Tuber aestivum and Tuber mesentericum). Firstly, screening for mycelial growth on agar media with commercial glucose and OMW (concentrations from 0 to 50%, v/v) revealed a strain-specific tolerance to phenolic toxicity. Although all tested strains could grow on OMW-based media, G. adspersum, T. mesentericum and T. aestivum presented the highest mycelial growth rates (Kr), exceeding 10 mm/day at elevated OMW levels (50%, v/v). Based on screening outcomes, seven strains were selected for further evaluation under static liquid fermentations in media with 15 and 35% (v/v) OMW. Growth kinetics, substrate consumption, phenolic removal, decolorization capacity, intracellular polysaccharide (IPS) and total lipid content were assessed. Tuber spp. and G. adspersum exhibited the highest tolerance to phenolic compounds, producing biomass exceeding 15 g/L at 35%, v/v OMW. Maximum IPS production reached up to 46.23% (w/w), while lipid content exceeded 15% (w/w) of dry biomass in F. velutipes and T. mesentericum, indicating an oleaginous microorganism-like behavior. Phenolic removal surpassed 80% in most cases, demonstrating efficient enzymatic degradation. Decolorization efficiency varied between strains, but remained above 70% for L. edodes, G. adspersum and F. velutipes. These findings highlight the potential of edible and/or medicinal fungi to simultaneously detoxify OMW and produce biomass and high-value metabolites, supporting a sustainable, low-cost agro-industrial waste management aligning with circular bioeconomy principles.

Graphical Abstract

1. Introduction

Olive oil production has been a longstanding and significant agricultural practice in Mediterranean countries since ancient times, playing a crucial role in their regional economies. Despite being a relatively small country, Greece ranks among the top three global producers of olive oil [1]. However, the extraction process also generates a significant amount of waste, with OMW in Greece alone reaching approximately 2.5 million cubic meters annually, a byproduct that not only contains elevated levels of polyphenols and exhibits an acidic pH ranging from 4.2 to 6.8 [2], but it is also abundant in reducing sugars and organic matter, as reflected by its high chemical oxygen demand (COD) that can exceed 220 g/L [3]. On a global scale, olive oil production reached 2,843,493 tons in 2007, with 2,178,774 tons originating from Europe. Countries such as Spain (2,400,000 ha), Italy (1,140,685 ha), Tunisia (1,500,000 ha) and Greece (765,000 ha) dominate olive cultivation [1]. The extraction of one metric ton of olive oil through conventional three-phase systems typically generates around 0.6 tons of olive mill solid waste and approximately 1.5 m3 of olive mill wastewater. These waste streams exert significant negative impacts on both soil and aquatic environments due to their high organic load and phytotoxicity [4,5]. For this reason, various treatment methods have been proposed, including both biological and physicochemical approaches. Physico-chemical methods can contain techniques such as simple evaporation, coagulation, ultrafiltration, reverse osmosis, oxidation, thermal processes and more. In contrast, biological treatments may include aerobic or anaerobic digestion, as well as fungal or enzymatic processes [6,7,8].
Liquid agro-industrial waste streams—such as concentrated glycerol water, molasses, expired glucose syrups and other inexpensive carbon-rich materials (e.g., commercial glucose), which are also environmental pollutants, have been biologically processed by higher fungi including Morchella spp., T. aestivum and L. edodes [9,10,11]. Regarding biological treatments, fungi are among the most efficient organisms for biodegrading and detoxifying a wide range of wastes and pollutants, including OMW. Since many organic compounds in OMW are identical or structurally similar to lignin degradation products, ligninolytic fungi have been employed for OMW biodegradation through phenolic content reduction, COD decrease and/or decolorization. Therefore, mushroom cultivation can serve as a biotechnological approach not only for detoxifying OMW-based substrates but also for converting them into useful biomass and metabolic products.
In this context, several studies have demonstrated the effectiveness of ligninolytic fungi in the bioremediation of olive mill effluents. These organisms, owing to their powerful oxidative enzyme systems, are capable of reducing the phenolic content and toxicity of such effluents to a significant extent. For instance, Pleurotus ostreatus achieved more than 90% reduction in phenolic compounds in flask-scale treatments of OMW, while L. edodes led to an 88% reduction under similar conditions [12]. Likewise, Pleurotus pulmonarius was able to decrease phenolic content by 66.2% when applied to three-phase olive mill waste in flask cultures [13]. These findings clearly illustrate the potential of edible and ligninolytic fungi as efficient biological agents for the detoxification of OMW, providing a sustainable alternative to conventional physico-chemical treatment methods.
Edible mushrooms are widely consumed due to their desirable organoleptic properties and nutritional composition. Their culinary and commercial significance primarily arises from their unique sensory characteristics, including texture, flavor and aroma. Their nutritional value is attributed to a high content of proteins, dietary fiber, essential vitamins and minerals, combined with low fat content. Throughout human history, mushrooms have also been utilized across various civilizations not only for their nutritional content but also for their therapeutic potential. Numerous studies have documented a wide range of therapeutic properties associated with mushrooms or their bioactive compounds [14].
Among the various bioactive compounds found in mushrooms, polysaccharides have attracted considerable scientific interest in recent years due to their wide range of health-promoting properties. Studies have shown that these substances possess antitumor, anti-inflammatory, antioxidant, antidiabetic and anti-obesity effects [15]. For instance, pleuran, a polysaccharide found in Pleurotus spp., exhibits pronounced immunostimulatory properties and has been clinically evaluated for respiratory health [16,17], while lentinan, a purified β-glucan from L. edodes, is one of the most studied mushroom polysaccharides, with documented anticancer and immunopotentiating activities [18,19]. Additionally, Ganoderma spp. polysaccharides have demonstrated strong immune-boosting and antioxidant properties [20], along with the ability to lower total cholesterol, triglycerides and LDL cholesterol levels [21]. Although less extensively studied, Agrocybe aegerita produces bioactive polysaccharides that exhibit significant antioxidant and anti-aging activities [22,23].
Expanding on the diversity of fungi with valuable biochemical properties, species within the phylum Ascomycota—in addition to the well-studied Basidiomycetes—also offer significant potential. Truffles (Tuber spp.), though primarily known as a high-value delicacy, are also especially recognized for their rich bioactive properties. Beyond their sensory characteristics, truffles possess a complex biochemical composition that provides both nutritional and therapeutic benefits. They are rich in essential nutrients, including carbohydrates, proteins, lipids, minerals and a diverse profile of amino acids [24]. Notably, polysaccharides isolated from fermentation cultures of T. aestivum, along with other Tuber species, displayed comparable chemical profiles (mannose, glucose, galactose) and demonstrated in vitro antitumor activity against cell lines including HepG2 and A549 [25].
In addition to their biologically active polysaccharides, edible mushrooms also exhibit a favorable lipid composition. Although their total lipid content is relatively low, they are enriched in nutritionally important mono- and polyunsaturated fatty acids (MUFAs and PUFAs) [26], including linoleic, oleic and linolenic acids, which typically represent the predominant lipid components [27,28]. Among these, linoleic acid plays a particularly significant role as the biochemical precursor of 1-octen-3-ol—the primary aroma compound responsible for the characteristic scent of mushrooms [29]—and also affects the physiological processes of fungi, plants and insects [30,31,32]. The quantity and metabolic profile of bioactive compounds are significantly affected by the type of substrate used for cultivation, as well as the genetic characteristics of the mushroom strain [28].
Given the environmental challenges of OMW disposal and the growing interest in sustainable biotechnological applications, this study aimed to investigate the effects of various OMW concentrations on the mycelial growth of several mushroom species (i.e., A. cylindracea, Pleurotus species, L. edodes, G. adspersum, T. aestivum and T. mesentericum). In addition, the feasibility of utilizing OMW as an alternative, eco-friendly substrate for fungal biomass production was assessed. The study also aimed to investigate the potential of OMW in supporting environmentally beneficial applications, such as decolorization and the sustainable production of high-value fungal metabolites, thereby contributing to circular bioeconomy strategies.

2. Materials and Methods

2.1. Fungal Strains

Eleven mushroom strains were utilized in this study (Table 1): A. cylindracea AMRL 101 & 104, L. edodes AMRL 125 & 126, P. sapidus AMRL 156-1 & 156-2, P. sajor-caju AMRL 197, F. velutipes AMRL 271, G. adspersum AMRL 315, T. aestivum AMRL 364 and T. mesentericum AMRL 365 belonging to the Laboratory of Edible Fungi/Institute of Technology of Agricultural Products/Hellenic Agricultural Organization—Dimitra. The strains were preserved on potato dextrose agar (PDA, Merck, Darmstadt, Germany) at 4 ± 1 °C and were sub-cultured regularly.

2.2. Measurement of Mycelial Linear Growth Rate

For screening purposes, the growth rate of the fungal strains was evaluated on Petri dishes containing commercial glucose (95% w/w purity; Hellenic Sugar Industry S.A., Thessaloniki, Greece), nitrogen source (yeast extract; Fluka, Steinheim, Germany) and essential mineral salts (KH2PO4; 7.00 g, Na2HPO4·2H2O; 2.50 g, MgSO4·7H2O; 1.50 g, (NH4)2SO4; 6.00 g, CaCl2·2H2O; 0.150 g, FeCl3·6H2O; 0.150 g, MnSO4·H2O; 0.060 g, ZnSO4·7H2O; 0.020 g), agar and 0% OMW (control), or OMW at concentrations of 5, 10, 15, 20, 25, 30, 35, 40, 45 and 50% (v/v). OMW was sourced from a three-phase olive oil processing facility located near the Holy City of Messolonghi, Western Greece and it was characterized by a glucose concentration of 30 g/L, total phenolic compounds 10 g/L and a pH value of 4.80. The culture medium was sterilized at 121 °C (1.1 atm) for 20 min. After cooling to room temperature, the Petri dishes were filled. Inoculation was performed by placing a 9 mm diameter mycelial plug at the center of each dish. Following inoculation, all Petri dishes were incubated in a growth chamber at 25 ± 1 °C. Five replicates were performed per substrate and strain. Measurements of colony diameter were taken daily along two perpendicular axes once the mycelial front reached 10 mm and the mycelial growth rate (Kr, expressed in mm/day) was subsequently determined.

2.3. Liquid Cultures Preparation and Mycelial Analyses

Selected fungal strains were further tested in liquid fermentations to evaluate biomass production, substrate consumption and their capacity for phenolic compound removal and decolorization. Furthermore, the recovered mycelial mass was analyzed for IPS and lipid synthesis. A liquid medium (as described above without agar) was used, containing 0 (control), 15 and 35% (v/v) OMW. These two OMW concentrations were selected based on the screening results, as they presented the most positive effects on fungal growth. A total volume of 30 mL was dispensed into 100 mL Erlenmeyer flasks, which were sterilized at 120 ± 0.5 °C for 20 min. After cooling to room temperature, flasks were inoculated with 9 mm mycelial plugs and incubated at 25 ± 1 °C under non-agitated conditions. Sampling, starting 6 days after inoculation, was performed every 5–6 days. The pH was measured with a Crison GLP 21 pH-meter (Barcelona, Spain) and the electrical conductivity (EC, S/cm) was assessed using a Hanna HI-8733 conductivity meter (Padova, Italy). The initial pH for all media after sterilization (121 ± 1 °C, 20 min) was 6.5–7.0. Glucose consumption (g/L) was estimated by the 3.5-dinitro-2-hydroxybenzoic acid (DNS) method [33]. Phenolic compounds were determined using the Folin–Ciocalteu (FC) method, as described by Slinkard and Singleton [34] and were measured at 760 nm. Total phenolic compounds (TPC, g/L) were expressed as gallic acid equivalents per L of medium. Decolorization was estimated by measuring the absorbance of both inoculated and non-inoculated OMW samples at 395 nm [35].
Mycelia were recovered using vacuum filtration with No. 2 Whatman filters (Kent, England) and washed twice with distilled water. Dry biomass (X, g/L) was determined gravimetrically after drying the mycelia at 60 ± 5 °C in an Elvem drying chamber (Athens, Greece) until a constant weight was achieved, using a Kern AGB balance (Balingen, Germany). IPS determination was carried out following acid hydrolysis, as described by Diamantopoulou et al. [36]. Dried mycelia (100 mg) were hydrolyzed with 20 mL of 2.5 M HCl (Merck, Darmstadt, Germany) at 100 ± 1 °C for 20 min. The mixture was then neutralized to pH 7 using 2.5 M NaOH (Merck, Darmstadt, Germany), adjusted to a final volume of 50 mL and filtered. From each filtrate, 0.5 mL was mixed with 0.5 mL of DNS reagent, vortexed, and incubated at 100 ± 1 °C for 5 min. After cooling the tubes to room temperature in a water bath, IPS content was quantified by measuring the absorbance (Jasco V-530 UV/VIS spectrophotometer, Tokyo, Japan) at 540 nm. Total cellular lipids were extracted from dry biomass using a chloroform/methanol mixture (2:1, v/v; Merck, Darmstadt, Germany) and quantified gravimetrically [37].

2.4. Data Analysis

All experiments were independently repeated twice. Each data point was derived from three replicate Petri dishes and three replicate flasks per experimental condition. Results are reported as mean values with standard deviations calculated and represented as error bars in the corresponding figures.

3. Results

3.1. Preliminary Experiment on Kinetics Growth in Petri Dishes with Different OMW Concentrations

Considering that the addition of OMW could act as an inhibitory factor for fungal growth, the addition of increasing concentrations up to the maximum level of 50% v/v was tested initially. Fungal growth (viz. mycelial growth rate, Kr, mm/d; Table 2) was subsequently monitored to determine the impact of OMW supplementation. Variation in OMW concentration resulted in species- and strain-specific changes in the Kr of the tested fungi. A. cylindracea strains AMRL 101 and 104 exhibited a steady decline in growth with increasing OMW concentrations; AMRL 101 decreased from 3.32 mm/day at 5% OMW to 2.05 mm/day at 50%, while AMRL 104 decreased from 3.40 mm/day at 5% to 2.27 mm/day at 50% OMW, indicating sensitivity to inhibitory compounds such as polyphenols. L. edodes strains presented variable tolerance to OMW. Strain AMRL125 demonstrated limited tolerance to OMW, with a peak growth rate of 4.76 mm/day at 5% OMW, followed by a significant reduction at higher concentrations, reaching only 1.55 mm/day at 50%. In contrast, strain AMRL126 demonstrated greater tolerance, achieving its highest growth rate of 5.22 mm/day at 10% OMW and sustaining moderate growth (2.31 mm/day) even at 50%, indicating a higher capacity to tolerate OMW stress. In contrast, P. sapidus strains maintained more stable growth. P. sapidus AMRL156-2 slightly increased from 4.58 mm/day at 0% OMW to 5.12 mm/day at 50%, while P. sapidus AMRL156-1 ranged from 2.86 to 3.79 mm/day over the same concentration range, demonstrating a higher tolerance. P. sajor-caju also performed well, with Kr values remaining above 4.0 mm/day across all concentrations, peaking at 6.45 mm/day at 5%. In addition, F. velutipes exhibited an initial decline in growth from 6.6 mm/day at 0% OMW to 3.52 mm/day at 25%, followed by a partial recovery at higher concentrations, reaching 4.87 mm/day at 40% OMW and maintaining 4.02 mm/day at 50%, suggesting moderate tolerance and potential adaptation to OMW-induced stress. Notably, G. adspersum demonstrated remarkable adaptability, achieving a peak growth rate of 14.57 mm/day at 10% OMW and sustaining rates above 10 mm/day even at 50%, highlighting its strong potential for bioremediation. Similarly, T. aestivum showed an increasing growth at higher OMW concentrations, from 6.21 at 5% OMW to 8.95 mm/day at 50%. T. mesentericum maintained high growth rates across all OMW concentrations, starting at 10.31 mm/day with no OMW (0% OMW) and still reaching 8.32 mm/day at the highest concentration tested (50% OMW), indicating a strong ability to tolerate OMW-related stress.
These findings are consistent with previous studies, emphasizing the importance of strain-level screening for biological treatment of agro-industrial waste. For example, Zervakis et al. [38] reported that fungal growth on raw two-phase olive mill waste (TPOMW) varied widely, with some species showing minimal or no growth (as low as 1.22 mm/day), while others, such as P. pulmonarius and P. ostreatus, achieved growth rates of 10.02–10.64 mm/day and 8.93–10.67 mm/day, respectively, on composted TPOMW (20:80 mixtures). Similarly, Lakhtar et al. [39] found out that L. edodes could grow on 10–20% OMW media, but growth was completely inhibited at 40%, suggesting sensitivity to phenolic compounds in OMW. Bevilacqua et al. [40] and Cibelli et al. [41] used a logistic model to assess fungal growth, calculating Δτ (the time to reach half of the maximum colony diameter) instead of direct radial growth rates. In those studies, Δτ values ranged from 3 to 5 days, offering a different but complementary measure of fungal response to OMW. These data further support the potential use of specific fungi, such as G. adspersum and Tuber species, in sustainable and effective OMW management strategies.

3.2. Growth Kinetics of Fungal Strains in Different OMW Concentrations

Although OMW is characterized by high toxicity due to elevated polyphenolic content [42], several studies have shown that various fungal strains can not only tolerate but also grow effectively in OMW-enriched media. The biomass production and substrate consumption profiles of L. edodes, P. sapidus, P. sajor-caju, F. velutipes, G adspersum, T. aestivum and T. mesentericum strains, cultivated under three OMW concentrations (0, 15 and 35%) are illustrated in Figure 1a–c, where increased biomass with decreasing glucose concentrations were observed, indicating effective carbon source assimilation. Under control conditions, T. aestivum and T. mesentericum produced the highest biomass amount, reaching approximately 16.5 and 17.5 g/L, respectively, on day 20, with glucose concentration in the substrates dropping from ~30 to less than 5 g/L. These two strains also maintained high metabolic activity at 15 and 35% OMW, suggesting good tolerance to phenolic compounds, as biomass levels remained above 15 g/L even at the highest OMW concentration. These data, concerning the ability of two Tuber strains to produce biomass on synthetic media and liquid wastes are very important for the maintenance and conservation of the wild-type mushrooms, since their cultivation on solid-state fermentations for carposome production is still not that feasible. G. adspersum and F. velutipes also performed well, particularly in 0 and 15% OMW, with biomass values being around 13–14 g/L with significant glucose reduction. In contrast, L. edodes and P. sajor-caju produced lower biomass (6–10 g/L) and showed slower substrate consumption, especially under 35% OMW, indicating greater sensitivity to phenolic presence. Among the different substrate concentrations tested, L. edodes showed a remarkable increase in biomass production at 15% OMW, whereas its growth was significantly lower under both control conditions (0% OMW) and at the highest concentration of OMW (35%). Similarly, 15% OMW concentration appeared to provide an optimal balance for P. sapidus, P. sajor-caju, and F. velutipes, supporting better biomass compared to control and higher OMW amount. This suggests that moderate levels of phenolic compounds and associated nutrients create a conducive environment for fungal growth, while excessive phenolics at 35% may impose stress that limits metabolism and biomass production. Such findings align with previous studies indicating that sublethal stress can enhance secondary metabolism and growth in fungi [43].
These findings are consistent with previous reports of lower biomass production under static conditions. For instance, other studies have confirmed the ability of certain fungi to thrive in phenolic-rich environments. For example, G. resinaceum showed enhanced biomass accumulation (14.46 g/L) in medium containing 0.8 g/L (25% v/v) of phenolic compounds [7], G. applanatum grew efficiently in 20% OMW [44] and G. carnosum demonstrated strong growth in 25% OMW [45]. Remarkably, even L. edodes, typically considered sensitive, displayed stimulated growth in the presence of phenolic compounds [39]. Agitated cultures significantly enhanced biomass yields in several strains according to Diamantopoulou et al. [46], F. velutipes reached up to 20 g/L under agitation and G. applanatum produced between 12 and 15 g/L. Comparatively, earlier studies reported lower values under similar conditions: ~7 g/L for G. applanatum in glucose-yeast extract media [47], 8.3 g/L for F. velutipes in shake flasks [48] and 11.6 g/L for A. aegerita in stirred-tank bioreactors, which increased to 25 g/L in shake-flask cultures [49]. Overall, these results highlight the superior growth capacity and substrate utilization efficiency of Tuber spp. and G. adspersum, making them promising candidates for OMW bioconversion processes. Moreover, since all fungal strains tested in the present investigation are edible and/or pharmaceutical, fungal mycelia could be directly used for human nutrition purposes [50].

3.3. Fungal Efficiency in Phenolics Compounds Removal and Decolorization

The tested fungal strains demonstrated a strong capability to effectively degrade phenolic compounds and achieve partial decolorization of OMW under liquid fermentation conditions, even at high initial phenolic concentrations. As shown in Figure 2a,b, the results demonstrate a consistent pattern of rapid phenolic removal (>60%) during the middle of each fermentation by all fungal strains, regardless of the initial phenolic load. More specifically, at the end of fermentation, L. edodes, P. sapidus, P. sajor-caju, G. adspersum, T. mesentericum and T. aestivum strains achieved over 80% phenolic removal in both concentrations of OMW (15 and 35% v/v). Increasing the initial phenolic concentration from 1.5 to 3.5 g/L did not affect phenol removal efficiency, suggesting that the fungal enzymatic systems maintained their activity under increased OMW concentrations. The ability of the fungal strains to reduce the phenolic compound concentrations of OMW may be attributed to their production of ligninolytic enzymes. This notable efficiency is largely due to the high solubility and reactivity of low-molecular-weight phenolic compounds, which are readily degraded by extracellular oxidative enzymes such as laccases and peroxidases. In contrast, decolorization proceeded more gradually and exhibited clear strain specificity. Notably, L. edodes, P. sapidus, G. adspersum, T. mesentericum and F. velutipes strains consistently achieved higher decolorization efficiencies (>74%), indicating a potentially broader oxidative enzyme system or enhanced metabolic adaptability. Such results align with earlier findings, including a phenolic reduction of 86.4% and decolorization of 79.1% by Pleurotus citrinopileatus LGAM 28,684 after 22 days of fermentation in 25% (v/v) OMW [51] and reductions of 74–81% and 64–67% in phenolic content by Pleurotus and Ganoderma spp., respectively, reported by Ntougias et al. [52]. Furthermore, the study by Diamantopoulou et al. [7] demonstrated the exceptional detoxification capacity of Ganoderma resinaceum, achieving a 94.5% reduction in phenolic compounds and a 76.5% decolorization efficiency under static cultivation in glucose-enriched OMW media. In addition, L. edodes has shown strong potential for phenolic degradation (88%), while concurrently reducing phytotoxicity and improving the agricultural reuse potential of treated effluents [53]. Pleurotus spp., particularly P. sajor-caju and P. pulmonarius, have previously shown dual efficacy in phenolics and decolorization removal. P. sajor-caju was reported to effectively degrade phenolic and non-phenolic constituents [54], while P. pulmonarius achieved up to 95% phenolic reduction and 85% decolorization, particularly under carbon-rich conditions that enhance ligninolytic enzyme production [6]. Ganoderma applanatum, known for its strong oxidative enzyme system, has also shown substantial potential in degrading phenolic compounds (94%) and color removal (73%) in OMW [44].
The difference in the degradation kinetics of phenolic compounds and chromophoric structures highlights the variable biodegradability of distinct pollutant fractions within OMW. However, a moderate decline in decolorization efficiency was observed at higher phenolic concentrations, potentially resulting from substrate inhibition, elevated phenolic toxicity, or partial deactivation of oxidative enzymes under elevated chemical stress. These findings highlight the complex nature of OMW bioprocessing; while phenolic compounds can be detoxified relatively quickly, the removal of chromophoric substances often requires longer treatment durations or the integration of additional technologies, such as advanced oxidation processes, enzymatic pretreatment, or adsorption-based methods. Biological treatment via submerged fungal fermentation has gained significant attention due to its environmental sustainability and efficacy in transforming phenol-rich effluents. White-rot and ligninolytic fungi, such as Pleurotus spp., L. edodes and Ganoderma spp., possess enzymatic systems capable of depolymerizing and mineralizing aromatic xenobiotics. Emerging fungal species like T. aestivum and T. mesentericum are still relatively unexplored in this application, but their ecological roles and enzymatic characteristics indicate promising potential, pending further experimental validation under submerged fermentation conditions.

3.4. Effect of OMW Concentration on Polysaccharide Production and Lipid Content of Fungi

The late stages of fermentation, corresponding to days 20 to 44 (depending on the fungal strain), showed considerable differences in IPS accumulation and lipid synthesis among the examined strains under the three different OMW concentrations (0, 15 and 35% v/v) (Table 3). IPS, a key indicator of fungal biomass and metabolic activity, generally peaked in the later cultivation days (days 20–44), particularly under high OMW concentration. The observed increase in IPS content during fermentation followed the progression of fungal biomass accumulation, indicating a positive correlation between cell growth and IPS biosynthesis.
T. mesentericum showed the highest IPS yield among all strains, reaching 45.53% w/w under control conditions (0% OMW) on day 28, whereas even at 15 and 35% OMW, IPS levels remained comparably high, with values of 42.55 and 43.92% w/w, respectively. Similarly, T. aestivum recorded IPS concentrations of 40–44% (w/w) on day 28, indicating a high tolerance to phenolic content and consistent polysaccharide biosynthesis. G. adspersum also demonstrated strong IPS production on day 33, with 46.23% w/w at 0% OMW, 42.81% w/w at 15% OMW and 39.89% w/w at 35% OMW. These values suggest that days 20 to 28 represent the optimal fermentation time for maximizing IPS production in these high-performing strains. The other strains, including P. sapidus, P. sajor-caju and F. velutipes, exhibited more moderate IPS production, with values generally ranging from 29.9 to 42.21% w/w, depending on the strain and OMW concentration. Compared to other reports, the IPS amount achieved by the tested fungal strains, although promising, was comparatively lower than values reported in several previous studies. For example, F. velutipes in the work of Diamantopoulou et al. [55] produced IPS levels reaching 51.4% w/w of dry biomass—higher than the corresponding values observed in this study. Likewise, P. ostreatus has been reported to yield exceptionally high polysaccharide content, ranging from 46.6 to 81.8% (w/w), as shown by Miles & Chang [56]. Furthermore, Tuber sinense cultures reported by Tang et al. [57] yielded an IPS content of 11.39% w/w, underscoring the high biosynthetic capacity of certain ectomycorrhizal fungi under optimized liquid culture conditions. Such discrepancies may be attributed to species- and strain-specific capabilities, as well as variations in culture conditions, including medium composition and carbon source availability. As highlighted in previous research [7,36,55], IPS accumulation often follows a distinct time profile, with maximum yields recorded within the first two weeks, followed by a decline, likely due to polysaccharide degradation or nutrient depletion. This trend was also observed in our experiments, supporting the hypothesis that extended cultivation may lead to IPS reduction after the 12th day, as documented for species like V. volvacea, F. velutipes, Morchella esculenta and G. resinaceum. Despite not reaching the highest yields, these fungi demonstrated consistent performance under the examined conditions, suggesting their potential use in bioprocesses that promote consistent over high polysaccharide production.
In contrast, lipid accumulation generally declined over time in most fungal strains, particularly under higher OMW concentrations (Table 3), indicating probable consumption of lipids as an energy source, especially under prolonged exposure to stress factors like the high phenolic concentrations. Several fungal species are known to metabolize intracellular lipids via β-oxidation when external carbon sources become limited or when cells need to maintain redox balance under stress [58,59]. However, certain species demonstrate notable resilience, maintaining elevated lipid levels despite the increased phenolic content. Lipid synthesis in fungi plays a fundamental role in the formation of cellular membranes and, in some species, in the production of extracellular compounds [60,61]. Total lipid content in the mycelium of fungal species has been reported to vary considerably, typically ranging from 0.6% to 22.54% (w/w) on a dry weight basis [55,56,62]. For example, F. velutipes displayed the highest lipid content on day 20 with 16.68% (w/w) under 0% OMW, followed by 14.87% (35% OMW) and 10.49% (15% OMW), suggesting a potential for lipid accumulation even under phenolic presence. Likewise, T. mesentericum 365 maintained high lipid levels on day 20, ranging from 15.63% (35% OMW) to 15.88% (0% OMW) and still retained 13.6% on day 20 under 35% OMW. G. adspersum AMRL 315 also exhibited relatively high lipid levels during the early stage of fermentation, particularly under elevated OMW concentrations (e.g., 12.70% on day 20 at 35% OMW), although a decline was observed by day 28. Contrarily, L. edodes, P. sapidus and P. sajor-caju showed a clear decrease in lipid content over time and with higher OMW concentrations, indicating lower tolerance to the stress conditions. The observed lipid levels suggest that certain mushroom strains—namely F. velutipes, T. aestivum and T. mesentericum—responded with oleaginous-like behavior under the tested conditions. Although most edible and pharmaceutical fungi are not classified as oleaginous microorganisms, several studies have reported appreciable lipid accumulation in their mycelial mass under specific culture conditions [63,64]. For instance, Kavishree et al. [65] reported relatively low fat content in P. djamor and P. sajor-caju, at 0.5 and 0.8% w/w dry weight, respectively. In contrast, significantly higher lipid contents (exceeding 25% w/w) have been reported for fungi such as F. velutipes and L. edodes in static liquid cultures [46]. These values are notable, considering that commonly studied genera like P. ostreatus, P. pulmonarius, P. sajor-caju and Phellinus spp. are generally not considered oleaginous, with typical lipid levels below 20% w/w [66,67,68]. Also, Diamantopoulou et al. [55] reported that A. aegerita and P. pulmonarius achieved a lipid content of 20% (w/w) by day 8 in static cultures, while similar or even higher values were observed under agitated conditions, further supporting the potential of these fungi to accumulate lipids at levels comparable to those of oleaginous species under favorable conditions. These results point to the potential of these fungi for lipid-focused biotechnological applications and highlight the need for further studies on lipid metabolism in basidiomycetes.

4. Conclusions

This study demonstrated the remarkable potential of diverse fungal strains in the bioremediation of OMW, highlighting their adaptability. Among the tested species, strains of G. adspersum, T. aestivum and T. mesentericum exhibited superior growth and metabolic activity, even under elevated OMW concentrations, suggesting strong tolerance to phenolic stress and suitability for industrial applications. All strains showed the ability to significantly reduce phenolic content (achieving over 80% reduction in most cases), and many also demonstrated noteworthy decolorization capabilities. Moreover, the examined fungi demonstrated valuable secondary metabolite profiles, particularly in the production of IPS and lipids. Notably, T. mesentericum, T. aestivum and G. adspersum achieved IPS levels exceeding 40 g/L and maintained stable production even in OMW-added media. Some strains, like T. mesentericum, T. mesentericum and F. velutipes, also exhibited oleaginous-like behavior, accumulating lipids above 15% (w/w), suggesting potential for integrated bioprocessing approaches. Overall, the findings support the dual role of these fungal species in environmental detoxification and the generation of high-value fungal biomass and metabolites. The integration of selected fungal strains into OMW management systems offers a sustainable, low-cost alternative to conventional treatments, aligning with principles of the circular bioeconomy and agro-industrial waste valorization. Future studies should explore scale-up fermentation, enzyme profiling and co-cultivation strategies in order to improve both the efficiency of pollutant degradation and the production of valuable bioproducts.

Author Contributions

I.D.: formal analysis, methodology, data curation, writing of original draft and editing; S.S.: formal analysis, methodology, data curation; E.-M.M.: methodology, writing of original draft; S.P.: funding acquisition, supervision; P.D.: conceptualization, supervision, writing—review. All authors have read and agreed to the published version of the manuscript.

Funding

The research work was supported by the Hellenic Foundation for Research and Innovation (HFRI) under the 4th Call for HFRI PhD Fellowships (Fellowship Number: 11399).

Data Availability Statement

All data presented in this paper are original for this study. The data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest

The authors declare no conflicts of interest.

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Biomass 05 00050 g001
Figure 1. Kinetics of substrate consumption (glucose remaining, g/L) and biomass production (g/L) of the medium during the cultivation of L. edodes, P. sapidus, P. sajor-caju, F. velutipes, G adspersum, T. aestivum and T. mesentericum strains in liquid cultures with (a) OMW 15% v/v, (b) OMW 35% v/v and (c) OMW 0% v/v (control). Each point is the mean value of at least three independent measurements, SD < 5%.
Figure 1. Kinetics of substrate consumption (glucose remaining, g/L) and biomass production (g/L) of the medium during the cultivation of L. edodes, P. sapidus, P. sajor-caju, F. velutipes, G adspersum, T. aestivum and T. mesentericum strains in liquid cultures with (a) OMW 15% v/v, (b) OMW 35% v/v and (c) OMW 0% v/v (control). Each point is the mean value of at least three independent measurements, SD < 5%.
Biomass 05 00050 g001
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Figure 2. Kinetics of % phenol reduction and % decolorization of the medium during the cultivation of L. edodes, P. sapidus, P. sajor-caju, F. velutipes, G adspersum, T. aestivum and T. mesentericum strains in liquid cultures with (a) with OMW 15% v/v and (b) OMW 35% v/v. Each point is the mean value of at least three independent measurements, SD < 5%.
Figure 2. Kinetics of % phenol reduction and % decolorization of the medium during the cultivation of L. edodes, P. sapidus, P. sajor-caju, F. velutipes, G adspersum, T. aestivum and T. mesentericum strains in liquid cultures with (a) with OMW 15% v/v and (b) OMW 35% v/v. Each point is the mean value of at least three independent measurements, SD < 5%.
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Table 1. Edible and/or medicinal mushroom strains tested in the present study.
Table 1. Edible and/or medicinal mushroom strains tested in the present study.
StrainAMRL
Agrocybe cylindracea101
A. cylindracea104
Lentinula edodes125
L. edodes126
Pleurotus sapidus156-1
P. sapidus156-2
Pleurotus sajor-caju197
Flammulina velutipes271
Ganoderma adspersum315
Tuber aestivum364
Tuber mesentericum365
Table 2. Linear growth rate (Kr, mm/day) of A. cylindracea, L. edodes, P. sapidus, P. sajor-caju, F. velutipes, G adspersum, T. aestivum and T. mesentericum strains on solid nutrient medium supplemented with OMW (0–50% v/v) at 25 (±1) °C.
Table 2. Linear growth rate (Kr, mm/day) of A. cylindracea, L. edodes, P. sapidus, P. sajor-caju, F. velutipes, G adspersum, T. aestivum and T. mesentericum strains on solid nutrient medium supplemented with OMW (0–50% v/v) at 25 (±1) °C.
OMW (% v/v)/
Species (Strains)		05101520253035404550
A. cylindracea
AMRL 101		3.20 ± 0.133.32 ± 0.053.11 ± 0.182.87 ± 0.10 2.54 ± 0.312.69 ± 0.052.86 ± 0.153.12 ± 0.042.63 ± 0.152.33 ± 0.162.05 ± 0.11
A. cylindracea
AMRL 104		3.11 ± 0.363.40 ± 0.033.57 ± 0.253.48 ± 0.113.18 ± 0.072.81 ± 0.092.98 ± 0.033.00 ± 0.042.67 ± 0.062.30 ± 0.032.27 ± 0.05
L. edodes
AMRL 125		3.63 ± 0.134.76 ± 0.113.83 ± 0.224.04 ± 0.684.59 ± 0.322.02 ± 0.143.33 ± 0.293.59 ± 0.092.80 ± 0.511.80 ± 0.031.55 ± 0.04
L. edodes
AMRL 126		3.84 ± 0.214.26 ± 0.275.22 ± 0.274.79 ± 0.054.59 ± 0.302.90 ± 0.323.52 ± 0.333.68 ± 0.043.17 ± 0.372.21 ± 0.102.31 ± 0.33
P. sapidus
AMRL 156-1		2.86 ± 0.204.96 ± 0.024.89 ± 0.144.90 ± 0.064.40 ± 0.553.83 ± 0.043.87 ± 0.033.89 ± 0.033.75 ± 0.063.76 ± 0.213.79 ± 0.13
P. sapidus
AMRL 156-2		4.58 ± 0.254.49 ± 0.034.42 ± 0.064.47 ± 0.054.72 ± 0.013.83 ± 0.073.84 ± 0.123.90 ± 0.073.88 ± 0.034.88 ± 0.245.12 ± 0.23
P. sajor-caju
AMRL 197		4.05 ± 1.226.45 ± 0.306.36 ± 0.156.10 ± 0.036.28 ± 0.075.06 ± 0.126.31 ± 0.935.45 ± 0.085.08 ± 0.084.26 ± 0.084.25 ± 0.10
F. velutipes
AMRL 271		6.60 ± 0.076.28 ± 0.466.44 ± 0.036.18 ± 0.485.86 ± 0.483.52 ± 0.104.73 ± 0.044.72 ± 0.074.87 ± 0.524.07 ± 0.064.02 ± 0.06
G. adspersum
AMRL 315		12.05 ± 0.0712.51 ± 0.0714.57 ± 0.4612.22 ± 0.0312.15 ± 0.4811.94 ± 0.4810.61 ± 0.1012.51 ± 0.0413.25 ± 0.0710.13 ± 0.5211.79 ± 0.06
T. aestivum
AMRL 364		7.39 ± 0.076.21 ± 0.076.24 ± 0.466.38 ± 0.035.97 ± 0.487.00 ± 0.486.98 ± 0.106.66 ± 0.047.24 ± 0.078.39 ± 0.528.95 ± 0.06
T. mesentericum
AMRL 365		10.31 ± 0.079.10 ± 0.078.16 ± 0.469.07 ± 0.0310.25± 0.489.41 ± 0.489.21 ± 0.109.30± 0.048.68 ± 0.078.88 ± 0.528.32 ± 0.06
Table 3. Production of total intracellular polysaccharides (IPS) and total lipids (L) (expressed as % w/w of dry biomass) on selected fermentation days by L. edodes, P. sapidus, P. sajor-caju, F. velutipes, G adspersum, T. aestivum and T. mesentericum strains. Representation of IPS and L (in g/L and w/w) at the following different fermentation points: (a) when the maximum IPS amount (g/L) was synthesized; (b) when the maximum IPS in dry weight (%, w/w) value was achieved and (c) when the maximum L in dry weight (%, w/w) value was recorded. Culture conditions: in static cultures supplemented with OMW (0, 15, 35%, v/v), growth on 100-cc flasks, initial pH = 5.6 ± 0.3, pH ranging between 4.0 and 5.7, incubation temperature T = 25 ± 1 °C. Each point represents the mean value of three independent measurements.
Table 3. Production of total intracellular polysaccharides (IPS) and total lipids (L) (expressed as % w/w of dry biomass) on selected fermentation days by L. edodes, P. sapidus, P. sajor-caju, F. velutipes, G adspersum, T. aestivum and T. mesentericum strains. Representation of IPS and L (in g/L and w/w) at the following different fermentation points: (a) when the maximum IPS amount (g/L) was synthesized; (b) when the maximum IPS in dry weight (%, w/w) value was achieved and (c) when the maximum L in dry weight (%, w/w) value was recorded. Culture conditions: in static cultures supplemented with OMW (0, 15, 35%, v/v), growth on 100-cc flasks, initial pH = 5.6 ± 0.3, pH ranging between 4.0 and 5.7, incubation temperature T = 25 ± 1 °C. Each point represents the mean value of three independent measurements.
StainsDays0% v/v OMW15% v/v OMW35% v/v OMW
LIPSLIPSLIPS
(% w/w)g/L(% w/w)g/L(% w/w)g/L(% w/w)g/L(% w/w)g/L(% w/w)g/L
L. edodes33c13.35 ± 0.221.18 ± 0.0427.58 ± 0.272.44 ± 0.036.74 ±
0.27		1.00 ± 0.1736.69 ± 0.505.46 ± 0.075.26 ± 0.240.41 ± 0.0432.49 ± 0.082.49 ± 0.08
AMRL 12644a, b7.11 ± 0.340.74 ± 0.0639.45 ± 0.164.13 ± 0.026.79 ±
0.31		1.02 ± 0.0822.99 ± 0.523.46 ± 0.084.23 ± 0.260.71 ± 0.0534.52 ± 0.034.52 ± 0.03
P. sapidus20c9.72 ± 0.160.48 ± 0.0237.82 ± 0.191.87 ± 0.018.19 ±
0.08		0.57 ± 0.1229.90 ± 0.482.09 ± 0.037.92 ± 0.080.19 ± 0.0131.06 ± 0.011.06 ± 0.01
AMRL 156-233a, b7.68 ± 0.090.40 ± 0.0338.10 ± 0.482.02 ± 0.033.12 ±
0.03		0.35 ± 0.0636.08 ± 0.104.00 ± 0.013.60 ± 0.020.48 ± 0.0533.92 ± 0.103.92 ± 0.10
P. sajor-caju26c13.61 ± 0.340.33 ± 0.0525.48 ± 0.010.61 ± 0.008.83 ±
0.07		0.51 ± 0.1436.05 ± 2.662.08 ± 0.155.60 ± 0.190.27 ± 0.0431.26 ± 0.011.26 ± 0.01
AMRL 19744a, b7.16 ± 0.290.45 ± 0.0139.36 ± 0.322.50 ± 0.027.13 ±
0.06		0.45 ± 0.1242.21 ± 0.452.66 ± 0.036.73 ± 0.120.65 ± 0.0833.42 ± 0.043.42 ± 0.04
F. velutipes20c16.68 ± 0.260.59 ± 0.0433.64 ± 0.751.19 ± 0.0310.49 ± 0.311.23 ± 0.0727.43 ± 0.353.22 ± 0.0414.87 ± 0.360.66 ± 0.0331.18 ± 0.061.18 ± 0.06
AMRL 27133a, b7.65 ± 0.130.31 ± 0.0244.13 ± 0.161.81 ± 0.106.90 ±
0.19		0.38 ± 0.0232.51 ± 1.091.78 ± 0.063.16 ± 0.110.39 ± 0.0133.92 ± 0.003.92 ± 0.00
G. adspersum20c10.68 ± 0.230.61 ± 0.0534.74 ± 0.682.00 ± 0.0311.69 ± 0.280.92 ± 0.0137.13 ± 0.832.91 ± 0.0912.70 ± 0.160.86 ± 0.0232.58 ± 0.932.21 ± 0.09
AMRL 31533a, b8.52 ± 0.201.07 ± 0.0646.23 ± 0.155.81 ± 0.109.90 ±
0.15		1.41 ± 0.0442.81 ± 0.856.09 ± 0.2011.62 ± 0.101.75 ± 0.0339.89 ± 0.916.00 ± 0.11
T. aestivum20c14.28 ± 0.161.60 ± 0.0832.88 ± 0.653.69 ± 0.0912.42 ± 0.331.47 ± 0.0331.42 ± 0.873.72 ± 0.0714.22 ± 0.331.71 ± 0.0731.83 ± 0.883.82 ± 0.21
AMRL 36428a, b9.85 ± 0.311.52 ± 0.0643.85 ± 0.206.78 ± 0.3010.93 ± 0.181.62 ± 0.0743.51 ± 1.206.46 ± 0.1113.26 ± 0.221.99 ± 0.0439.87 ± 0.925.98 ± 0.31
T. mesentericum20c15.88 ± 0.301.90 ± 0.0734.42 ± 0.584.14 ± 0.1413.99 ± 0.291.68 ± 0.0532.52 ± 0.833.90 ± 0.0915.63 ± 0.281.96 ± 0.0341.82 ± 0.895.26 ± 0.28
AMRL 36528a, b12.65 ± 0.122.01 ± 0.0745.53 ± 0.177.23 ± 0.3011.83 ± 0.161.94 ± 0.0842.55 ± 1.036.98 ± 0.2913.60 ± 0.212.25 ± 0.0543.92 ± 0.837.27 ± 0.22
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Diamantis, I.; Stamatiadis, S.; Melanouri, E.-M.; Papanikolaou, S.; Diamantopoulou, P. From Screening to Laboratory Scale-Up: Bioremediation Potential of Mushroom Strains Grown on Olive Mill Wastewater. Biomass 2025, 5, 50. https://doi.org/10.3390/biomass5030050

AMA Style

Diamantis I, Stamatiadis S, Melanouri E-M, Papanikolaou S, Diamantopoulou P. From Screening to Laboratory Scale-Up: Bioremediation Potential of Mushroom Strains Grown on Olive Mill Wastewater. Biomass. 2025; 5(3):50. https://doi.org/10.3390/biomass5030050

Chicago/Turabian Style

Diamantis, Ilias, Spyridon Stamatiadis, Eirini-Maria Melanouri, Seraphim Papanikolaou, and Panagiota Diamantopoulou. 2025. "From Screening to Laboratory Scale-Up: Bioremediation Potential of Mushroom Strains Grown on Olive Mill Wastewater" Biomass 5, no. 3: 50. https://doi.org/10.3390/biomass5030050

APA Style

Diamantis, I., Stamatiadis, S., Melanouri, E.-M., Papanikolaou, S., & Diamantopoulou, P. (2025). From Screening to Laboratory Scale-Up: Bioremediation Potential of Mushroom Strains Grown on Olive Mill Wastewater. Biomass, 5(3), 50. https://doi.org/10.3390/biomass5030050

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