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Article

Preliminary Screening of Non-Conventional Yeasts for Olive Mill Wastewater Valorization

by
Gabriella Siesto
1,2,
Rocchina Pietrafesa
1,*,
Antonio Caporusso
3,
Giorgia La Rocca
1,
Grazia Alberico
1,
Vito Valerio
3 and
Angela Capece
1,2
1
Department of Agricultural, Forest, Food and Environmental Sciences, University of Basilicata, Via dell’Ateneo Lucano 10, 85100 Potenza, Italy
2
Spin-off StarFInn S.r.l.s., Department of Agricultural, Forest, Food and Environmental Sciences, University of Basilicata, Via dell’Ateneo Lucano 10, 85100 Potenza, Italy
3
ENEA C.R. Trisaia, Laboratory for Processes and Technologies for Biorefineries and Green Chemistry, Italian National Agency for New Technologies, Energy and Sustainable Economic Development (ENEA), S.S. 106 Jonica, 75026 Rotondella, Italy
*
Author to whom correspondence should be addressed.
Fermentation 2026, 12(4), 188; https://doi.org/10.3390/fermentation12040188
Submission received: 28 February 2026 / Revised: 1 April 2026 / Accepted: 5 April 2026 / Published: 8 April 2026
(This article belongs to the Special Issue Biotechnological Strategies for Agro-Industrial Food Waste Management)
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Abstract

Olive mill wastewater (OMWW) is a highly polluting agro-industrial effluent characterized by elevated organic load, low pH, and high concentrations of phenolic compounds responsible for its phytotoxicity and dark coloration. In this study, 41 non-conventional yeast strains belonging to the University of Basilicata Yeast Collection (UBYC), were tested for both the oleaginous potential traits and OMWW detoxification capacity in comparison to two commercial oleaginous controls, Yarrowia lipolytica ATCC 46483 and Lipomyces tetrasporus Li-0407. Primary screening in synthetic medium under nitrogen-limited conditions revealed widespread intracellular lipid accumulation. Quantitative analysis showed lipid contents above 20% (w/w) in some strains, with Candida tropicalis AII122 (33.3%) and Pichia manshurica ML-3 (29.4%) exhibiting the highest values in synthetic medium. The cultivation of eight selected strains in synthetic medium supplemented with 15% (v/v) of OMWW reduced intracellular lipid accumulation, with the highest value of 6.48% for the 2R1 strain. Levels of phenol reduction and color removal were highly different among all the analyzed strains, and C. tropicalis AII122 achieved the highest phenolic reduction and decolorization ability. These findings demonstrate that indigenous non-conventional yeasts represent a source of natural biodiversity, supporting sustainable waste valorization strategies based on the use of selected microorganisms within a circular bioeconomy framework.

1. Introduction

Olive mill wastewater (OMWW) is widely recognized as one of the most problematic agro-industrial effluents produced by the olive oil industry, particularly in Mediterranean countries where olive cultivation plays a major economic and cultural role [1]. Large volumes of OMWW are generated annually (between 7 and over 30 million m3) during olive oil extraction, creating significant environmental management challenges due to their seasonal production and high pollutant load [2,3,4]. OMWW is characterized by elevated chemical oxygen demand (COD), high organic content, low pH (typically 3–6), dark coloration, and significant concentrations of phenolic compounds [5]. These phenolics are primarily responsible for the effluent’s phytotoxicity, antimicrobial activity, and resistance to biodegradation, which contribute to its persistence in soils and aquatic environments [4,6,7]. Among the major phenolic compounds identified in OMWW, hydroxytyrosol, tyrosol, oleuropein derivatives, and other polyphenols are the main compounds that contribute both to the effluent’s toxicity and its coloration [8]. They inhibit microbial growth, interfere with enzymatic activities and reduce the efficiency of traditional wastewater treatment processes. Therefore, the reduction in total phenolic content and decolorization of OMWW are widely regarded as key indicators of effective detoxification, reduced environmental pollution and improved remediation [9,10]. Biological treatment strategies, based on the use of microorganisms, have gained attention as environmentally sustainable alternatives to physicochemical approaches [11,12]. Within this context, yeasts are promising candidates for OMWW detoxification due to their metabolic versatility, physiological robustness, and tolerance to inhibitory compounds. Several yeast species have been shown to grow in OMWW-based media, reduce phenolic concentrations, and decrease color intensity, mitigating the effluent’s inhibitory effects [13,14]. Beyond their role in bioremediation, many yeast species, particularly oleaginous yeasts, can accumulate intracellular lipids, commonly referred to as single-cell oils (SCOs), under conditions of carbon excess and nitrogen limitation. These lipids are mainly stored as triacylglycerols and display fatty acid profiles comparable to those of vegetable oils, making them attractive for food, feed, and biotechnological applications [15,16,17]. Integrating OMWW detoxification with microbial lipid production represents a challenging strategy within a circular bioeconomy framework, allowing for the transformation of a problematic waste stream into value-added products [15].
Among oleaginous yeasts, Yarrowia lipolytica is one of the most extensively studied species due to its high metabolic flexibility, capacity to utilize diverse carbon sources, and production of extracellular enzymes such as lipases. Its growth on OMWW contributes to reductions in organic load and phenolic content, highlighting its potential for combined bioremediation and biomass valorization [16,18]. Similarly, Lipomyces tetrasporus is widely used as a reference oleaginous yeast due to its high lipid accumulation potential [16]. However, focusing exclusively on well-characterized model species may limit the discovery of strains with enhanced or complementary traits. Non-conventional yeasts represent a largely untapped microbial resource. Several non-conventional yeasts, including species of Candida, Pichia, Metschnikowia, Hanseniaspora, Cutaneotrichosporon, and Debaryomyces, have demonstrated promising performance in lipid accumulation and OMWW valorization [15,19]. Despite growing interest in yeast-mediated OMWW treatment, systematic studies combining detoxification (phenol reduction and decolorization) with oleaginous potential screening, particularly focusing on indigenous non-conventional yeasts, remain limited. Addressing this gap is essential to identify multifunctional strains capable of coupling environmental remediation with the production of value-added microbial biomass.
In this context, the present study aimed to screen 41 non-conventional yeast strains for their ability to accumulate intracellular lipids and produce lipase enzyme, in comparison with two collection oleaginous yeasts (Yarrowia lipolytica ATCC 46483 and Lipomyces tetrasporus Li-0407). Eight selected strains were further evaluated for their capacity to detoxify OMWW through decolorization and total phenolic compound reduction while simultaneously accumulating microbial lipids. This integrated approach provides new insights into the potential use of non-conventional yeasts for sustainable OMWW management within a circular bioeconomy.

2. Materials and Methods

2.1. Yeast Strains

In this study, forty-one non-conventional yeasts belonging to the University of Basilicata Yeast Collection (UBYC) (Potenza, Italy) and two collection strains Yarrowia lipolytica ATCC 46483 (DSM 8218, DSMZ, Germany) and Lipomyces tetrasporus Li-0407 (DSM 70314, DSMZ, Germany), used as positive controls, were analyzed for their oleaginous properties (Table 1).
The yeast strains were maintained at −20 °C in Yeast Peptone Dextrose broth (YPD, 20 g/L Glucose, 20 g/L Peptone, 10 g/L Yeast Extract, Oxoid, Hampshire, UK) supplemented with 50% glycerol (Merk, Darmstad, Germany) as a protective agent until further analysis.

2.2. Primary Screening for Lipid Accumulation Capacity

To evaluate the intracellular lipids or single cell oils (SCOs) accumulation, the 41 strains were pre-cultured in 20 mL Yeast Malt Broth medium (YMB, yeast extract 3 g/L; malt extract 3 g/L; peptone 5 g/L and glucose 10 g/L, Oxoid, Hampshire, UK) and incubated at 28 °C for 48 h with shaking at 150 rpm.
For each strain, an amount of the pre-culture corresponding to about 1 × 107 cells/mL was transferred into 50 mL Glycerol Yeast Peptone medium (GYP, glycerol 4% (w/v) Merk, Darmstad, Germany; yeast extract 1% (w/v); peptone 1% (w/v) Oxoid, Hampshire, UK) and incubated at 28 °C under agitation (150 rpm). After 120 h, the yeast cells were collected by centrifugation at 4700× g for 10 min (Eppendorf Centrifuge 5920 R, Hamburg, Germany) and washed three times with NaCl (0.85% w/v, Merk, Darmstad, Germany) to remove the culture medium residues [20].
The harvested yeasts biomass fixed on the microscopic slide surface was stained with Sudan Black B solution (3 g/L) prepared in ethanol (70% v/v; Merk, Darmstad, Germany), and observed under the ECLIPSE 50i microscope (Nikon, Tokyo, Japan), using 100X objective with direct light to detect the presence of lipid droplets in the cells [21].

2.3. Evaluation of Lipase Activity

The strains were tested for their ability to produce lipase enzyme. To determine the production of lipase enzyme, a qualitative test was performed on Tributyrin Agar medium (TBA), containing 15 g/L of bacteriological agar, 3 g/L of yeast extract, 5 g/L of peptone (Oxoid, Hampshire, UK), and tributyrin 1% v/v (Merk, Darmstad, Germany), added to MgSO4 (5.0 mM, Merk, Darmstad, Germany), following the method of Carrazco-Palafox et al. [22].
An aliquot (40 µL) of a fresh 24 h yeast culture was spotted in wells (diameter 0.6 cm) on TBA, and the plates were incubated at 28 °C for 48 and 72 h. The presence of lipolytic activity was evaluated on the basis of the halo size (mm) developed around the inoculated wells.

2.4. Accumulation and Extraction of SCOs from Yeast Cells in Synthetic Medium

The strains were pre-cultured in 100 mL Erlenmeyer flasks, containing 20 mL of YMB medium and incubated in a shaker incubator at 150 rpm, 28 °C for 24 h. An aliquot of pre-inoculum (1 × 107 cell/mL) was transferred into 100 mL of a nutrient culture media containing glucose 20 g/L, peptone 0.22 g/L (Oxoid, Hampshire, UK), glycerol 40 g/L and trace elements, namely KH2PO4 0.55 g/L, KCl 0.425 g/L, CaCl2 0.125 g/L, MgSO4 0.125 g/L, FeCl3 0.0025 g/L, MnSO4 0.0025 g/L (Merk, Darmstad, Germany). According to previous work [23], 0.18 g/L of yeast extract was added in order to obtain the initial C/N ratio at 60 g/g. After 144 h of incubation (stationary phase) at 28 °C, the yeast biomass was harvested by centrifugation at 4700 rpm for 10 min, washed twice with NaCl solution (0.85% w/v, Merk, Darmstad, Germany), dried overnight at 105 °C and then determined gravimetrically.
To determine the intracellular lipid content, the extraction of lipids was performed following the protocol reported by Thancharoen et al. [21], with some modifications. Briefly, the dried pellet collected from 1 g of biomass was added to 10 mL HCl solution (4 M, Merk, Darmstad, Germany) and incubated at 60 °C for 2 h in order to disrupt the cell membranes. Then, 20 mL of hexane:methanol mixture (1:1 ratio, Merk, Darmstad, Germany) was supplemented in the hydrolyzed cell biomass and stirred at room temperature for 3 h. The upper organic phase containing lipids was collected using a syringe and evaporated under vacuum at 40 °C in a rotary evaporator until a constant weight was achieved. Total lipid content was then quantified gravimetrically. Recovery yields, verified using the certified reference material BCR 163 (beef–pork fat blend), were approximately 98%. Lipid content was expressed as the percentage of extracted lipids on the dry biomass.

2.5. Evaluation of Selected Strain Performance in Olive Mill Wastewater-Supplemented Medium

Eight yeast strains, selected on the basis of previous steps, were analyzed in terms of growth ability in YMB supplemented with pasteurized olive mill wastewater (OMWW) (heated to 90 °C for 10 min) at different concentrations (15%, 25%, 50%, v/v). At this aim, 5 mL of OMWW medium was inoculated with the cell suspension of each strain and incubated at 28 °C for 48 h. Cell growth was assessed by calculating the difference between the optical density value (at 600 nm) recorded at the beginning and at the end of the experiments [24].
On the basis of cell growth, 15% OMWW addition was selected for further growth and lipid accumulation experiments. After 144 h of incubation, the cell biomass was harvested by centrifugation and analyzed for lipids content according to the protocol previously described.
The obtained supernatant was used to evaluate the OMWW decolorization capacity of the selected strains and the total polyphenol content. To evaluate OMWW decolorization, the absorbance of the supernatant was measured at 395 nm using the spectrophotometer Spectrostarnano (BMG LABTECH, Ortenberg, Germany) following the method of Sayadi and Ellouz [9].
The determination of the total polyphenol content in the supernatant was carried out following the Folin–Ciocalteau method (FC) as reported by Lindner et al. [7], with some modifications.
Specifically, 0.2 mL of Folin–Ciocalteau reagent (Merk, Darmstad, Germany) and 1.8 mL of deionized water were added to 0.2 mL of supernatant previously centrifugated (4700× g for 5 min), stirred and kept in the dark for five minutes. Then, 2 mL of Na2CO3 (7%, Merk, Darmstad, Germany) and 0.8 mL of deionized water were added to the samples, vortexed and kept in the dark for 90 min.
Total polyphenol content was spectrophotometrically determined at ʎ = 765 nm in microplates, using the calibration curve prepared with gallic acid solutions at known concentrations.
The results were reported as the reduction percentage of phenolic compounds in comparison to the control sample:
%   Phenol   Reduction   =   ( T P c − T P s ) T P c × 100
where
TPc = Total polyphenol content in the control
TPs = Total polyphenol content in the sample.

2.6. Analytical Methods

Reducing sugars (glucose, fructose, xylose, arabinose, mannose) were determined using an HPIC DX 300 chromatographic system (Dionex, Sunnyvale, CA, USA) equipped with a Nucleogel Ion 300 OA column (Macherey-Nagel, Düren, Germany) operating at 60 °C with 10 mN H2SO4 solution as a mobile phase and a flux of 0.4 mL/min. The detector used was a Shodex RI101 refractive index detector (ECOM, Chrastany u Prahy, Czech Republic). Each analysis was performed in triplicate. All the quantifications were performed through the external standard technique.

2.7. Data Analysis

All the experiments were carried out in duplicate and the results were analyzed by Paleontological Statistics (PAST) software 4.09 [25]. One-Way Analysis of Variance (ANOVA), followed by Tukey’s HSD test, were performed, and differences with p values < 0.05 were considered statistically significant.

3. Results and Discussion

3.1. Chemical Composition of Olive Mill Wastewater

Olive mill wastewater (OMWW) exhibits a highly variable composition, influenced by several factors including the oil extraction process, the degree of fruit ripeness, and the olive cultivar. It is generally characterized by an acidic pH and contains substantial amounts of sugars, lipids, proteins, and phenolic compounds [26,27]. Because of this compositional variability, a comprehensive biochemical characterization of OMWW is essential prior to its use as a cultivation medium for the production of microbial metabolites (Table 2).
The values obtained in the present work are consistent with those of the reference literature [24,28].

3.2. Qualitative Screening of Yeast Strains for SCOs Accumulation

The SCOs accumulation ability of the forty-one strains was investigated using the Sudan Black B technique. Although this technique did not provide precise information on intracellular lipid content, it proved to be a useful preliminary screening method for assessing the ability of some microorganisms to accumulate lipid droplets as carbon storage. Microorganisms were cultivated under conditions of high carbon availability and nitrogen limitation, in accordance with the reference literature [29,30,31]. Results showed that many yeasts were able to accumulate lipids, although at different levels. The presence of blue-stained lipid cytoplasmic inclusions, observed by the microscope analysis (Figure 1A,B), was correlated to the ability of each strain to accumulate lipids.
In particular, on the basis of the presence of lipid droplets in the yeast cells, the tested strains were arbitrarily classified into two groups:
−
Group 1, characterized by lipid droplets inside some cells, which include the following strains: K (Y. lipolytica); AII176 (Cut. curvatus); 1RP9; 2-13; 4-2; 4-15; 4R11 (M. pulcherrima); 4-22 (P. kudriavzevii); and RB1 (P. nakasei).
−
Group 2, composed of strains containing oil droplets in all the cells. These strains were the following: 1P1, 1RP2, 1SPR1, 1SPR14, 2-1, 2-7, 2R1, 4R1, AII 37, AII 68, AII 136, AII 181, AII 49 (M. pulcherrima); M, C, A, RB3 (Y. lipolytica); PD 36-2 (C. zeylanoides); AII 122 (C. tropicalis; RB5, PD 12-1, PD 49-1 (D. hansenii); ND1 (H. osmophila); AII184 (L. kluyveri); AII 40 (Mon. pollinis); AII171 (Mey. caribbica); 2-21, 4-16, 2R-17 (P. kudriavzevii); AII110 (P. kluyveri); ML-3 (P. manshurica); and D (P. nakasei). As expected, the control strains (L. tetrasporus and Y. lipolytica) showed a high intracellular lipid accumulation ability. The strains included in group 2 can be considered as potential lipid producers.

3.3. Quantitative Production of SCOs

The Sudan Black technique provides partial information on the lipid accumulation capacity of the tested yeast strains [32] without detailed insight into the cellular lipid content of the yeasts. In order to confirm the results obtained by qualitative screening and select the potential oleaginous strains, lipid production was evaluated among the strains grown in nitrogen limited conditions (C/N = 60 g/g) to favor intracellular lipid accumulation over biomass production. Figure 2 summarizes the main results.
In particular, AII122 and ML-3, identified as Candida tropicalis and Pichia manshurica, respectively, exhibited the highest lipid contents (33.3% and 29.4% of CDW). These values fall in the range typically reported for oleaginous yeasts, which are conventionally defined as microorganisms able to accumulate more than 20% of intracellular lipids under appropriate culture conditions. Previous studies have shown that C. tropicalis is a versatile oleaginous yeast of great scientific and industrial interest. Dey P. and Maiti [33] demonstrated that C. tropicalis showed a maximum lipid production at C:N 150:1, at which the lipid yield was 58.6% with glucose as the carbon source and ammonium sulfate as the nitrogen source.
The literature data on Pichia manshurica are very limited, as this species is not traditionally classified among the model oleaginous yeasts. However, a recent work [34] indicates that selected strains, isolated from sugarcane bagasse, may accumulate lipids in the range of 25–40% under optimal conditions. In this context, the 29.4% lipid content observed for strain ML-3 suggests a promising metabolic capability to produce lipids under fine-tuned conditions.
Strains AII171 (Meyerozyma caribbica) and RB5 (Debaryomyces hansenii) also showed lipid contents of 25.0% and 22.2%, respectively.
Mey. caribbica has mainly been investigated for its biocontrol activity and fermentative versatility [35], while reports on its ability to accumulate lipid are comparatively limited. Therefore, the 25% lipid content observed in strain AII171 indicates a noteworthy storage capacity under the tested conditions.
Similarly, D. hansenii is widely recognized for its halotolerance, osmotolerance, and application in food fermentations [36] rather than for high lipid production. Although lipid accumulation in this species is usually reported at moderate levels, strain-dependent variability has been described, particularly under stress or nutrient-imbalanced conditions. The lipid content recorded for RB5 (22.2%) suggests that, under the cultivation parameters adopted in this study, this strain was able to redirect carbon flux toward storage lipid biosynthesis beyond its structural requirements.
Finally, the other tested strains, including 1SPR14, 1RP2, 1P1, 4R1, AII49, 2R1 and 1SPR1 (identified as Metschnikowia pulcherrima), accumulated lipids in a range between 6.25% and 19.05%. Although these values do not classify them as strictly oleaginous, moderate lipid accumulation has reported. M. pulcherrima is a species commonly recognized for its biocontrol activity [37], pigment production [38], oenological applications [39] and intracellular polysaccharides accumulation [40] rather than for high lipid yields. Therefore, its lipid biosynthetic capacity, even if limited compared to highly oleaginous yeasts, may represent an additional interesting trait for this species.
Moderate lipid accumulation was also recorded for strains M, A, D, AII110 and 2-21 (11.8–14.3%). Such values are consistent with non-oleaginous or weakly oleaginous yeasts, where lipid accumulation remains primarily associated with structural membrane synthesis. Finally, strains belonging to Hanseniaspora osmophila (ND1), Cutaneotrichosporon curvatus (AII176), and Pichia nakasei (RB1) exhibited lipid contents below 10%. While C. curvatus is recognized in the literature as a highly oleaginous species [41], the low lipid content observed in strain AII176 suggests that cultivation parameters, strain-specific variability, or nutrient balance likely limited storage lipid biosynthesis under the adopted experimental setup.
Overall, these findings highlight that lipid accumulation is not only species-dependent but also influenced by strain-level metabolic variability and culture conditions.

3.4. Determination of the Lipase Activity

To assimilate exogenous lipids, microorganisms must produce lipases that hydrolyze triglycerides into free fatty acids, which can then be taken up by the cells [42]. As OMWW contains variable quantities of olive oil, it can be used as a substrate by yeast for the production of lipolytic enzymes. This step is fundamental for ex novo lipid synthesis, a pathway in which yeasts accumulate lipids starting from hydrophobic substrates, such as oils and fats. Although the OMWW analyzed in this study did not contain lipids (Table 2), our aim was to submit different strains to a wide screening for different characteristics, potentially useful for OMWW valorization.
In contrast, during de novo lipid synthesis, yeasts utilize hydrophilic carbon sources, including polysaccharides and glycerol, which are metabolized through central carbon metabolism and the fatty acid biosynthetic pathway to generate lipid bodies.
Lipase activity was evaluated in TBA medium using the well diffusion assay. The formation of a clear zone (mm) around the inoculated wells indicated lipid hydrolysis. Analyses were performed after two different incubation times, 48 and 72 h. Figure 3 shows that the lipolytic activity was expressed at different levels by the tested yeasts. In most of the strains, this activity had already been exhibited after 48 h of incubation, similarly to the control strains (L. tetrasporus and Y. lipolytica), while few strains showed this activity only after 72 h of incubation. This was the case of almost all Pichia kudriavzevii strains (with the exception of 4-22), as well as the AII 171, AII 40, and 4-2 strains. However, in the majority of strains, lipase activity increased with incubation time, with the highest values found after 72 h of incubation. Conversely, in some strains (RB3, AII 122, PD36-2, AII 184, 4R1, AII 168, 1P1, 2-7) this activity was not affected by incubation time. The highest lipolytic activity was found in three strains (M, RB5 and D) after 72 h, with the diameter of the clear zone being 10, 9 and 6 mm respectively. Finally, lipase activity was lacking in two strains, ND1 and ML-3.
The ability to produce lipases has been extensively studied and well-documented for Y. lipolytica [43,44] and D. hansenii [44,45] strains. For instance, Papagora et al. [45] investigated and optimized lipase production by D. hansenii isolated from dry-salted olives. Their study demonstrated that sugar and lipid concentrations, as well as pH, are key factors influencing the production of highly active lipases. Under optimized conditions, lipase activity reached 7.44 U/mL, corresponding to an increase of approximately 2.3-fold compared to non-optimized conditions. Furthermore, other studies have shown that the lipolytic activity of D. hansenii is strongly influenced by the type of fatty acids present in the culture medium, with higher activities reported in the presence of tristearin (0.68 U/mg), oleic acid (0.56 U/mg), and soybean oil (0.36 U/mg) [46]. Furthermore, Y. lipolytica has been reported to produce three main lipases, namely Lip2, Lip7, and Lip8, all of which are secreted extracellularly [47]. Among these enzymes, Lip7 and Lip8 are membrane-anchored, whereas Lip2 is released into the culture medium and exhibits the highest catalytic activity. Colacicco et al. [43] investigated lipase activity in Y. lipolytica W29 cultivated with different concentrations of waste cooking oil (WCO) over time. The highest lipase activity (0.633 ± 0.015 U/mL) was observed after 72 h of cultivation at 10% (v/v) WCO [43]. Conversely, lipase activity in Pichia nakasei (D strain) has not been previously documented in the literature. The findings obtained suggest an interesting catalytic activity of this enzyme; further studies are needed to determine the activity under different conditions and to clarify the underlying enzymatic mechanisms.

3.5. Lipid Production by Yeast Strains Grown in OMWW-Based Medium

Based on the results obtained during the screening phase, eight strains were selected for further characterization on a substrate supplemented with olive mill wastewater (OMWW), with the aim of identifying potential candidates for agro-industrial waste valorization. Specifically, four strains (AII122, ML-3, AII171, and RB5) showing the highest lipid accumulation capacity (above 20%) were selected as promising oleaginous yeasts. In addition, four other strains, including M (Yarrowia lipolytica), AII110 (Pichia kluyverii), D (Pichia nakasei), and 2R1 (Metschnikowia pulcherrima), were selected based on their good combination of lipase activity, lipid accumulation and representativity of species poorly investigated for this specific application.
The eight selected strains were cultivated in YMB supplemented with increasing concentrations of OMWW (15%, 25%, and 50% v/v; C/N g/g = 20, 25, and 38, respectively). Growth performance was evaluated by optical density change, calculated on the basis of the difference between optical density values measured at the beginning (0 h) and at the end (48 h) of the experiments under all tested conditions, in comparison to control (YMB without OMWW addition).
The results reported in Figure 4 showed significant statistical differences among treatments within each strain. The inclusion of OMWW in the substrate did not inhibit the growth of yeasts, and in most cases, the better growth level was obtained by using YMB medium supplemented with OMWW at 15% v/v, which was chosen as the substrate for the further experiments. In fact, at 15% OMWW, several strains exhibited ΔAbs values comparable to or higher than those recorded in YMB, indicating effective adaptation to low OMWW concentrations. In particular, strains D and AII110 showed markedly enhanced growth at 15% OMWW, with statistically significant differences compared to higher concentrations. Conversely, 50% OMWW generally resulted in reduced biomass accumulation, confirming the inhibitory impact of high content of phenolic compounds in OMWW used in this study (Table 2).
Table 3 reports the intracellular lipid content and dry cell weight (DCW) of the eight selected strains cultivated in YMB supplemented with 15% (v/v) OMWW. Regarding biomass production, no statistically significant differences were observed among the tested strains compared to Y. lipolytica, although strains D, AII110, and ML-3 showed significantly lower DCW than L. tetrasporus (8.69 ± 0.28 g/L), which exhibited the highest biomass yield under the tested conditions. Furthermore, for some strains (i.e., RB5 and ML-3) inconsistent values for DCW (Table 3) and OD600 (Figure 4) were found in OMWW 15%; indeed, the RB5 strain resulted in 9.05 g/L DCW (Table 3) and 1.9 OD600 approximately (Figure 4), while the ML-3 resulted in 6.73 g/L DCW and 4.0 OD600. These results might be due to the use of two methods to evaluate the growth performance. Optical density evaluation is a fast method for the quantification of biomass yield, while direct measurements of DCW are generally more accurate than OD-based estimations.
Overall, intracellular lipid accumulation was consistently low in all strains, ranging between approximately 1% and 6%. These values are far below the threshold commonly adopted to define oleaginous microorganisms, namely 20% of dry cell weight [15]. The highest lipid contents were detected in AII110 (4.6 ± 0.07%), AII171 (4.47 ± 0.72%), and 2R1 (6.48 ± 0.27%), which were significantly higher than those of the control strains, yet remain quantitatively modest. Such low percentages most likely correspond to structural membrane lipids rather than the accumulation of storage triglycerides.
Comparable results were reported by Arous et al. [48], who observed lipid contents ranging from 2.8% to 5% in yeasts cultivated on undiluted OMWW. Under those conditions, biomass composition was characterized by a high protein fraction (up to 39%), indicating a metabolic direction for cellular growth and protein synthesis rather than lipid storage.
In contrast, other investigations have reported substantially higher lipid production from OMWW [49]. For example, L. starkeyi and Y. lipolytica were shown to reach lipid contents of approximately 25% when OMWW was supplemented with additional carbon sources [50]. Similarly, Sayın et al. [51] reported 41% of lipid accumulation in Y. lipolytica cultivated on diluted OMWW.
In the present work, the limited lipid production could be explained by a metabolic imbalance induced by OMWW. The presence of phenolic compounds and other inhibitory molecules can impose significant physiological stress. Therefore, the limited lipid accumulation observed in this study is likely due to substrate inhibition rather than an unfavorable nutrient balance. Under such conditions, cells likely redirect metabolic energy toward stress-protective mechanisms, medium detoxification, rather than intracellular lipid production. Taken together, these findings highlight the intrinsic complexity of inducing lipid synthesis in complex wastewaters such as OMWW. They also confirm the high interstudy variability reported in the literature, which is strongly influenced by strains, wastewater composition, and process parameters.
Indeed, the two control strains (L. tetrasporus and Y. lipolytica), previously tested in different substrates and culture conditions [52], gave lipid yield higher than levels found in this study. This might be due to the severe effect of process parameters, such as substrate, temperature, pH and dissolved oxygen (DO), on yeast lipid production [53]. As a consequence, further characterization of the indigenous yeasts showing lipid production higher than control strains, such as AII 110, AII 171 and 2R1, are necessary in order to evaluate the possibility of increasing lipid production by optimizing fermentative conditions.

3.6. Effect of the Potential Oleaginous Yeasts on OMWW-Based Medium Decolorization and Polyphenol Content Reduction

Olive mill wastewaters (OMWWs) represent a polluting liquid effluent, mainly due to their elevated concentration of phenolic compounds. Numerous studies have demonstrated that the toxicity of OMWWs is largely associated with their phenolic fraction [54,55]. In fact, polyphenols represent the main class of phytotoxic compounds present in this waste and seem to be responsible for the dark color of OMWWs [24].
Therefore, the identification of microorganisms capable of valorizing these effluents is of particular interest, as it offers a dual advantage: the detoxification of the wastewater and the simultaneous production of value-added bio-based products. The capacity of several microorganisms to metabolize phenolic compounds is well-established. For instance, Candida tropicalis ATCC 750, Rhodotorula spp., and Y. lipolytica, have been reported to metabolize phenolic compounds present in OMWWs [24,49,56]. The ability of fungi to break down phenolic compounds is correlated to the secretion of enzymes, such as the extracellular oxidases (ligninolytic enzymes) laccase, lignin peroxidase and peroxidase, although the secretion of these enzymes is strain-dependent and influenced by culture conditions [57].
Table 4 reports the percentage of phenol reduction and the corresponding medium discoloration for each tested strain. In general, the values of decolorization in comparison to the non-inoculated sample ranged between 1.04 and 41.78%, while the range of reduction in phenolic compounds was between 11.60 and 40.35%.
The results obtained for the phenolic compounds removal showed statistical differences in all the analyzed strains compared to both control strains, except for the strains AII 171 and M, whereas for the sample inoculated with AII 122, the phenolic reduction (40.35%) was significantly higher than the sample inoculated with Y.l. (31.90%). Finally, the decolorization capacity in the strains ML-3, AII171, 2R1 and AII 122 was significantly different from the values obtained for both control strains. Conversely, no statistical differences were observed for the strains RB5 and AII 110 in comparison to Y.l. and L.t.
The strain AII122 (C. tropicalis) showed the highest values for both of the desired characteristics, the reduction in phenolic content (40.35 ± 3.05) and OMWW decolorization (41.78 ± 1.80%). The strain AII 171 (Mey. caribbica) led to a significant reduction in total phenolic content (37.07%), but no appreciable decolorization of the olive mill wastewater was observed. This result suggests that Mey. caribbica selectively metabolized specific phenolic compounds that do not contribute to the chromatic properties of the OMWW. Indeed, the color of olive mill wastewater is mainly associated with complex and polymerized phenolic structures, which may remain unaffected or be transformed into still chromophoric derivatives [58]. Although no decolorization was observed, this finding is noteworthy, since, to the best of our knowledge, the ability of Mey. caribbica to remove phenolic compounds from olive mill wastewater has not been previously reported in the literature. This result confirms the high metabolic versatility and ecological adaptability of this yeast species, which, over the past two decades, has been identified as a yeast with significant relevance to biotechnology, agriculture, environmental remediation, and food applications [35].

4. Conclusions

This study demonstrated that non-conventional yeasts isolated from different ecological niches represent a valuable resource for the valorization of olive mill wastewater. The preliminary screening using Sudan Black B staining revealed that several strains were able to accumulate intracellular lipids. Quantitative analyses confirmed that some strains exhibited significant oleaginous potential, with lipid contents exceeding 20% of dry biomass. In particular, Candida tropicalis AII122 and Pichia manshurica ML-3 showed the highest lipid accumulation in synthetic medium, while strains such as Meyerozyma caribbica AII171 and Debaryomyces hansenii RB5 also displayed promising lipid yields.
When cultivated in OMWW-based medium (15% v/v), the selected strains were able to grow efficiently, confirming their adaptation to this inhibitory substrate. Although overall lipid accumulation remained low (1–6% of DCW), M. pulcherrima 2R1 exhibited significantly higher lipid accumulation (6.48% of DCW) compared to the control strains Yarrowia lipolytica ATCC 46483 and Lipomyces tetrasporus Li-0407. Furthermore, the strain C. tropicalis AII122 achieved the highest phenolic compound reduction and color removal, outperforming at least one of the control strains. Overall, the results highlight the feasibility of coupling OMWW detoxification with microbial production, supporting a circular bioeconomy approach. Among the tested strains, C. tropicalis AII122 emerged as the most promising multifunctional candidate, combining high biomass production and effective phenolic reduction in OMWW with the highest lipid accumulation observed under synthetic medium conditions. Although lipid production on OMWW remained limited under the tested conditions, the strong detoxification capacity and robust biomass formation displayed by this strain suggest that, through a process optimization, the lipid synthesis on OMWW could be significantly enhanced. These findings provide new insights into the biotechnological approach based on use of indigenous non-conventional yeasts for sustainable OMWW management and open perspectives for further optimization and scale-up studies.

Author Contributions

G.S.: Conceptualization, Data curation, Investigation, Methodology, Writing—original draft, Writing—review and editing. R.P.: Conceptualization, Investigation, Data curation, Formal analysis, Methodology, Software, Writing—original draft. A.C. (Antonio Caporusso): Methodology, Data curation, Writing—review and editing. G.A.: Conceptualization, Investigation, Methodology, Writing—original draft. V.V.: Investigation, Methodology, Validation. G.L.R.: Validation, Writing—review and editing. A.C. (Angela Capece): Conceptualization, Project administration, Supervision, Writing—review and editing. All authors have read and agreed to the published version of the manuscript.

Funding

This study was carried out within the Agritech National Research Center and received funding from the European Union Next-Generation EU (PIANO NAZIONALE DI RIPRESA E RESILIENZA (PNRR)—MISSIONE 4 COMPONENTE 2, INVESTIMENTO 1.4—D.D. 1032 17/06/2022, funding number: CN00000022). This manuscript reflects only the authors’ views and opinions; neither the European Union nor the European Commission can be considered responsible for them.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in the study are included in the article; further inquiries can be directed to the corresponding author.

Acknowledgments

The JRU MIRRI-IT (http://www.mirri-it.it/, accessed on 15 January 2026) is greatly acknowledged for scientific support.

Conflicts of Interest

Gabriella Siesto and Angela Capece were employed by Spin-off StarFInn S.r.l.s. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The authors declare that this study received funding from European Union Next-Generation EU. The funder was not involved in the study design, collection, analysis, interpretation of data, the writing of this article or the decision to submit it for publication.

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Figure 1. Cells stained with Sudan Black B of potential oleaginous (A) and non-oleaginous yeasts (B) observed with optical microscope at 100X magnification. The (A) refers to cells of Lipomyces tetrasporus, while the strain K (Yarrowia lipolytica) is reported in (B).
Figure 1. Cells stained with Sudan Black B of potential oleaginous (A) and non-oleaginous yeasts (B) observed with optical microscope at 100X magnification. The (A) refers to cells of Lipomyces tetrasporus, while the strain K (Yarrowia lipolytica) is reported in (B).
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Figure 2. Lipid content (%) in the strains grown in nitrogen limited conditions. Triangles indicate the strains showing the highest lipid content.
Figure 2. Lipid content (%) in the strains grown in nitrogen limited conditions. Triangles indicate the strains showing the highest lipid content.
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Figure 3. Lipolysis halo (in mm) in TBA by diffusion test in wells of the 41 yeast strains. Data are reported as mean ± SD of two independent experiments.
Figure 3. Lipolysis halo (in mm) in TBA by diffusion test in wells of the 41 yeast strains. Data are reported as mean ± SD of two independent experiments.
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Figure 4. OD values at 600 nm (expressed as Abs48h − Abs0h) by the yeast strains grown in OMWW (15%, 25%, 50%, v/v) in comparison to YMB medium without OMWW addition. Letters (a, b, ab, ac) indicate significant differences among treatments within each strain (p < 0.05).
Figure 4. OD values at 600 nm (expressed as Abs48h − Abs0h) by the yeast strains grown in OMWW (15%, 25%, 50%, v/v) in comparison to YMB medium without OMWW addition. Letters (a, b, ab, ac) indicate significant differences among treatments within each strain (p < 0.05).
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Table 1. Origin of the non-conventional yeasts tested in this study.
Table 1. Origin of the non-conventional yeasts tested in this study.
SpeciesStrain CodeSource
Pichia kudriavzevii2-21, 4-16, 2R17, 4-22Grape must
Hanseniaspora osmophilaND1
Metschnikowia pulcherrima1P1, 1RP2, 1RP9,1SPR14, 1SPR1, 2-1, 2-7, 2-13, 2R1, 4-2, 4-15, 4R1, 4R-11
Metschnikowia pulcherrimaAII 37, AII 68, AII 136, AII 181, AII 49Honey bees
Candida tropicalisAII 122
Meyerozyma caribbicaAII 171
Pichia kluyveriAII 110
Lachancea kluyveriAII 184
Moniliella pollinisAII 40
Cutaneotrichosporon curvatusAII 176
Yarrowia lipolyticaRB3, K, M, C, ACheese
Pichia nakaseiD, RB1
DebaryomyceshanseniiRB5, PD 12-1, PD 49- 1
Candida zeylanoidesPD 36-2
Pichia manshuricaML-3Olive mill wastewater
Yarrowia lipolytica ATCC 46483 (DSM 8218)Y.l.Control
Lipomyces tetrasporus Li-0407 (DSM 70314)L.t.
Table 2. Biochemical parameters of OMWW.
Table 2. Biochemical parameters of OMWW.
ParametersValue (In Present Work)Value from Reference [28]Value from Reference [24]
pH4.75.45.1
Total reducing sugars (g/L)9.99.410.1
Phenolic compounds (g/L)5.13.44.8
Lipids-0.76%-
Table 3. Intracellular lipid accumulation (%) and dry cell weight (DCW, g/L) of selected yeast strains grown in OMWW (15% v/v).
Table 3. Intracellular lipid accumulation (%) and dry cell weight (DCW, g/L) of selected yeast strains grown in OMWW (15% v/v).
StrainLipid Production (%)DCW (g/L)
RB5 (Debaryomyces hansenii)2.88 ± 0.069.05 ± 0.04
ML-3 (Pichia manshurica)1.84 ± 0.056.73 ± 0.13 (#)
M (Yarrowia lipolytica)2.78 ± 1.478.31 ± 0.14
D (P. nakasei)2.71 ± 0.525.43 ± 0.73 (#)
2R1 (Metschnikowia pulcherrima)6.48 ± 0.27 (#;*)8.30 ± 0.36
AII 171 (Meyerozyma caribbica)4.47 ± 0.72 (#;*)8.13 ± 0.20
AII 110 (P. kluyverii)4.60 ± 0.07 (#;*)6.11 ± 0.06 (#)
AII 122 (Candida tropicalis)2.68 ± 0.159.86 ± 0.24
Y.l. (Y. lipolytica)1.80 ± 0.367.54 ± 1.93
L.t. (Lipomyces tetrasporus)1.67 ± 0.028.69 ± 0.28
Data are expressed as mean ± SD values of two independent experiments. In each column, strains significantly different (Tukey’s test, p < 0.05) from L.t. are indicated by (#) and those significantly different from Y.l. by (*).
Table 4. Percentage of decolorization and reduction in phenolic compounds by selected yeasts grown in OMWW-based medium.
Table 4. Percentage of decolorization and reduction in phenolic compounds by selected yeasts grown in OMWW-based medium.
StrainsPhenolic Compounds
Reduction (%) 		Decolorization (%)
RB5 (Debaryomyces hansenii)20.42 ± 2.02 (#;*)26.39 ± 1.94
ML-3 (Pichia manshurica)22.95 ± 0.46 (#;*)17.25 ± 0.52 (#;*)
M (Yarrowia lipolytica)34.17 ± 1.4322.22 ± 1.22 (*)
D (P. nakasei)23.20 ± 1.64 (#;*)18.98 ± 2.76 (*)
2R1 (Metschnikowia pulcherrima)15.51 ± 0.71 (#;*)3.47 ± 0.02 (#;*)
AII 171 (Meyerozyma caribbica)37.07 ± 3.111.04 ± 0.16 (#;*)
AII 110 (P. kluyverii)11.6 ± 0.14 (#;*)25.00 ± 0.33
AII 122 (Candida tropicalis)40.35 ± 3.05 (*)41.78 ± 1.80 (#;*)
Y.l. (Y. lipolytica)31.90 ± 2.8529.97 ± 0.49 (#)
L.t. (Lipomyces tetrasporus)32.53 ± 2.3224.07 ± 0.89 (*)
Data are expressed as mean ± SD values of two independent experiments. In each column, strains significantly different (Tukey’s test, p < 0.05) from L.t. are indicated by (#) and those significantly different from Y.l. by (*).
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MDPI and ACS Style

Siesto, G.; Pietrafesa, R.; Caporusso, A.; La Rocca, G.; Alberico, G.; Valerio, V.; Capece, A. Preliminary Screening of Non-Conventional Yeasts for Olive Mill Wastewater Valorization. Fermentation 2026, 12, 188. https://doi.org/10.3390/fermentation12040188

AMA Style

Siesto G, Pietrafesa R, Caporusso A, La Rocca G, Alberico G, Valerio V, Capece A. Preliminary Screening of Non-Conventional Yeasts for Olive Mill Wastewater Valorization. Fermentation. 2026; 12(4):188. https://doi.org/10.3390/fermentation12040188

Chicago/Turabian Style

Siesto, Gabriella, Rocchina Pietrafesa, Antonio Caporusso, Giorgia La Rocca, Grazia Alberico, Vito Valerio, and Angela Capece. 2026. "Preliminary Screening of Non-Conventional Yeasts for Olive Mill Wastewater Valorization" Fermentation 12, no. 4: 188. https://doi.org/10.3390/fermentation12040188

APA Style

Siesto, G., Pietrafesa, R., Caporusso, A., La Rocca, G., Alberico, G., Valerio, V., & Capece, A. (2026). Preliminary Screening of Non-Conventional Yeasts for Olive Mill Wastewater Valorization. Fermentation, 12(4), 188. https://doi.org/10.3390/fermentation12040188

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