Economic production and characterization of fungal SCOs

The microbial lipids or SCOs, are categorized among the most promising natural feedstock for biofuel and nutraceuticals production. Remarkably, the proximity of their structure with fish oil or even vegetable oils had gained a momentum, especially those derived from fungi. That could be attributed to their large quantity of biomass with high lipids yield in a short growth cycle and facile biomass collection. Remarkably, the versatility in fungal growth conditions facilitated their cultivation on low-cost culture medium based on different wastes, which were continuously accumulated in the environment causing severe pollution. Such dual tasks, of investing the environmental contaminants in biotechnological products, consider being the fundamental pillar of sustainable green techno-economic productivity [9]. Several species of filamentous fungi were recorded in bioconversion of food processing wastes into value-added by-products such as Aspergillus, Rhizopus and Trichosporon [17]. Interestingly, Drechslera sp. and Alternaria sp. demonstrated significant potential for lipid production, especially under using of agro-industrial wastes as substrates [20, 21]. Hereupon, the current study focused on mycological production of SCOs from both fungal strains in an economic process via utilizing by-product of the cheese manufacturing industries (i.e., whey), as a sole nutrition source. Generally, it was recorded as highly nutritive waste stream full of proteins, vitamins, sugars, minerals, and other growth factors [29] and also reported previously as supporting material in fungal lipid production [30, 31]. Herein, the whey was utilized as a cultivation media without any addition of other nutrient sources. It facilitated the lipid accumulation yield in both filamentous fungi by 3.22 and 4.33 g/L, which representing 45.3 and 48.2% lipid content in Drechslera sp. and Alternaria sp., respectively. In comparison, the lipid content of Drechslera sp. and Alternaria sp. reached to 33.18 and 29%, with lipid yield evaluated by 3.65 and 5.6 g/L, respectively, upon replacing carbon source in their optimized media with agricultural wastes (i.e., orange peel and molasses). However, on optimized microbiological media (i.e., Czapek-Dox’s medium), their lipid contents were assessed by 40.75 and 50.3% for Drechslera sp. and Alternaria sp., respectively, reflecting the higher potentiality of whey stream as the main cultivation and production media in the lieu of carbon or nitrogen source [20, 21].

The characterstic features of SCOs from both oleaginous fungi were determined initially by identifying their components through investigating the fatty acid methyl esters profiles after acidic transesterification as shown in (Fig. 1) and (Table 1), which manifested the qualitative and quantitative differences in both examined profiles. Notably, the results of gas chromatography–mass spectrometry (GC-MS) showed common features among both profiles. Wherein, unsaturated fatty acids (USFAs) represented the dominate constituent in the profiles of both fungi by 62.18 and 53.15% for Alternaria sp. and Drechslera sp. respectively. Meanwhile, Palmitic acid-C16 (SFAs) was the most prevalent FA by 29.0 and 28.9% for Alternaria sp. and Drechslera sp. respectively, followed by Oleic acid -C18 (MUSFAs), which was evaluated by 24.7 and 28.8%, % for Alternaria sp. and Drechslera sp., correspondingly. On the other hand, the omega-6 (ɷ-6) poly unsaturated fatty acid (PUFAs) (i.e., Linolenic acid and γ-linolenic (C18) was the third major constituent in both profiles with the values of 26.89% and 15.72% for Alternaria sp. and Drechslera sp. respectively. Interestingly, an obvious percent of PUFAs (30.02%) was detected for Alternaria sp. compared to 17.93% for Drechslera sp. In accordance with our result, Lauryn et al. [32] demonstrated that cheese whey was ideal choice for Mucor circinelloides in SCOs production with GC-MS-profile contained predominantly oleic acid (41%), palmitic acid (23%), linoleic acid (11%), and γ-linolenic acid (9%).

Fig. 1

GC-Ms analysis of fatty acid produced by (A) Alternaria sp. B Drechslera sp. cultivated on whey

Table 1 Showing fatty acids (FAs) patterns of both SCOs extracted from Alternaria sp.and Drechslera sp.after their cultivation on whey media

The acquisition of infrared spectra of lipids has been attained senior concern thank to providing opulent information on their chemical components, besides it is a fast and economical technique [33]. Fig. 2 demonstrates the FTIR spectrum of both examined SCOs. Generally, the peaks at wavenumbers of 3742 and 3359 cm−1 of A-OS and 3200 cm−1 of D-OS indicated the existence of hydrate hydroxyl group (-OH). Similarly, as signposted by Nandiyanto et al. [34], a broad absorption band in the range of 3650 and 3250 cm−1 represented the sign of hydrogen bond. Besides, the band at 3010 cm−1 in both SCOs samples pointed out to the C = CH- vibration originated from unsaturated fatty acids; interestingly, such band could be used for examining the degree of unsaturation in oils as referred by Shapaval et al. [35]. In the same sense, Nandiyanto et al. [34] stated that the bands above 3000 cm−1 are representative of unsaturated compounds. Accordingly, this could reflect the superior unsaturation of fatty acids in Alternaria sp. than that observed in Drechslera sp., as more peaks were detected in the area above 3000 cm−1, which harmonized with results of GC-MS that confirmed the higher unsaturation degree in A-OS comparing to D-OS.

Fig. 2

FTIR spectrum of oil samples from (A) Alternaria sp. (B) Drechslera sp

Notably, the spectral bands at 2889 cm−1 detected in D-OS characterizes the C-H stretching vibrations of lipids [36]. Meanwhile, Nandiyanto et al. [34] mentioned that the double bond groups such as carbonyl (C = C) were present in the region of 1500–2000 cm−1. So, remarkably, both fungal patterns confirmed the presence of carbonyl (C = C) peaks as detected at 1994 and 1648 cm−1 of A-OS and 1662 cm−1 of D-OS. While the presence of spectral band at that regions reflected the presence of crystallizable FAs [33]. However, the signature of CH2 asymmetric bending and CH2 vibration could be detected by the presence of peaks at wave numbers of 1403 and 1435 cm−1 [33, 37]. Whereas, typical bands at 1252, 1114, 1050, 986 and 948 cm−1 of A-OS-FTIR pattern and also 1273, 1096 and 993 cm−1 of D-OS FTIR pattern referred to C-O stretching of phospholipids [38,39,40]. Regarding to the spectral bands at 718 and 611 cm−1 of D-OS and A-OS profiles, respectively, could be attribute to alkyne C-H bend and CH2 rocking vibration, respectively [34, 41]. Likewise, Forfang et al. [42] elucidated that biological material (e.g., carbohydrates, proteins and lipids) exhibits CH stretching vibrations due to the presence of -CH3 and -CH2. Arguably, based on the above mentioned characterization techniques, the results of FTIR were deemed as strong evidence for the presence of functional groups that were related to intracellular lipids, which were detected and quantified by GC-MS analysis; emphasizing hereby the obvious discrepancies between SCOs of Alternaria sp. and Drechslera sp. in the content and structures.

Antibiofilm activity of SCOs

The presence of biofilm represents a serious threat to human health and surrounding ecosystem in various sectors. The biofilms injure medical equipment such as catheters, contact lenses, prosthetic devices, heart pacemakers, endoscopes, colonoscopes, dental plaques and dental irrigation units. Let alone their capability to invade human tissues causing severe infections [43]. However, the biofilms also adhere to food manufacturing equipment, air-conditioning units, petroleum pipelines and cooling towers, which symbolizes as evidences on industrial risk of biofilm. In the same sense, biofilms fixed themselves on external surfaces of marine vessels, water pipes, stones in a stream and sewage treatment plants, facilitating the accumulation of organic and inorganic materials with other organism such as algae, plants and protozoa in a phenomenon called biofouling. The problem of biofouling lies behind deteriorating the aqueous flow system with its fauna; causing the prevalence of microbial contamination and accelerated corrosion.

Against this backdrop, various mechanical removal approaches and chemical biocides were utilized to eliminate biofilms and prevent their hazard [44]. Nonetheless, the extensive use of antimicrobial agents generates multidrug resistance (MDR) phenomenon that led to ecological balance disruption and epidemic diseases. Therefore, modern insights are directed toward employing natural bioproducts as ecofriendly, biocompatible, safe and economic agents in defeating water/foodborne pathogens, which harmonized with aims of recent international events like COP28. Hence, the current investigation is undertaken to determine the in vitro antibiofilm activity of SCOs extracted from Alternaria sp. and Drechslera sp. against some MDR microbes-forming biofilm. This target was implemented through detecting the effect of SCOs on biofilm formation, viability, biochemical composition and hydrophobicity.

In fact, S. aureus, P. aeruginosa and C. albicans were opted due to their ubiquitous occurrence and concomitance with nosocomial/community-acquired infections. Besides, they exhibit the capability to colonize vast array of surfaces either abiotic or cellular interfaces, which lead to significant environmental and health threats. Therefore, crystal violet assay (CV) was employed to detect the antibiofilm influence of different doses of SCOs (1–100 µg/mL), which deemed as reliable and facile assay in staining the biofilm biomass entirely [45, 46]. As observed in Figs. (3 and 4), the inhibitory patterns of both SCOs samples displayed significant differences (P ≤ 0.05) in the biofilm development after treatment, as unveiled by ANOVA. Besides, the inhibitory power of both samples showed notoriously variation against examined pathogens. Namely, SCOs of Alternaria sp. (A-OS) suppressed the growth of P. aeruginosa biofilm at concentrations ranged from 1 µg /mL to 10 µg/mL by 7.01 ± 0.3% to 50.85 ± 2.68%, respectively; while SCOs of Drechslera sp. (D-OS) enhanced the growth of P. aeruginosa biofilm by 33.41 ± 6.24% and 5.83 ± 2.83% at exact concentrations, respectively. Remarkably, the antibiofilm potency increased with elevation of applied doses of both examined oil samples. Wherein, the biofilm of P. aeruginosa was inhibited significantly (P ≤ 0.05) at 100 µg/mL of A-OS and D-OS by 84.10 ± 0.445 and 47.41 ± 2.83%, correspondingly. On the other hand, about 90.37 ± 0.065% and 62.63 ± 5.82% inhibition was observed for S. aureus biofilm at 100 µg/mL of both SCOs in the same order. Furthermore, a pronounced and significant (P ≤ 0.05) fungicidal potency was noticed in blocking the biofilm formation of C. albicans that reached to 94.96 ± 0.21% and 78.67 ± 0.23% upon treating with A-OS and D-OS (100 µg/mL), respectively. Generally, as inferred from these results, there is an inter-species variation phenomenon in a dose dependent performance exerted by the examined SCOs samples. In agreement with our results, Murugan et al. [47], found a variation in the biofilm growth of Proteus sp., E. coli, Bacillus sp. and S. aureus; assigning that to the differences in the physiological behavior of different microbial species. Seemingly, the cell wall architecture, microbial physiology with varied metabolic performance and uptake/regulation systems are considered being the fundamental parameters in managing the tolerance and susceptibility profiles among inter and intra-species of the microbes in their response to any antagonistic agent [48].

Fig. 3

The Impact of A-OS on biofilm development by P. aeruginosa, S. aureus and C. albicans. A-Biofilm biomass suppression, B-Metabolic performance, C- EPS inhibition, D-Protein inhibition and E-Hydrophobicity inhibition. All values were expressed as mean ± SEM. Treatments at various doses were comparing to untreated control with significance at *P ≤ 0.05

Fig. 4

The Impact of D-OS on biofilm development by P. aeruginosa, S. aureus and C. albicans. A-Biofilm biomass suppression, B-Metabolic performance, C- EPS inhibition, D-Protein inhibition and E-Hydrophobicity inhibition. All values were expressed as mean ± SEM. Treatments at various doses were comparing to untreated control with significance at *P ≤ 0.05

Effect of SCOs samples on biofilm metabolic activity

Actually, CV firmly stains the entire biofilm biomass, which includes polysaccharides in the mucilaginous mat conjugated with other biomolecules that are disseminated in an even manner on the live as well as dead cells surface. Subsequently, the overall metabolic performance of adhered microbial cells, which were treated with different concentrations of oil samples relative to untreated, was assessed calorimetrically using MTT assay. It is worth noting that tetrazolium salts (such as MTT (3-[4, 5- dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide), XTT (2,3-bis(2-methoxy-4-nitro-5-sulfo-phenyl)- 2 H-tetrazolium-5-carboxanilide) and TCC (2,3,5- triphenyl tetrazolium chloride) are frequently utilized in biological assays to investigate the viability of living cells. That occurs through enzymatic reduction of tetrazolium salt by the cellular NADH of metabolically active cells, which leads to the formation of colored formazan. Hence, different tetrazolium-based dyes were employed in various studies to determine biofilm viability in accompanying with other means like CV approach and microscale analysis [45, 46, 49].

Interestingly, both types of oil samples impacted on the viability of cells in the biofilm structure adversely, progressively and significantly (P ≤ 0.05) with increasing the doses of applied SCOs. Figs. (3 and 4) indicate that A-OS frustrated the propagation of active cell in P. aeruginosa biofilm matrix in all applied concentrations in the range of 2.35 ± 0.885% − 83.08 ± 0.235%. Conversely, D-OS flourished the growth of P. aeruginosa biofilm at low concentrations (1 µg/mL − 5 µg/mL) by the range of 16.72 ± 1.36% − 4.43 ± 1.36%; whereas, the viability of live cells curtailed to 48.12 ± 3.0% upon increasing the concentration to 100 µg/mL. Regarding to the biofilm of S. aureus, both oil samples inhibited the growth of active cells in linear concentration-dependent behavior; reaching to the maximum suppression by 85.9 ± 0.375 and 62.48 ± 1.52% for A-OS and D-OS, respectively at 100 µg/mL. In the same sense, the survival of C. albicans cells in biofilm network arrested by 86.54 ± 0.86% and 64.94 ± 1.48% under the treatment of A-OS and D-OS (100 µg/mL), respectively. As noticed, the results of biofilm inhibition were harmonized with that of metabolic activity. It is plausible to mention that viability and metabolic activity of all tested biofilm-forming pathogens correlated significantly with biofilm biomass (r ≥ 0.9, P = 0.00); reflecting hindrance impact of oil samples on active cells that are distributed within multilayer architecture of biofilm.

The effect of oil samples on biofilm’s carbohydrate and protein content

Carbohydrates or exopolysaccharides (ePs) and proteins represent the intrinsic constituents of EPS scaffold of the biofilm from both structure and function. As denoted by Gunn et al. [50] and Mosharaf et al. [51], the secreted proteins, adhesion proteins (e.g., lectins, Baplike proteins) and motility organelles configure the biofilm matrix proteins. However, galactose, mannose, glucose, arabinose, xylose, rhamnose, fucose, cellulose nanofibers, galacturonic acid and N-acetyl-glucosamine are the most abundant carbohydrates detected in slimy matrix of S. aureus, Enterococcus faecalis, Klebsiella pneumoniae, and P. aeruginosa [52]. As declared by Gunn et al. [50] the chemical constituents of the biofilm differ rely on the organism and was influenced by environmental parameters. Intriguingly, such specific components control biofilm integrity, maintain biofilm stability, configure its morphology, mediate cell-cell signaling, contribute in cell colonization/adherence and preserve its cells from adverse external stressors [23, 51].

Figure (3) depicts the effect of A-OS on biofilm content of carbohydrates or ePS, which diminished from 7.515 ± 0.13, 5.541 ± 0.147 and 6.59 ± 0.33 mg/mL in control untreated samples of S. aureus, P. aeruginosa, and C. albicans biofilms to 3.61 ± 0.34, 3.14 ± 0.335 and 2.99 ± 0.19 mg/mL at 10 ug/mL of A-OS, respectively; representing by such way inhibition percentages of 51.87 ± 4.55, 43.26 ± 5.9, and 54.5 ± 2.84%, respectively. However, the same concentration of D-OS reduced carbohydrate content of S. aureus, P. aeruginosa, and C. albicans biofilms to 5.100 ± 0.47, 4.98 ± 0.22, and 4.20 ± 0.335 mg/mL by 32.09 ± 0.536, 9.94 ± 4.04, and 36.23 ± 5.04% inhibition percentages, respectively (Fig. 4). In the same context, 10 ug/mL of A-OS reduced protein content of S. aureus, P. aeruginosa and C. albicans biofilms from 9.64 ± 0.095, 9.75 ± 0.065 and 9.34 ± 0.1 mg/mL in the control untreated samples to 5.92 ± 0.11, 6.94 ± 0.17 and 4.72 ± 0.16 mg/mL, which symbolize 38.54 ± 0.94, 33.47 ± 1.74 and 49.43 ± 1.69% inhibition percentages, respectively. Whereas, D-OS (10 ug/mL) diminished protein content to 7.44 ± 0.255, 8.40 ± 0.065 and 5.61 ± 0.37 mg/mL causing 22.81 ± 2.63, 13.84 ± 0.59 and 39.92 ± 3.89% inhibition.

Notably, significant and dramatic changes were observed in both contents upon elevating the concentrations till reached to the highest values at 100 ug/mL. Wherein, such concentration of A-OS caused in lowering the carbohydrate/ePS content of S. aureus, P. aeruginosa and C. albicans biofilms by 76.54 ± 7.04, 66.81 ± 2.51 and 80.54 ± 1.77%, respectively. While the carbohydrate/ePS reduction percentage reached 60.22 ± 2.02, 50.03 ± 1.95 and 64.26 ± 4.7% by D-OS for S. aureus, P. aeruginosa and C. albicans biofilms, respectively. Regarding the protein content, it lessened significantly to 63.94 ± 3.89, 57.27 ± 5.43 and 74.87 ± 2.49% upon applying 100 ug/mL of A-OS; however, D-OS (100 ug/mL) reduced it to 49.39 ± 1.96, 47.61 ± 2.03 and 59.07 ± 1.78% for S. aureus, P. aeruginosa and C. albicans biofilms, respectively.

Effect of oil samples on biofilm hydrophobicity

The adhesion capability of microbial cell deemed as intrinsic property to colonize any substrate and boost biofilm lifestyle easily. Interestingly, cell surface traits in the formed of ePS and hydrophobicity are decisive parameters that manage the entire adhesion process. In addition, surface characteristics and ambient environmental conditions rule the adhesion of the cells with the surface through number of interactions like hydrophobic, van der Waals and electrostatic. Remarkably, the cells with higher biofilm forming capacity possess higher hydrophobic nature that leads to potent adhesion and vice versa [53]. Given that the biofilm-forming cells with hydrophobic characteristics exhibit affinity to hydrocarbons (e.g., hexadecane, octene, xylene, etc.), the cells retained in the organic phase; generating low turbidity of aqueous phase and by such method (i.e., MATH), the hydrophobicity nature of the biofilm is detected [54].

In the current study, the hydrophobicity index (HI) recorded 65.72 ± 2.3, 51.61 ± 1.12 and 59.12 ± 5.43% for P. aeruginosa, S. aureus and C. albicans respectively; denoting a higher hydrophobicity property of P. aeruginosa than that exhibited by S. aureus and C. albicans. Upon applying different concentrations (10–100 µg/mL) of both oil, a noticeable reduction in HI was shown (Figs. 3 and 4); implying progressively transition to hydrophilicity state, which reached to the maximum at the highest applied doses. Wherein, A-OS (10 µg/mL) lessened hydrophobicity to 64.18 ± 4.23, 49.18 ± 1.73 and 58.62 ± 6.94% for P. aeruginosa, S. aureus and C. albicans, by 1.54 ± 4.23, 2.43 ± 1.73 and 0.498 ± 6.91% inhibition percentage in the respective order. However, A-OS (100 µg/mL) switched S. aureus and C. albicans biofilms to weak hydrophobicity state (i.e., became hydrophilic) by recording 18.5 ± 1.22 and 22.18 ± 0.94% HI, respectively; implementing 33.1 ± 1.22 and 36.94 ± 0.94% inhibition. While, the HI of P. aeruginosa biofilm altered to be moderate by recording 41.32 ± 1.65% and inhibition percentage recorded 24.04 ± 1.65%. On the other hand, D-OS (10 and 20 µg/mL) insignificantly enhanced the hydrophobicity of P. aeruginosa biofilm by 0.046 ± 2.55 and 0.032 ± 2.364%, respectively. Whereas, at 100 µg/mL inhibited its hydrophobicity by 15.96 ± 0.74%; maintaining HI in the potent region by recording 49.78 ± 0.74%. Regarding S. aureus and C. albicans biofilms, D-OS promoted their hydrophilic affinity in a dose-dependent behavior, reaching to the maximum at 100 µg/mL by recording 25.34 ± 0.422 and 37.89 ± 0.826%, correspondingly, which all remained in the moderate phase of hydrophobicity. Generally, albeit distinct structural variation in cells surface among the examined strains in our study, both oil samples substantiated their efficacy in influencing on hydrophobicity adversely. It is important to highlight that hydrophobicity reflects the microbial attachment or adhesiveness ability, which varies even from strain to strain and influenced by microbial age, microbial surface charge and growth medium [55]. In study performed by Kim et al. [56], 10 µg/ml of antibiofilm FAs (e.g., tricosanoic acids, palmitoleic, myristoleic acid, lauric acid, stearic, heptadecanoic and α-linolenic) reversed the biofilm of Cutibacterium acnes from hydrophobic region to hydrophilic region (hydrophobic index < 20%) simultaneously with biofilm inhibition, which agreed with our results.

Intriguingly, the results of the current investigation declared the existence of significant positive correlation between all examined variables (i.e., inhibition of biofilm, protein, ePS, viability and hydrophobicity) with SCOs concentrations, as signified by Pearson’s correlation coefficients (Table 2) (Figs. 5 and 6). Wherein, oil samples influenced negatively on the biofilm development through modulating microbial-surface interactions, in particular hydrophobic interactions through impacting on surface-associated exopolysaccharides and proteins. In consistent with our results, Pompilio et al. [57] attributed the higher hydrophobicity of Stenotrophomonas maltophilia biofilm to its higher exopolysaccharides content, which was positively correlated with biofilm development. Also, Mu et al. [58] manifested and explained the same finding in S. epidermidis biofilm. Otherwise, several reports documented the independence of biofilm formation on hydrophobicity [53, 59]. Nonetheless, there is a consensus among all studies regarding that the cell surface properties and overall physiological properties of microbes govern the process of biofilm development and maturation. Noteworthy mention that the hydrophobicity is fostered by the action of microbial appendages (e.g., pilli, fimbriae, fibrils, etc.) that scattered on the cell surface. Such organelles contain hydrophobic amino acid residues that expedite noncovalent attachment of the cells on any substratum [54, 55]. However, exopolysaccharides facilitate irreversible adhesion and sheltering the developed cells within the backbone of biofilm [23, 52].

Table 2 Representing the correlation between biofilm inhibition with other studied factors (i.e., viability, ePS, protein and hydrophobicity) by the action of SCOsFig. 5

Contour plot showing the correlation of biofilm suppression by A-OS versus EPS inhibition (left panel) and protein inhibition (right panel) with cell surface hydrophobicity. The diagram was plotted by Minitab 14 software. Different colors elucidate different levels of biofilm suppression. A & B - P. aeruginosa, C & D- S. aureus and E & F- C. albicans

Fig. 6

Contour plot showing the correlation of biofilm suppression by D-OS versus EPS inhibition (left panel) and protein inhibition (right panel) with cell surface hydrophobicity. The diagram was plotted by Minitab 14 software. Different colors elucidate different levels of biofilm suppression. A & D - P. aeruginosa, B & E- S. aureus and C & F- C. albicans

Based on the previous results, it is conspicuous that the antibiofilm potency of SCOs samples appeared more evident against S. aureus. That could be attributed to its physiological and metabolic sensitivity, besides the nature and architecture of its cell wall, which seemed to contribute intrinsically in its susceptibility. Namely, the hydrophilic nature of gram-positive bacteria’s cell wall with their low content of lipids (1–4%) trigger the adsorption and penetration processes of exogenous materials interiorly easier. In contrast, the more complex structure of gram-negative bacterial cell wall with its abundant hydrophobic moieties (11–22% lipid content) serves as potent entry barrier toward detergents and hydrophobic molecules, hindering by such way the internal transportation of SCOs [60]. Strikingly, the superior biocidal potency of fatty acids, especially those of longer chain FAs (i.e., ≥ C12) against gram-positive bacteria such as B. subtilis, Micrococcus luteus, Propionibacterium acnes, Listeria monocytogenes and Clostridium difficile was reported tremendously by several research groups [56, 61,62,63,64,65], which coincident with our results. Additionally, Shukla et al. [62] reported that gram-negative bacteria displayed more resistance to medium- and long-chain FAs than gram-positive bacteria, which also agreed with our results; however, gram-negative bacteria were more susceptible to FAs with C6 or less in their chain. In the same context, [