Exploring the potential of microbial biomass and microbial extracted oils in tribology: a sustainable frontier for environmentally acceptable lubricants

Green Chemistry Letters and Reviews ISSN: (Print) (Online) Journal homepage: www.tandfonline.com/journals/tgcl20 Exploring the potential of microbial biomass and microbial extracted oils in tribology: a sustainable frontier for environmentally acceptable lubricants Szymon Bernat, Francesca Di Bartolomeo, Sergio Armada, Emil Valaker, Nemailla Bonturi, Deni Koseto, Tone Haugen, Ann-Karin Kvernbråten, Petr Stavárek, Marek Večeř & Ladislav Zelenka To cite this article: Szymon Bernat, Francesca Di Bartolomeo, Sergio Armada, Emil Valaker, Nemailla Bonturi, Deni Koseto, Tone Haugen, Ann-Karin Kvernbråten, Petr Stavárek, Marek Večeř & Ladislav Zelenka (2024) Exploring the potential of microbial biomass and microbial extracted oils in tribology: a sustainable frontier for environmentally acceptable lubricants, Green Chemistry Letters and Reviews, 17:1, 2330644, DOI: 10.1080/17518253.2024.2330644 To link to this article: https://doi.org/10.1080/17518253.2024.2330644 © 2024 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group Published online: 22 Mar 2024. Submit your article to this journal Article views: 733 View related articles View Crossmark data Full Terms & Conditions of access and use can be found at https://www.tandfonline.com/action/journalInformation?journalCode=tgcl20 Exploring the potential of microbial biomass and microbial extracted oils in tribology: a sustainable frontier for environmentally acceptable lubricants Szymon Bernat a , Francesca Di Bartolomeo a , Sergio Armada a , Emil Valaker a , Nemailla Bonturi b , Deni Koseto a , Tone Haugen a , Ann-Karin Kvernbråten a , Petr Stavárek c , Marek Večeř d and Ladislav Zelenka e a SINTEF Industry, Trondheim, Norway; b Department of Chemistry and Biotechnology, Tallinn University of Technology, Tallinn, Estonia; c CPF CAS, v. v. i., Prague, Czech Republic; d VŠB-TU Ostrava, FMT, Ostrava, Czech Republic; e Biona Jersín s. r. o., Jersín 45, Czech Republic ABSTRACT Mineral oil-derived lubricants, extracted from fossil fuels, account for approximately 90% of the lubricant market. A large proportion of these lubricants end up in the environment through usage, spillage, and disposal, leading to contamination of aquatic systems, ecosystems, and agricultural lands. To address this, new regulations were released (e.g. Vessel General Permit 2013) to promote the use of Environmentally Acceptable Lubricants (EALs) over conventional, toxic, non-biodegradable mineral oils. Today, the range of EAL is limited, particularly affecting the maritime sector. Since 2013, the variety and effectiveness of EALs have improved, but further advancements and cost reductions are essential. This study focuses on developing sustainable bio-based additives from microbial processes to enhance EALs. These additives, sourced through fermentation, avoid using fossil fuels and do not require arable land, preserving water resources and food production areas. The research reveals that yeast, high in sulfur and phosphorus, and microbial oils, mainly carboxylic acids, effectively stabilize EAL formulations, reducing friction and wear in water-based lubricants. Microbial oils are superior in reducing friction, while yeast offers better wear protection. This study opens the possibility of incorporating various bio-based products into EALs, providing a sustainable, environmentally friendly option. ARTICLE HISTORY Received 15 December 2023 Accepted 11 March 2024 KEYWORDS Environmentally acceptable lubricants; microbial biomass; lubricant additives; Saccharomyces cerevisiae; oleaginous yeast 1. Introduction Environmental concerns and non-renewable resource depletion are pushing research towards renewable sources like microbial oils for tribology applications. The conventional lubricants and materials used in tribology are primarily derived from petroleum resources. Given the environmental concerns and the threatening resource shortage, it is necessary to explore sustainable alternatives (1–3). The adoption of Environmentally Acceptable Lubricants (EALs) is increasingly required in certain applications (4), while their potential is being actively explored on a global scale in various other applications. However, one of the main limitations of the EALs is the limited amount of chemicals compounds that can be used to improve the performance of the base oils. For lubricants, several classification lists exist, determining their safety for humans and the environment or potential risks. The FDA’s 21 CFR § 178.3570 details ‘Lubricants with incidental food contact’ including H1 food-grade lubricant components, unchanged since 1977 (5). European Commission’s LuSC-list includes substances and base oils, focusing on their EU Ecolabel Lubricant (EEL) biodegradability and aquatic toxicity (6). Substances on the LuSC-list must have 100% EEL © 2024 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The terms on which this article has been published allow the posting of the Accepted Manuscript in a repository by the author(s) or with their consent. CONTACT Szymon Bernat [email protected] SINTEF Industry, Richard Birkelands vei 3, 7034 Trondheim, Norway GREEN CHEMISTRY LETTERS AND REVIEWS 2024, VOL. 17, NO. 1, 2330644 https://doi.org/10.1080/17518253.2024.2330644 biodegradability and be non-toxic. The OSPAR List, initiated in 2004, targets chemicals hazardous to marine environments, including organometallics, organohalogens, biocides, and phenols (7). Taken all above, the use of microorganisms might open the possibility of expanding the number of additives that can be used in EALs formulations. Microorganisms, through their versatile metabolic pathways, can produce a wide range of compounds including lipids, polysaccharides, proteins, and nucleic acids as part of their biomass (8). Particularly, certain microbial strains are known to synthesize lipids. These microbial oils, often termed single cell oils (SCOs), have shown promise in several applications including biodiesel production, cosmetics, and nutraceuticals (9). Studies have also established that certain lipids might serve as potent additives, significantly enhancing the frictional performance of lubricants, however the tested lipids, were acquired in a purified commercial form and not from natural or microbial origin (10–12). These microbial lipids are particularly effective when dispersed in waterbased media, underscoring their utility in improving lubricant efficiency in reducing both friction and wear. The potential role of microorganisms in tribology represents a largely unexplored area of research, and so far, very few studies have explored using microorganisms in tribology (3,13). Microbial biomass and oils provide a promising avenue for sustainable alternatives, leveraging the diverse and versatile metabolic capabilities of microorganisms. Yeasts such as Saccharomyces cerevisiae and Rhodotorula toruloides are known for their roles in biofuel production and broader industrial uses (14). Notably, both these yeasts have the capacity to grow on lignocellulosic hydrolysate, a byproduct derived from the breakdown of lignocellulosic biomass (15–18). This property enhances their industrial relevance and sustainability. Lignocellulosic biomass, the most abundant renewable organic material on Earth, primarily consists of lignin, cellulose, and hemicellulose. The hydrolysate of this biomass serves as an inexpensive, readily available, and environmentally friendly substrate for microbial growth and fermentation. The yeast S. cerevisiae is renowned for its capacity to ferment sugars to ethanol, and despite the primary focus on ethanol, the process also results in a substantial yield of microbial biomass, which is often overlooked and discarded. However, this biomass represents a potential source of valuable biomaterials that could be harnessed in tribology and other industrial applications. The repurposing of this ‘by-product’ not only minimizes environmental footprint but also aligns with the principles of a circular bio-economy, thereby enhancing the overall sustainability of industrial operations (Figure 3). The spent yeast biomass, a byproduct of ethanol production, contains various valuable compounds including proteins, nucleic acids, vitamins, and minerals. Given the multi-faceted nature of tribological systems, the yeast biomass could potentially serve as a biodegradable, renewable, and sustainable material alternative. On the other hand, R. toruloides, a basidiomycetous yeast, has been identified as an efficient cell factory for the production of lipids (Figure 4). These single-cell oils (SCOs) have garnered attention in the biofuel industry as potential replacements for plant-derived oils. The advantage of SCOs lies in their renewable nature, the ability to tailor their production to specific lipid profiles, and the possibility of cultivating these yeasts on a variety of substrates, including industrial waste. Conventionally, lubricants are composed of oils derived from either fossil or plant-based sources, each with specific characteristics that influence their performance in tribological applications. The prospect of replacing these traditional oils with microbial SCOs, offers an environmentally friendly alternative, potentially transforming the composition and production of lubricants in the tribological field and could signal a shift towards sustainable practices in the field. This study explores the potential of S. cerevisiae and R. toruloides biomass and oils in tribology and it aims to encourage more research towards using microbial products for eco-friendly lubrication and reduced fossil fuel reliance. The process involves transforming residual forest biomass into yeast biomass (YB) and extracted oils from oleaginous yeast (EOO) through fermentations, showcasing a promising avenue for sustainable lubricant production (Figure 1). 2. Experimental methods 2.1. Ethanol and yeast biomass production from lignocellulosic hydrolysate This paragraph outlines the experimental approach taken to cultivate Saccharomyces cerevisiae on a laboratory scale. The objective was to produce ethanol, employing lignocellulosic hydrolysate as the chosen fermentation substrate. The study documents the collection and processing of Yeast Biomass (YB), referred to as Route 1 in Figure 1. 2.1.1. Yeast strain Saccharomyces cerevisiae Ethanol Red TM industrial strain for bioethanol production was obtain from Leaf by Lesaffre (Marcq-en-Barœul, France) and was used in this study. 2 S. BERNAT ET AL. 2.1.2. Preparation of the Norwegian spruce hydrolysates used for bio-ethanol production process The Norwegian spruce hydrolysates, Excello-90 lignocellulosic hydrolysate provided by biorefinery Borregaard (Sarpsborg, Norway) and BALI TM pulp hydrolysate were used in the study. Excello-90 hydrolysate contains monomeric sugars mixture, with glucose as the main component. BALI TM hydrolysate was produced in the following way: the enzymatic saccharification of sulfite pulped Norway spruceBALI TM pulp was carried out in 3 L bioreactors (Applikon, Schiedam, the Netherlands; working volume, 1.5 litres) during the fermentation in a simultaneous saccharification and fermentation (SSF) process. The enzyme CTec3 Novozyme dosed at 6% w/ w was added as catalyst to initiate the hydrolysis. 2.1.3. Bioreactor cultivations for bio-ethanol production with Saccharomyces cerevisiae Batch cultivations were carried out in 3 L bioreactors (Applikon, Schiedam, the Netherlands; working volume, 1.5 litres) equipped with off-gas O 2 and CO 2 sensors (BlueSens GmbH, Herten, Germany), pH, temperature and dissolved oxygen sensors. 10% of the preculture was used as inoculum. For the preparation of the pre-culture a glycerol stock of S. cerevisiae Ethanol Red TM was used to prepare YPD plated (yeast extract [10 g/L], bacto peptone [20 g/L], dextrose [20 g/L], bacto Agar [20 g/L], pH5.5 HCl adjusted). The YPD plates were incubated at 35°C for 48 h; single colonies from the plates were used to inoculate the pre-culture growing in 150 ml in non-baffled flask and incubated at 200 rpm, 35°C overnight. The starting pure glucose concentration, and glucose concentration in the respective lignocellulosic hydrolysate equivalent, was 40 g/L. In addition, each medium contained the following nutrients: 7.5 g/L (NH 4 )2S0 4 , 14.4 g/L KH 2 P0 4 , 0.5 g/L MgS0 4 -7H 2 0, 2 mL/L trace metals solution, and 1 mL/L vitamins. The trace metals solution contained per liter: 4.5 g CaCl 2 ·2Η 2 0, 4.5 g ZnS0 4 -7Η 2 0, 3 g FeS0 4 -7Η 2 0, 1 g H 3 BO 3 , 1 g MnCl 2 - 4H 2 0, 0.4 g Na 2 Mo0 4 *2H 2 0, 0.3 g CoCl 2 -6H 2 0, 0.1 g CuS0 4 -5H 2 0, 0.1 g KI, 15 g EDTA. The trace metals solution was prepared by dissolving all the components except EDTA in 900 mL ultra-pure water at pH 6 followed by gentle heating and addition of EDTA. Finally, the trace metal solution pH was adjusted to pH 4, and the solution volume was adjusted to 1 L and autoclaved (121°C in 20 min). Trace metals solution was stored at +4°C. The vitamins solution contained per liter: 50 mg biotin, 200 mg p-aminobenzoic acid, 1 g nicotinic acid, 1 g Ca-pantotenate, 1 g pyridoxine-HCl, 1 g thiamine-HCl, 25 g myo-inositol. Biotin was dissolved in 20 mL 0.1 M Figure 1. Residual forest biomass is used to produce yeast biomass (YB) and Extracted Oils from Oleaginous Yeast (EOO) through Saccharomyces cerevisiae and Rhodotorula toruloides fermentations, respectively. In this study, YB and EOO are evaluated for their potential as advanced lubricating ingredients. GREEN CHEMISTRY LETTERS AND REVIEWS 3 NaOH and 900 mL water is added. pH was adjusted to 6.5 with HCl and the rest of the vitamins was added. pH was re-adjusted to 6.5 just before and after adding m-inositol. The final volume of the vitamin solution was adjusted to 1 1 and sterile filtered before storage at 4°C. For all the fermentation conditions, run in duplicates at 35°C and pH 5, dissolved oxygen levels were maintained at a minimum of 30% by automatic adjustment of the stirrer speed (agitation 800 rpm). The aeration rate was constant 0.67 L sterile air/L culture/min. Dissolved oxygen, agitation speed, pH and CO2 concentration in off-gas were measured and logged on-line. Foaming was controlled by adding 1 g/L antifoam A (10% antifoam; Sigma-Aldrich, St. Louis, MO, USA). The fermentation run that was terminated after 24 hours. 2.1.4. Preparation of yeast biomass (YB) for tribological analysis and measurements The yeast biomass was separated from the culture supernatant by centrifugation (10 min, 2000rpm), and washed with distilled water using the Millipore vacuum filtration system. For the monitoring of glucose and ethanol concentrations, supernatant was analysed on Cedex® Bio Analyzer (Roche) and used according to manufacturing instructions. As indication of the strain growth the optical density at 600 nm (OD600) was measured using a benchtop spectrophotometer. 2.2. Lipid production from Rhodotorula toruloides with lignocellulosic This paragraph describes the scaled-up cultivation process of Rhodotorula toruloides aimed at lipid production, with lignocellulosic hydrolysate serving as the fermentation medium. This segment, designated as Route 2 in Figure 1, focuses on the systematic approach to extracting Oleaginous Yeast Oils (EOO) from the cultivated biomass. 2.2.1. Oleaginous yeast strain Rhodotorula toruloides CCT0783 (synonym IFO10076) was obtained from Coleção de Culturas Tropicais (Fundação André Tosello, Campinas, Brazil). 2.2.2. Preparation of the Estonian Birch hemicellulosic hydrolysate The pentose-sugars enriched lignocellulosic hydrolysate stream of Birch (Betula pendula), here named as C5-Birch, was produced and provided by Fibenol OÜ (Tallinn, Estonia). The hexoses were in g/L: 113.5 of glucose, 13.3 of galactose, and 23.4 of mannose. While the pentoses were in g/L: 315.9 of xylose and 9.3 of arabinose. Acetic acid and 5-HMF concentrations were 4.7 and 2.8 g/L, respectively. The amounts of nitrogen and furfural were negligible. The pH of C5-Birch was adjusted to 6.0 with NaOH and the formed solids were removed by centrifugation at 12.000 rcf for 30 min, here in called clarified C5. 2.2.3. Rhodotorula toruloides cultivation conditions for the lipid production and extraction Pre-inoculum and inoculum were prepared as described by Monteiro de Oliveira et al. (16). The 50 liters reactor (New Brunswick™ BioFlo® 610) was filled with 27 liters of water, (NH 4 ) 2 SO 4 at 5 g/l, KH 2 PO 4 at 3 g/l, MgSO 4 .7H 2 O at 0.5 g/l, and antifoam at 0.075 ml/l. The bioreactor was autoclaved by 20 min at 121 °C. After cooling down, the clarified C5 was added to 50 g/l of total sugars and carbon to nitrogen (C/N) ratio of 20 and inoculation performed right after. Cultivation was carried out at 30 °C, dissolved oxygen maintained at 25% by varying stirring (140-360 rpm) and aeration (0.3–1 vvm), pH at 6 by the addition of NaOH or HCl 6 M. Two pulses of clarified C5 were done at 21 and 41 h of cultivation aiming at 50 and 90 g/l of sugars, respectively. Cultivation finished at 90 h. The cells were harvested by centrifugation at 12,000 rcf and 20 min. The wet cells were resuspended in water (1:2 mass ratio) and homogenized at 1300 bar for 15 passages. The slurry was mixed with hexane and ethanol to reach (1:0.5:2, mass ratio). After mixing, the phase separation was done by gravity, and the lipid rich phase was recovered, and hexane was evaporated using rotary evaporator. 2.2.4. Analytical methods Microbial growth was estimated by OD600 nm and measured gravimetrically by dry cell mass. Sugars and sugar alcohols were quantified by HPLC using Rezex RPM Monosaccharide column (Phenomenex, United States) and LC-grade H2O as a mobile phase at a flow rate of 0.6 mL/min at 85°C. Organic acids and other metabolites were also measured by HPLC using Rezex ROA Organic Acid column (Phenomenex, United States) at 45°C and 5 mM sulfuric acid as a mobile phase. Total lipid was done by an adapted Folch method (19). The original Folch methodology (20) was developed for extracting lipids from brain cells which are homogenized using chloroform:methanol at 2:1 vol ratio, followed by adding NaCl 0.9% at 0.2 vol ratio for washing polar lipids and proteins. Meanwhile in this work we used dried intact yeast cells and no washing step was performed. 2.3. Preparation of bio-lubricants In the present study, Yeast Biomass (YB) and Extracted Oleaginous Yeast Oils (EOO) were explored as potential 4 S. BERNAT ET AL. lubricant additives. These additives were dissolved in a water-glycol media, which was formulated by combining distilled water, Diethylene glycol (99% purity and a molar mass of 106.12 g·mol −1 ), and 2-Dimethylaminoethanol (≥99.5% purity and a molar mass of 89.138 g·mol −1 ) in a precise ratio of 60:39:1, respectively. All chemicals in these experiments were used as received without further purification. In the experimental procedure, a water-based fluid (BF) was formulated with biobased additives utilizing a magnetic stirrer, ensuring thorough mixing over a duration of 4 hours at a controlled temperature of 50°C. For the ensuing tribological investigations, additive concentrations were methodically varied: 0.5 wt.% to 5 wt.% forYB additives and 1 wt.% to 3 wt.% for EOO additives. The selection of YB and EOO additive concentrations was determined through a systematic series of iterative experiments. Initially, we chose a starting concentration of 2 wt.% based on preliminary literature review insights. The concentration range was then adjusted from 0.5 wt.% to 5 wt.% for YB and 1 wt.% to 3 wt.% for EOO through iterative testing to evaluate performance implications. These adjustments were guided by two main criteria: the observation of increased friction, indicating reduced lubricant performance, and the identification of a performance plateau, suggesting an optimal concentration level. This process allowed us to refine our concentration choices to those that provided the most beneficial tribological outcomes without compromising the lubricant’s overall performance. The chosen percentages represent a balance between maximizing lubricative benefits and ensuring formulation stability, based on empirical evidence rather than arbitrary selection. 2.4. Testing and characterization methods 2.4.1. Chemical analysis of additives Chemical analysis of obtained bio-base additives was performed with the ICP-MS. Samples were diluted with HNO 3 in a Milestone UltraWAVE microwave oven, subsequently being subjected to analysis via an Agilent 8900 Triple Quadrupole Inductively Coupled Plasma Mass Spectrometry (ICP-QQQ), complemented with an SPS 4 Autosampler. The elements selected for analytical scrutiny encompassed Lithium (Li), Sodium (Na), Magnesium (Mg), Phosphorus (P), Sulfur (S), Potassium (K), Chromium (Cr), Manganese (Mn), Iron (Fe), Nickel (Ni), Copper (Cu), Selenium (Se), and Lead (Pb). Quantification was executed against standards procured from Inorganic Ventures, employing Indium-115 (115In) as the internal standard, ensuring precision and reliability in the analytical data obtained. 2.4.2. Tribological testing Samples of mild carbon steel (ST-52) were cut from a steel bar with a diameter of 30 mm, forming disks with a thickness of 5 mm to be utilized in tribological testing. The disks underwent a grinding process with SiC paper and were subsequently polished using diamond paste, achieving a surface finish characterized by a roughness average (Ra) of 0.1 ± 0.003 μm. A Mitutoyo SJ-301 surface roughness stylus profilometer was employed to measure the roughness, with the average surface roughness (Ra) being represented in 0°, 90°, 180°, and 270° directions to comprehensively describe the surface condition. Following the grinding and polishing process, the disks were subjected to ultrasonic cleaning in a 1:1 solution of ethanol and distilled water for a duration of 5 minutes. The tribological performance of formulated lubricants, under harsh boundary conditions, was assessed with a unidirectional ball-on-disk tribometer from Phoenix Tribology. An alumina ball (fused ceramic) with a diameter of 6 mm, delivered by Precision Ball and Gauge Co., Ltd., was pressed against the ST-52 disk under a dead-weight load of 20 N, corresponding to a maximum Hertzian contact pressure of 2.1 GPa. The disk was rotated at a velocity of 44 mm/s. All lubricants underwent testing at ambient room temperature, with each tribological test lasting for 30 minutes. To validate the repeatability of the outcomes, three experiments were conducted for each condition. Average and standard deviation of data points were calculated and plotted for the last 15 min of the test. Wear volume was quantitatively analyzed utilizing an Alicona Infinite Focus optical 3D microscope and Gwyddion software, dedicated to surface imaging, analysis, and metrology. Measurements of the wear volume were taken from four distinct locations along the wear track for each sample, and subsequently, an average value was computed. 2.4.3. Weartrack investigation The wear track morphology was investigated utilizing a Scanning Electron Microscope-Energy-Dispersive Xray Spectroscopy (FEI Quanta FEG 650). SEM images were captured with an Everhart–Thornley Detector (ETD), while EDX enabled the analysis of the chemical properties of the tribofilms. Throughout this study, a working distance of 10 mm ±1 mm was maintained, and an accelerating voltage of either 10 or 15 keV was applied. It is estimated that, with these voltages, the beam enables the detection of chemical information to a depth of approximately 1 micron within the samples. GREEN CHEMISTRY LETTERS AND REVIEWS 5 3. Results 3.1. Yeast biomass and oils from yeast results 3.1.1. Yeast biomass production process as bioproduct of ethanol fermentation In our exploration of sustainable tribological alternatives, we conducted a lab-scale cultivation of the yeast Saccharomyces cerevisiae (Figure 2), using environmentally friendly and locally sourced substrates. As illustrated in Figure 3, the growth kinetics of S. cerevisiae in a glucose substrate, which served as our reference cultivation condition (Figure 3(a)) in comparison to the cultivation using lignocellulosic hydrolysates, were precisely recorded. This reference cultivation condition not only demonstrated the yeast’s robust growth but also its glucose utilization Figure 2. SEM images of Saccharomyces cerevisiae in YB at 10 kV and 10,000x magnification. Figure (a) shows dense clusters of intact cells. Figure (b) reveals damaged cells with compromised walls and changed shapes. Figure 3. Lab-scale Saccharomyces cerevisiae ethanol production from residual forest biomass. (a) Growth kinetics on glucose. (b) Feedstock utilization and ethanol yield using the feedstocks: BALI pulp, Excello substrate (Norway spruce), and glucose. 6 S. BERNAT ET AL. and ethanol production trajectories. Intriguingly, the biomass derived from this condition was subsequently employed in our tribological experiments, underscoring its potential utility. Furthermore, substrate utilization patterns, particularly in the BALI pulp simultaneous saccharification and fermentation (SSF) process, revealed an initial glucose concentration of 10.78 g/L just 2.5 hours into the experiment. Ethanol production profiles across test conditions were also elucidated, with the BALI pulp SSF process showcasing a peak ethanol production of 8.72 g/L, 9 hours post-inoculation. Notably, the Excello 90 substrate, sourced from Norway spruce, stood out for its efficacy, registering a remarkable ethanol production peak of 20.95 g/L. These findings underscore the potential of S. cerevisiae. While this yeast is celebrated for its role in biofuel production via ethanol synthesis, these findings highlight its alignment with circular economy principles and recyclability. The biomass, often discarded as a byproduct, holds significant promise for tribological applications. 3.1.2. Production of oils from oleaginous yeast (EOO) through fermentation The cultivation of R. toruloides resulted in 36 gdcw/l with a lipid yield on biomass of 0.41 g/gdcw in a process which included a batch (from 0 to 20 hours) and a fed-batch phase (from 20 to 120 hours) (Figure 4). The yield of biomass produced per consumed substrate was 0.5 gdcw/g substrate in the batch phase of the cultivation and it drop after each pulse of C5, reaching 0.2 gdcw/g substrate. An opposite trend was observed for the lipid content in biomass, which increased after each pulse (lipid content, %, Figure 4). As for lipid yield per consumed substrate maintained itself relatively stable over the batch and fed batch phases (0.07-0.1 g lipid/g substrate). 3.2. Lubricant stability While the OEE additives did not show any precipitation, the YB is known to have limited solubility in water-based solutions due to its complex and heterogeneous nature. This limited solubility can lead to sedimentation issues, Figure 4. Physiological profile of R. toruloides cultivated in C5 hydrolysate and lipidomics profile of the produced EOO. GREEN CHEMISTRY LETTERS AND REVIEWS 7 as yeast biomass particles tend to settle down over time. To assess the dispersion stability, a series of experiments were conducted in which 3 wt.% of yeast biomass was added to the lubricant and mixed thoroughly and placed in a glass container. Photographs of the lubricant were taken every 5 days to monitor any changes (Figure 5). After 15 days of testing, sedimentation was observed, as yeast biomass particles settled at the bottom of the container. This sedimentation was indicative of the limited solubility of yeast biomass in the water-based lubricant. The settling of yeast biomass particles over time can have adverse effects on the performance of the lubricant, potentially causing uneven distribution of additives and hindering the intended tribological improvements. However, it is clearly seen that the color of the lubricant changed from a transparent paleyellow solution to a blurrier solution. This is an indication that some of the yeast was dissolved in the solution. 3.3. Tribological testing In the pursuit of enhancing the tribological properties of water-based lubricants, the incorporation of additives has emerged as a common strategy. This study delves into an investigation of the frictional behavior of water-based lubricants, assessed by the pin-on-disc tribometer, when modified with two distinct additives: YB and EOO. The overarching objective is to study the concentration-dependent effects of these additives on friction reduction. When YB is used as additive in the water-based lubricant, a discernible shift in the frictional response of the system became evident when the YB concentration exceeded 0.5 wt%. Before reaching this threshold, the friction coefficient remained relatively stable, hovering around 0.18 (Figures 6 and 7). However, as YB concentrations surpassed 0.5 wt%, a substantial reduction in friction transpired, manifesting as a decline in the Figure 5. Photographs depicting the results of a sedimentation test on a BF mixed with 3 wt.% YB, conducted over a period of 15 days. The test tube on the left (labeled as ‘a’) contains the BF with a 3 wt.% YB mixture, while the test tube on the right (b) contains only the BF for comparison Figure 6. The role of additive concentration on friction of water-based fluid modified with yeast biomass additives (YB) and extracted oils from Oleaginous yeast (EOO) measured with the use of Pin On Disc tribometer. 8 S. BERNAT ET AL.