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Palm oil, widely used in various products, poses environmental and climate change risks. "Yeast oil" produced by Lipomyces starkeyi, an oil-producing yeast, is one of the sustainable alternatives for palm oil and was successfully produced as an edible substitute for palm oil. However, the high cost of the culture medium for oil production remains a challenge for practical applications. Okara is a by-product of tofu and soymilk production. Because yeast extract contributes to the high cost of the culture medium, we considered using okara, a cheap and nitrogen-rich substitute, to reduce costs. In the initial study with okara, the production of yeast oil was confirmed, but its productivity was low due to the high viscosity caused by its insoluble solids. To overcome this, we extracted and concentrated nitrogen components in okara using the membrane concentration process. Using NF (nanofiltration) membrane concentration, oil production increased 1.69 and 1.44 times compared to the unconcentrated extract solution (added 90% (v/v) in the culture medium) and yeast extract (added 5% (w/v) in the culture medium), respectively. These findings indicate the potential for a significant cost reduction in the culture medium and high oil yield in yeast oil production. [ABSTRACT FROM AUTHOR]

The oleaginous yeast Lipomyces starkeyi is an attractive industrial yeast that can accumulate high amounts of intracellular lipids. Identification of genes involved in lipid accumulation contributes not only to elucidating the lipid accumulation mechanism but also to breeding industrially useful high lipid-producing strains. In this study, the suppressed lipid accumulation-related gene (SLA1) was identified as the causative gene of the sr22 mutant with decreased lipid productivity. Suppressed lipid accumulation-related gene mutation reduced gene expression in lipid biosynthesis and increased gene expression in β-oxidation. Our results suggest that SLA1 mutation may leads to decreased lipid productivity. Suppressed lipid accumulation-related gene deletion also exhibited decreased gene expression in β-oxidation and increased lipid accumulation, suggesting that SLA1 deletion is a useful tool to improve lipid accumulation in L. starkeyi for industrialization. [ABSTRACT FROM AUTHOR]

ABSTRACT The oleaginous yeast Lipomyces starkeyi is recognized for its remarkable lipid accumulation under nitrogen‐limited conditions. However, precise control of microbial lipid production in L. starkeyi remains challenging due to the complexity of nutrient media. We developed a two‐stage fed‐batch fermentation process using a well‐defined synthetic medium in a 5‐L bioreactor. In the first stage, the specific growth rate was maintained at a designated level by maximizing the cell density through optimizing the feeding rate, molar carbon‐to‐nitrogen (C/N) ratio, and phosphate concentration in feeding media, achieving a high cell density of 213 ± 10 × 107 cells mL−1. In the second stage, we optimized the molar C/N ratio in the feeding medium for lipid production and achieved high biomass (130 ± 5 g L−1), lipid titer (88 ± 6 g L−1), and lipid content (67% ± 2% of dry cellular weight). Our approach yielded a high lipid titer, comparable to the highest reported value of 68 g L−1 achieved in a nutrient medium, by optimizing cultivation conditions with a synthetic medium in L. starkeyi. This highlights the importance of well‐established yet powerful bioprocess approaches for the precise control of microbial cultivation.

Only a few reports available about the assimilation of hydrophobic or oil-based feedstock as carbon sources by Lipomyces starkeyi. In this study, the ability of L. starkeyi to efficiently utilize free fatty acids (FFAs) and real biomass like palm acid oil (PAO) as well as crude palm kernel oil (CPKO) for growth and lipid production was investigated. PAO, CPKO, and FFAs were evaluated as sole carbon sources or in the mixed medium containing glucose. L. starkeyi was able to grow on the medium supplemented with PAO and FFAs, which contained long-chain length FAs and accumulated lipids up to 35% (w/w) of its dry cell weight. The highest lipid content and lipid concentration were achieved at 50% (w/w) and 10.1 g/L, respectively, when L. starkeyi was cultured in nitrogen-limited mineral medium (-NMM) supplemented with PAO emulsion. Hydrophobic substrate like PAO could be served as promising carbon source for L. starkeyi. • Lipomyces starkeyi could grow on specific types of hydrocarbon or plant-oil-based feedstocks and accumulate lipid. • Palm acid oil (PAO) and long-chain length fatty acids such as C16:0 and C18:1 could be efficiently utilized by L. starkeyi. • L. starkeyi was not able to use carbon sources consisting of C12:0 such as CPKO and sodium laurate. • Addition of Tween 20 as a surfactant to emulsify PAO increases the availability of PAO in the culture medium. [ABSTRACT FROM AUTHOR]

The shift from fossil fuels to renewable energy sources is crucial in addressing environmental challenges. Vegetable oils have been focused on as the main potential source for biodiesel and biolubricant production. However, due to their fatty acid (FA) composition they are characterized by low stability to oxidation and variable viscosity. Single-cell oils (SCOs) from oleaginous microorganisms are a possible alternative to vegetable oils: their composition is more suitable, and it can further be improved by controlling the fermentation's physiological conditions. In the present study, the production of SCOs with targeted technological properties from Lipomyces starkeyi in fermentation under controlled temperatures was assessed. A dairy effluent (scotta) was used as the fermentation substrate to improve the economic sustainability of the process. Batch aerobic fermentations were carried out in a fermenter at two different temperatures (25 °C and 30 °C). The fermentation yields and SCO FA profiles were analyzed. The highest yields of biomass (9.76 g L−1) and microbial oil (1.83 g L−1) were obtained from fermentations carried out at 30 °C. Furthermore, a significantly lower content (46% vs. 55%) of unsaturated FAs and higher content (11% vs. 1.5%) of shorter-chain saturated FAs, with myristic acid almost matching stearic acid, were detected at 30 °C in comparison to 25 °C. Very low peroxide values were also found (0.14 meq O2 kg−1 at 30 °C and 0 meq O2 kg−1 at 25 °C). These results indicate that these SCOs were highly oxidation-resistant, and that a higher fermentation temperature improves their oxidative stability and tribophysical features. The biodiesels' technological properties, calculated from the FA composition, were within the limits of both U.S. standards and E.U. regulations. Then, SCOs produced from L. starkeyi by fermentation of dairy effluents carried out under controlled temperature can be considered a suitable alternative to vegetable oils to produce biodiesel and biolubricants. [ABSTRACT FROM AUTHOR]

The oleaginous yeast, Lipomyces starkeyi can have diverse industrial applications due to its remarkable capacity to use various carbon sources for the biosynthesis intracellular triacylglycerides (TAGs). In L. starkeyi , TAG synthesis is enhanced through upregulation of genes involved in citrate-mediated acyl-CoA synthesis and Kennedy pathways through the transcriptional regulator LsSpt23p. High expression of LsSPT23 can considerably enhance TAG production. Altering the regulatory factors associated with lipid production can substantially augment lipid productivity. In this study, we identified and examined the L. starkeyi homolog sucrose nonfermenting 1 SNF1 (LsSNF1) of YlSNF1 , which encodes a negative regulator of lipid biosynthesis in the oleaginous yeast Yarrowia lipolytica. The deletion of LsSNF1 enhanced TAG productivity in L. starkeyi , suggesting that LsSnf1p is a negative regulator in TAG production. The enhancement of TAG production following deletion of LsSNF1 can primarily be attributed to the upregulation of genes in the citrate-mediated acyl-CoA synthesis and Kennedy pathways, pivotal routes in TAG biosynthesis. The overexpression of LsSPT23 enhanced lipid productivity; strain overexpressing LsSPT23 and without LsSNF1 exhibited increased TAG production capacity per cell. LsSnf1p also has a significant role in the utilization of carbon sources, including xylose or glycerol, in L. starkeyi. Our study results elucidated the role of LsSnf1p in the negative regulation of TAG synthesis in L. starkeyi , which has not previously been reported. • Lipomyces starkeyi SNF1 homolog (LsSNF1) was identified and the LsSNF1 deletion mutant was constructed. • LsSnf1p had important roles in lipid production and carbon-source utilization in L. starkeyi. • The overexpression of LsSpt23p and without LsSnf1p exhibited increased TAG production capacity per cell. [ABSTRACT FROM AUTHOR]

Palm oil, widely used in various products, poses environmental and climate change risks. “Yeast oil” produced by Lipomyces starkeyi, an oil-producing yeast, is one of the sustainable alternatives for palm oil and was successfully produced as an edible substitute for palm oil. However, the high cost of the culture medium for oil production remains a challenge for practical applications. Okara is a by-product of tofu and soymilk production. Because yeast extract contributes to the high cost of the culture medium, we considered using okara, a cheap and nitrogen-rich substitute, to reduce costs. In the initial study with okara, the production of yeast oil was confirmed, but its productivity was low due to the high viscosity caused by its insoluble solids. To overcome this, we extracted and concentrated nitrogen components in okara using the membrane concentration process. Using NF (nanofiltration) membrane concentration, oil production increased 1.69 and 1.44 times compared to the unconcentrated extract solution (added 90% (v/v) in the culture medium) and yeast extract (added 5% (w/v) in the culture medium), respectively. These findings indicate the potential for a significant cost reduction in the culture medium and high oil yield in yeast oil production.

The clean energy demand and limited fossil fuel reserves require an alternate source that is sustainable and eco-friendly. This demand for clean energy steered the introduction of biofuels such as bioethanol and biodiesel. The third-generation biodiesel is promising as it surpasses the difficulties associated with food security and land usage. The third-generation biodiesel comprises biodiesel derived from oil produced by oleaginous microbes. The term oleaginous refers to microbes with the ability to accumulate lipids to about 20% of the biomass and is found in the form of triacylglycerols. Yeasts can be grown easily on a commercial scale and are amenable to modifications to increase single-cell oil (SCO) productivity. The oleaginous yeast L. starkeyi is a potential lipid producer that can accumulate up to 70% of SCO of its cell dry weight under optimum conditions. Compared to other oleaginous organisms, it can be grown on a wide range of feedstock and a good part of the lipid produced can be converted to biodiesel. This review presents the recent advances in single-cell oil production from L starkeyi and strategies to increase lipid production are analyzed.

Sugar-rich waste streams, generated in very high quantities worldwide, constitute an important source of environmental pollution. Their eco-friendly conversions into a plethora of added-value compounds through the use of microbial fermentations is currently a very "hot" scientific topic. The aim of this study, was to assess the potential of single cell oil (SCO), microbial mass and citric acid (CA) production by non-conventional yeast strains growing on expired ("waste") glucose. Six yeast strains (viz. Rhodosporidium toruloides DSM 4444, Rhodotorula glutinis NRRL YB-252, R. toruloides NRRL Y-27012, Yarrowia lipolytica LFMB Y-20, Y. lipolytica ACA-DC 50109 and Lipomyces starkeyi DSM 70296) were initially grown in shake flasks with expired glucose used as substrate under nitrogen limitation, in order to "boost" the cellular metabolism towards the synthesis of SCO and CA, and their growth response was quantitatively evaluated. Initial glucose concentration (Glc0) was adjusted at c. 50 g/L. Besides Y. lipolytica, all other yeast strains produced noticeable SCO quantities [lipid in dry cell weight (DCW) ranging from 25.3% w/w to 55.1% w/w]. Lipids of all yeasts contained significant quantities of oleic acid, being perfect candidates for the synthesis of 2nd generation biodiesel. The highest DCW production (=13.6 g/L) was obtained by L. starkeyi DSM 70296, while both Y. lipolytica strains did not accumulate noticeable lipid quantities, but produced non-negligible CA amounts. The most promising CA-producing strain, namely Y. lipolytica ACA-DC 50109 was further studied in stirred-tank bioreactor systems, while the very promising DCW- and SCO-producing L. starkeyi DSM 70296 was further studied in shake flasks. Both strains were grown on media presenting higher Glc0 concentrations and the same initial nitrogen quantity as previously. Indeed, L. starkeyi grown at Glc0 = 85 g/L, produced DCWmax = 34.0 g/L, that contained lipid =34.1% w/w (thus SCO was =11.6 g/L). The strain ACA-DC 50109 in stirred tank bioreactor with Glc0 ≈ 105 g/L produced CA up to 46 g/L (yield of CA produced on glucose consumed; YCA/Glc ≈ 0.45 g/g). Finally, in fed-batch bioreactor experiment, the significant CA quantity of 82.0 g/L (YCA/Glc = 0.50 g/g) was recorded. Concluding, "waste" glucose proved to be a suitable substrate for a number of non-conventional yeast strains. Y. lipolytica ACA-DC 50109 produced significant quantities of CA while L. starkeyi DSM 70296 was a very interesting DCW- and SCO-producing candidate. These strains can be used as potential cell factories amenable to convert glucose-based residues into the mentioned metabolic compounds, that present high importance for food, chemical and biofuel facilities. [ABSTRACT FROM AUTHOR]

The shift from fossil fuels to renewable energy sources is crucial in addressing environmental challenges. Vegetable oils have been focused on as the main potential source for biodiesel and biolubricant production. However, due to their fatty acid (FA) composition they are characterized by low stability to oxidation and variable viscosity. Single-cell oils (SCOs) from oleaginous microorganisms are a possible alternative to vegetable oils: their composition is more suitable, and it can further be improved by controlling the fermentation’s physiological conditions. In the present study, the production of SCOs with targeted technological properties from Lipomyces starkeyi in fermentation under controlled temperatures was assessed. A dairy effluent (scotta) was used as the fermentation substrate to improve the economic sustainability of the process. Batch aerobic fermentations were carried out in a fermenter at two different temperatures (25 °C and 30 °C). The fermentation yields and SCO FA profiles were analyzed. The highest yields of biomass (9.76 g L−1) and microbial oil (1.83 g L−1) were obtained from fermentations carried out at 30 °C. Furthermore, a significantly lower content (46% vs. 55%) of unsaturated FAs and higher content (11% vs. 1.5%) of shorter-chain saturated FAs, with myristic acid almost matching stearic acid, were detected at 30 °C in comparison to 25 °C. Very low peroxide values were also found (0.14 meq O2 kg−1 at 30 °C and 0 meq O2 kg−1 at 25 °C). These results indicate that these SCOs were highly oxidation-resistant, and that a higher fermentation temperature improves their oxidative stability and tribophysical features. The biodiesels’ technological properties, calculated from the FA composition, were within the limits of both U.S. standards and E.U. regulations. Then, SCOs produced from L. starkeyi by fermentation of dairy effluents carried out under controlled temperature can be considered a suitable alternative to vegetable oils to produce biodiesel and biolubricants.

Recently, microbial oil has become one of the promising next-generation feedstocks for producing biodiesel. While microbial oil can be extracted from different sources, there is only limited work on microbial production from fruits and vegetables. In this work, biodiesel was extracted through a two-step process: microbial conversion of vegetable waste into microbial oil using Lipomyces starkeyi, followed by transesterification of microbial oil into biodiesel. The lipid accumulation, composition of microbial oil, and the fuel properties of biodiesel were evaluated. The microbial oil consisted mainly of C16:0, C18:0 and C18:1, which were close to the properties of palm oil. The fuel properties of biodiesel comply with the EN14214:2012 standard. Thus, the vegetable waste can be a good biodiesel feedstock. Three blends (MOB10, MOB20 and MOB30 with 10, 20, and 30% of biodiesel) were tested for engine performance and emission characteristics in a 3.5 kW VCR research engine. At full load, MOB20 reduced the pollutant emissions of CO and HC by 47.8% and 33.2% with the penalty of increased NOx by 3.9%, while BTE reduced by 0.8% with the increased BSFC by 5.2%. Thus, the addition of vegetable waste biodiesel blends reduced the emissions of CO and HC significantly with slight reduction of brake thermal efficiency. [ABSTRACT FROM AUTHOR]

The clean energy demand and limited fossil fuel reserves require an alternate source that is sustainable and eco-friendly. This demand for clean energy steered the introduction of biofuels such as bioethanol and biodiesel. The third-generation biodiesel is promising as it surpasses the difficulties associated with food security and land usage. The third-generation biodiesel comprises biodiesel derived from oil produced by oleaginous microbes. The term oleaginous refers to microbes with the ability to accumulate lipids to about 20% of the biomass and is found in the form of triacylglycerols. Yeasts can be grown easily on a commercial scale and are amenable to modifications to increase single-cell oil (SCO) productivity. The oleaginous yeast L. starkeyi is a potential lipid producer that can accumulate up to 70% of SCO of its cell dry weight under optimum conditions. Compared to other oleaginous organisms, it can be grown on a wide range of feedstock and a good part of the lipid produced can be converted to biodiesel. This review presents the recent advances in single-cell oil production from L starkeyi and strategies to increase lipid production are analyzed. [ABSTRACT FROM AUTHOR]

The production of palm oil, which is used in various foods, is associated with environmental destruction and climate change risks; therefore, there is an urgent need for sustainable alternatives. "Yeast oil" produced by Lipomyces starkeyi, an oil-producing yeast, is expected to solve these problems because its fatty acid composition is similar to that of palm oil. To date, we have successfully developed yeast oil as an edible alternative to palm oil. However, conventional processes, including cell collection and lyophilization, are difficult to industrialize in terms of equipment and cost. Therefore, a method for extracting yeast oil from the emulsified liquid generated by crushing the culture was investigated. It is presumed that the emulsified state is stable owing to the components derived from yeast cells and metabolites; thus, solid–liquid filtration separation was attempted before extraction. The extraction recovery ratio of yeast oil was 98.2% when a hexane/ethanol mixture (3:1) was added to the residue after filtration. Furthermore, the energy consumption and processing cost of this new process were estimated to be 26% and 34%, respectively, of that of conventional methods, suggesting that the new process has potential for practical applications. [ABSTRACT FROM AUTHOR]

Pine needles are relatively less explored lignocellulosic forest waste which can be saccharified to fermentable sugars and be used in subsequent conversion to biofuels. Microbial lipids were produced from pine needles hydrolysate and recovered lipid liquor. Ultrasound assisted pretreatment increased the cellulose content by 20.679%, 27.17%, 32.21%, and 35.61% when treated for 60 min at varying NaOH concentrations (1–4%). The 4-feed batch sachharification was performed to investigate the effect of increasing substrate loadings and fixed initial concentration (5% w/v) and yielded maximum increase of 70.23% in reducing sugars. The pine needle hydrolysate yielded 19.581 g/L total lipids, 35.888 g/L biomass concentration and lipid concentration at 67.46% g/g. Simultaneously, at equimolar ratios of HCl and liquor resulted in highest yields for total lipids (6.92 g/L) and traces of inhibitors. The fatty acids distribution and biodiesel properties revealed a similar biodiesel quality to that of palm oil. Findings from this study facilitate the complete utilisation of pine needles for microbial lipid production to resolve forest fires, and in keeping view of climate, fuels and inorganic resources towards developing a circular bioeconomy. [Display omitted] [ABSTRACT FROM AUTHOR]

The oleaginous yeast Lipomyces starkeyi is a promising triacylglycerol (TAG) producer for biodiesel fuel. However, it is necessary to further improve TAG productivity in L. starkeyi from a mixed sugar of glucose and xylose. This study aimed to construct an L. starkeyi mutant with increased TAG productivity from glucose/xylose mixed-sugar and to elucidate the causes underlying increased lipid productivity. Ultra-violet (UV) mutagenesis combined with enrichment culture with ethanol and H 2 O 2 and selection of low-density cells was applied to L. starkeyi to obtain the L. starkeyi mutant strain UMP47, which exhibited higher TAG production from glucose/xylose. Transcriptome analysis revealed high expression of genes involved in transporter activity and carbohydrate metabolism, whereas genes involved in DNA replication exhibited lower expression in the mutant strain UMP47 than in the wild-type strain. Altogether, the lipid productivity of L. starkeyi was successfully improved by UV mutagenesis. Transcriptome analysis suggested the importance of previously unidentified genes in TAG production. This study provides information on potential target genes for improving TAG production through the genetic modification of oleaginous yeast. • Lipomyces starkeyi is a promising triacylglycerol producer for biodiesel fuel. • UV mutagenesis successfully improved the lipid productivity of L. starkeyi. • UMP47 strain showed increased TAG productivity from glucose/xylose mixed-sugar. • Transport and carbohydrate metabolism exhibited higher expression in the mutant. • DNA replication exhibited lower expression in the mutant. [ABSTRACT FROM AUTHOR]

Microorganisms have a high potential as biofuel sources. Co-culture of microalgae and yeasts can result in high lipid production as a modification treatment. The goal of this study was to see how the co-culture of the Glagah consortium (diversity of associated microalgae and bacteria from Glagah Lagoon, Yogyakarta) and Lipomyces starkeyi affected the production of biomass, lipids, proteins, and carbohydrates. The culture was performed under airtight conditions on a shaker at 127 rpm, with a light intensity of 27.75 mol/m²/s and a temperature of 30°C. The culture was subjected to a dark: light (6:18) treatment. Biomass was measured by dry weight, lipids by the Bligh and Dyer method, proteins by the Bradford method and carbohydrates by the phenol- sulfuric acid method. On day 3, L. starkey culture produced the most biomass, yielding 2.21 g/L with a productivity of 0.49 g/ L/day. On day 4, the highest lipids produced from co-culture treatment yielded 1.03 g/g with a productivity of 0.21 g/L/day. The highest protein yield was obtained from L. starkeyi culture treatment on day 4, yielding 0.60 g/g with a productivity of 0.12 g/L/ day. On day 6, co-culture produced the total carbohydrates, yielding 4.78 g/g with a productivity of 0.68 g/L/day. The co-culture treatment produced the highest lipids and carbohydrates production (1.03 g/g and 4.78 g/g) and productivity (0.21 g/L/day and 0.68 g/L/day), while L. starkeyi culture produced the highest total biomass and protein production (2.21 g/L and 0.6 g/g) and productivity (0.49 g\L\day and 0.12 g/L/day). In microalgae culture, CO2 generally given directly through the aeration process. In this study, the source of CO2 was yeast, whereas yeast also obtained O2 from microalgae in the consortium for their metabolic process. This mutualism symbiosis will help in providing benefits in reducing the costs for the cultivation process, especially in optimizing the production of biomass an lipids. [ABSTRACT FROM AUTHOR]

The oleaginous yeast Lipomyces starkeyi has considerable potential in industrial application, since it can accumulate a large amount of triacylglycerol (TAG), which is produced from sugars under nitrogen limitation condition. However, the regulation of lipogenesis in L. starkeyi has not been investigated in depth. In this study, we compared the genome sequences of wild-type and mutants with increased TAG productivity, and identified a regulatory protein, LsSpt23p, which contributes to the regulation of TAG synthesis in L. starkeyi. L. starkeyi mutants overexpressing LsSPT23 had increased TAG productivity compared with the wild-type strain. Quantitative real-time PCR analysis showed that LsSpt23p upregulated the expression of GPD1, which encodes glycerol 3-phosphate dehydrogenase; the Kennedy pathway genes SCT1, SLC1, PAH1, DGA1, and DGA2; the citrate-mediated acyl-CoA synthesis pathway-related genes ACL1, ACL2, ACC1, FAS1, and FAS2; and OLE1, which encodes ∆9 fatty acid desaturase. Chromatin immunoprecipitation-quantitative PCR assays indicated that LsSpt23p acts as a direct regulator of SLC1 and PAH1, all the citrate-mediated acyl-CoA synthesis pathway–related genes, and OLE1. These results indicate that LsSpt23p regulates TAG synthesis. Phosphatidic acid is a common substrate of phosphatidic acid phosphohydrolase, which is used for TAG synthesis, and phosphatidate cytidylyltransferase 1 for phospholipid synthesis in the Kennedy pathway. LsSpt23p directly regulated PAH1 but did not affect the expression of CDS1, suggesting that the preferred route of carbon is the Pah1p-mediated TAG synthesis pathway under nitrogen limitation condition. The present study contributes to understanding the regulation of TAG synthesis, and will be valuable in future improvement of TAG productivity in oleaginous yeasts. Key points: LsSpt23p was identified as a positive regulator of TAG biosynthesis LsSPT23 overexpression enhanced TAG biosynthesis gene expression and TAG production LsSPT23M1108Toverexpression mutant showed fivefold higher TAG production than control [ABSTRACT FROM AUTHOR]

The oleaginous yeast Lipomyces starkeyi has a high capacity for starch assimilation, but the genes involved and specific mechanisms in starch degradation remain unclear. This study aimed to identify the critical carbohydrate-active enzyme (CAZyme) genes contributing to starch degradation in L. starkeyi. Comparative transcriptome analysis of cells cultured in glucose and soluble starch medium revealed that 55 CAZymes (including transcript IDs 3772, 1803, and 7314) were highly expressed in soluble starch medium. Protein domain structure and disruption mutant analyses revealed that 3772 encodes the sole secreted α-amylase (LsAmy1p), whereas 1803 and 7314 encode secreted α-glucosidase (LsAgd1p and LsAgd2p, respectively). Triple-gene disruption exhibited severely impaired growth in soluble starch, dextrin, and raw starch media, highlighting their critical role in degrading polysaccharides composed of glucose linked by α-1,4-glucosidic bonds. This study provided insights into the complex starch degradation mechanism in L. starkeyi., (© The Author(s) 2024. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

The purpose of this study is to evaluate the effect of high hydrostatic pressure (HHP) as a novel approach for yeast cell disruption and lipid extraction from Lipomyces starkeyi DSM 70295 grown in glucose medium (40 g/L and C/N:55/1) at initial pH of 5.0, 25°C, and 130 rpm for 8 days. HHP extraction conditions including pressure, time, and temperature were optimized by response surface methodology. The high speed homogenizer‐assisted extraction (HSH) was also used for comparison. The biomass subjected to HHP was examined under scanning electron microscopy and light microscope. A maximal lipid yield of 45.8 ± 2.1% in dry cell basis (w/w) was achieved at 200 MPa, 40°C, and 15 min, while a minimum yield of 15.2 ± 0.9% was observed at 300 MPa, 40°C, and 10 min (p < 0.05). The lipid yield decreased with increasing pressure. It was demonstrated that low pressure (200 MPa) collapsed the cells, while high pressure (400 MPa) created protrusions on the cell wall and cell fragments spread in the environment. This study favors HHP as a promising method for Lipomyces oil extraction. Practical Application: Single‐cell oils are considered future alternatives to plant‐based oils as food additives and dietary supplements. Oleaginous microorganisms accumulate oils in their cell plasma, which makes extraction essential. One of the main obstacles with existing methods is the utilization of strong acids to destroy cell walls. This study aims to demonstrate high hydrostatic pressure as a rapid method for lipid extraction from oleaginous yeast Lipomyces starkeyi. [ABSTRACT FROM AUTHOR]

Sugar-rich waste streams, generated in very high quantities worldwide, constitute an important source of environmental pollution. Their eco-friendly conversions into a plethora of added-value compounds through the use of microbial fermentations is currently a very “hot” scientific topic. The aim of this study, was to assess the potential of single cell oil (SCO), microbial mass and citric acid (CA) production by non-conventional yeast strains growing on expired (“waste”) glucose. Six yeast strains (viz. Rhodosporidium toruloides DSM 4444, Rhodotorula glutinis NRRL YB-252, R. toruloides NRRL Y-27012, Yarrowia lipolytica LFMB Y-20, Y. lipolytica ACA-DC 50109 and Lipomyces starkeyi DSM 70296) were initially grown in shake flasks with expired glucose used as substrate under nitrogen limitation, in order to “boost” the cellular metabolism towards the synthesis of SCO and CA, and their growth response was quantitatively evaluated. Initial glucose concentration (Glc0) was adjusted at c. 50 g/L. Besides Y. lipolytica, all other yeast strains produced noticeable SCO quantities [lipid in dry cell weight (DCW) ranging from 25.3% w/w to 55.1% w/w]. Lipids of all yeasts contained significant quantities of oleic acid, being perfect candidates for the synthesis of 2nd generation biodiesel. The highest DCW production (=13.6 g/L) was obtained by L. starkeyi DSM 70296, while both Y. lipolytica strains did not accumulate noticeable lipid quantities, but produced non-negligible CA amounts. The most promising CA-producing strain, namely Y. lipolytica ACA-DC 50109 was further studied in stirred-tank bioreactor systems, while the very promising DCW- and SCO-producing L. starkeyi DSM 70296 was further studied in shake flasks. Both strains were grown on media presenting higher Glc0 concentrations and the same initial nitrogen quantity as previously. Indeed, L. starkeyi grown at Glc0 = 85 g/L, produced DCWmax = 34.0 g/L, that contained lipid =34.1% w/w (thus SCO was =11.6 g/L). The strain ACA-DC 50109 in stirred tank bioreactor with Glc0 ≈ 105 g/L produced CA up to 46 g/L (yield of CA produced on glucose consumed; YCA/Glc ≈ 0.45 g/g). Finally, in fed-batch bioreactor experiment, the significant CA quantity of 82.0 g/L (YCA/Glc = 0.50 g/g) was recorded. Concluding, “waste” glucose proved to be a suitable substrate for a number of non-conventional yeast strains. Y. lipolytica ACA-DC 50109 produced significant quantities of CA while L. starkeyi DSM 70296 was a very interesting DCW- and SCO-producing candidate. These strains can be used as potential cell factories amenable to convert glucose-based residues into the mentioned metabolic compounds, that present high importance for food, chemical and biofuel facilities.

Oleaginous yeasts have received significant attention due to their substantial lipid storage capability. The accumulated lipids can be utilized directly or processed into various bioproducts and biofuels. Lipomyces starkeyi is an oleaginous yeast capable of using multiple plant-based sugars, such as glucose, xylose, and cellobiose. It is, however, a relatively unexplored yeast due to limited knowledge about its physiology. In this study, we have evaluated the growth of L. starkeyi on different sugars and performed transcriptomic and metabolomic analyses to understand the underlying mechanisms of sugar metabolism. Principal component analysis showed clear differences resulting from growth on different sugars. We have further reported various metabolic pathways activated during growth on these sugars. We also observed non-specific regulation in L. starkeyi and have updated the gene annotations for the NRRL Y-11557 strain. This analysis provides a foundation for understanding the metabolism of these plant-based sugars and potentially valuable information to guide the metabolic engineering of L. starkeyi to produce bioproducts and biofuels. Key points: • L. starkeyi metabolism reprograms for consumption of different plant-based sugars. • Non-specific regulation was observed during growth on cellobiose. • L. starkeyi secretes β-glucosidases for extracellular hydrolysis of cellobiose. [ABSTRACT FROM AUTHOR]

The efficient valorization of lignocellulosic biomass into valuable chemicals represents a cornerstone for advancing sustainable bioeconomy practices. This study focuses on the innovative conversion of oil palm empty fruit bunch (OPEFB) into lignin monomeric alcohols and lipids through a novel two-step process combining alkaline nitrobenzene depolymerization and bioconversion by Lipomyces starkeyi. Initially, OPEFB undergoes alkaline nitrobenzene depolymerization to break down complex lignin structures into monomeric aldehydes. The resulting derivates serve as substrates for the bioconversion by L. starkeyi , which is competent at metabolizing lignin-derived compounds into higher-value lignin monomeric alcohols (syringyl, vanillyl, and 4-hydroxybenzyl alcohol) and lipids, simultaneously. The strategy delineates the optimization of depolymerization conditions, sodium hydroxide concentration, nitrobenzene effect, and reaction time to maximize the yield of depolymerized lignin suitable for bioconversion. Subsequently, the bioconversion process toward cell growth, lipid production, and fatty acid profiles in various fermentations by L. starkeyi was explored. The highest syringaldehyde and vanillin obtained reached up to 4.81 mM and 0.64 mM, respectively. Then, it successfully produced 3.57 mM and 0.40 mM of syringyl alcohol and vanillyl alcohol, respectively, with 5.86 g L−1 of the lipid titer. The study demonstrates the potential of this integrated approach to convert OPEFB into valuable lignin-derived biochemicals and lipids, thereby contributing to the development of alternative strategies for biomass utilization efficiently. [Display omitted] • Lignin monomeric alcohols and lipids were produced from OPEFB. • The two-step utilization process of OPEFB lignin valorization was demonstrated. • Syringaldehyde, vanillin, and 4-hydroxybenzaldehyde were extracted from OPEFB. • Lignin alcohol production and lipid biosynthesis via L. starkeyi were evaluated. • Bioconversion achieved significant amounts of lipid and lignin alcohol production. [ABSTRACT FROM AUTHOR]

Lignin carries great potential as an attractive alternative to petroleum-based products. In particular, biological lignin conversion to various value-added products has recently gained attention owing to its promising benefits in supporting the future bioeconomy. The oleaginous yeast Lipomyces starkeyi is a promising organism due to its impressive native abilities to tolerate and metabolize various lignin monomer derivatives and its ability to synthesize single-cell oil (lipids) inside the cells. Herein, we investigated the coupling abilities of lignin monomer metabolism and lipid biosynthesis via L. starkeyi. Three representatives of lignin derivative aldehydes (4-hydroxybenzaldehyde (H-lignin), vanillin (G-lignin), and syringaldehyde (S-lignin)) were converted into their alcohol forms (4-hydroxybenzyl alcohol, vanillyl alcohol, and syringyl alcohol, respectively) without the formation of undesirable byproducts, and each was maintained in its alcohol form for the remaining cultivation. In addition, a double concentration of syringaldehyde in individual lignin fermentation promoted cell growth (17.01 g L−1) and lipid accumulation (30.72 % (w w−1)). However, mixed fermentation of all lignin aldehydes resulted in heavy inhibition of the cells. Hence, combination fermentation strategies of high initial inoculum size using a medium with a high C:N ratio were beneficial to improving the fermentation time and lipid titer increment up to 2.6-fold. Overall, this study describes the first prospective of lignin alcohol and lipid production from lignin derivative aldehydes via L. starkeyi. [Display omitted] • Lignin derivative alcohols were produced from lignin aldehydes by L. starkeyi. • Lignin derivative alcohols metabolism via L. starkeyi was evaluated. • L. starkeyi lipid biosynthesis while fermented under lignin derivative aldehydes. • High inoculum size and high C:N ratio improved fermentation time and lipid titer. [ABSTRACT FROM AUTHOR]

[Display omitted] • Combination of SSF of corn stover with CBP of cassava starch was firstly developed. • The combination strategy showed synergistic effect for improving lipid production. • Rapid hydrolysis of starch into fermentable sugars eliminated a pre-hydrolysis step. • The enzyme cost could be significantly decreased via the combination strategy. • Biodiesel from the combination strategy showed high-quality fuel properties. Highly integrated processes are crucial for the commercial success of microbial lipid production from low-cost substrates. Here, combination of simultaneous saccharification and fermentation (SSF) of corn stover with consolidated bioprocessing (CBP) of cassava starch by Lipomyces starkeyi was firstly developed as a novel strategy for lipid production. Starch was quickly hydrolyzed within 24 h by the amylolytic enzymes secreted by L. starkeyi to provide adequate fermentable sugars at the initial stage of culture, which eliminated the pre-hydrolysis step. More interestingly, synergistic effect for achieving higher lipid production by combined utilization of corn stover and cassava starch at relatively low enzyme dosage was realized, in comparison with the separate utilization of these two substrates. The fatty acid profiles indicated that lipid prepared by the combination strategy was suitable precursor for biodiesel production. The combined SSF&CBP strategy offers a simplified, highly-efficient, and economical route for co-valorization of low-cost substrates into lipids. [ABSTRACT FROM AUTHOR]

Single cell oil (SCO) produced by oleaginous yeasts represents an intriguing source for second generation biofuels or other biobased oleochemicals. This study aimed at improving SCO production from giant reed (Arundo donax L.). Five different yeast species were used to evaluate the lipid yield and fatty acids profile obtained in six media prepared with two enzymatic hydrolysates of mild alkali pre-treated giant reed fibers (washed or not after filtration) and variable nitrogen supplementation (three levels). After pre-treatment and hydrolysis, up to 489 ± 9 mg of reducing sugars per g of initial untreated biomass were obtained. C/N ranged widely (38−258) among the different formulated media, depending on both washing and nitrogen supplementation, and strongly affected SCO accumulation by most of the strains. Lipomyces starkeyi, Rhodosporidiobolus azoricus, and Cutaneotrichosporon oleaginosum reached the highest lipid contents (54–68%) and concentrations (4.8–5.6 g/L). The highest lignocellulosic biomass-to-lipid conversions were obtained with C. oleaginosum, R. azoricus, and L. starkeyi (102 ± 9, 95 ± 9, and 89 ± 3 mg of lipids per g of untreated giant reed biomass, respectively). Oleic, palmitic, linoleic, and stearic acid were predominant in the fatty acids profile, similarly to seed oils. In conclusion, hydrolysates derived from the alkali pre-treated biomass of giant reed are suitable to obtain high lipid yields with selected yeasts, to be used for the biobased industry. • High C/N enzymatic hydrolysates can be obtained from alkali pre-treated giant reed. • Extensive washing of the pre-treated fibers before hydrolysis can be avoided. • Remarkable giant reed-to-lipid yields up to 10% were obtained with oleaginous yeasts. • Highest yields with Rhodosporidiobolus azoricus and Cutaneotrichosporon oleaginosum. • Oleic, palmitic, linoleic, and stearic acid were predominant in the fatty acids profile. [ABSTRACT FROM AUTHOR]

Direct conversion of starch materials into lipid has been rarely reported as most oleaginous species are unable to efficiently produce amylolytic enzymes. Herein, consolidated bioprocessing (CBP) of cassava starch was described efficiently for lipogenesis by Lipomyces starkeyi. Lipid concentration and yield reached 13.99 g/L and 0.187 g/g, respectively, from 75 g/L cassava starch under optimal conditions. The CBP strategy featured zero enzyme cost, simpler process, and higher oleaginicity compared with the separate hydrolysis and fermentation (SHF) strategy. Amylolytic enzymes with α-amylase, maltase and glucoamylase activities were secreted into extracellular space and the amylase activity reached 3.65 U/mL as maximum. The genome of L. starkeyi was sequenced and a small-scale metabolic model was established. The mechanism of starch hydrolysis for lipogenesis by L. starkeyi was estimated accordingly. The estimated biodiesel parameters of the CBP-derived lipid samples indicated high-quality fuel properties. The CBP strategy provides a highly integrated and techno-economic route for starch-to-lipid conversion. [Display omitted] • CBP of cassava starch into lipid was promising by the amylolytic yeast L. starkeyi. • Lipid titer and lipid yield reached 13.99 g/L and 0.187 g/g, respectively. • CBP was superior to SHF with simpler process, lower cost, and higher oleaginicity. • Starch hydrolysis and lipogenesis were assessed using a small-scale metabolic model. • Biodiesel prepared from the CBP-derived lipids showed high-quality fuel properties. [ABSTRACT FROM AUTHOR]