Economic and environmental bottlenecks in the industrial-scale production of lipid-derived biofuels from oleaginous yeasts: A review of the current trends and future prospects

Abstract

Concerns about climate change and the reliance on fossil fuel reserves have motivated researchers to identify new renewable sources of energy. Biomass holds the potential to replace fossil-derived products with biofuels and bio-based chemicals. Plant-derived lipids are promising sources of biofuels; however, the production of plant oil often leads to the release of massive amounts of carbon dioxide due to deforestation and land-use change. The production of biofuels via plant oils (such as soybean) also competes with food production and in turn, impacts biodiversity. To mitigate these issues, the production of lipids from oleaginous yeasts could be an excellent alternative by incorporating these microbes into biorefineries utilizing agricultural or forest residues. Eventually, these microbial lipids could be potential sources for producing lipid-derived biofuels. However, the current conventional methods for the production and recovery of lipids from oleaginous microbes suffer from economic and ecological challenges that affect its industrial-scale expansion. This review highlights the major economic and environmental bottlenecks for the production of lipid-derived biofuels from oleaginous yeasts. It also provides perspectives on the strategies that could be adapted on economic and ecological fronts to assist the expansion of the production of microbial lipid-derived biofuels at an industrial scale.

INTRODUCTION

The increasing global demand for fossil-based resources has concerns over greenhouse gas (GHG) emissions associated with increased global warming, air pollution and acid rain. Efforts are being made to minimize these effects by identifying and utilizing renewable resources for a sustainable future. The higher demand for fossil fuels has motivated researchers to explore alternative sustainable biofuels. Lipid-derived biofuels have a higher energy density and better compatibility with the existing automobile when compared to other biofuels (Wang et al., 2022). Biodiesel is among the group of lipid-derived biofuels that has attracted the attention of researchers worldwide. However, to mitigate the reliability of vegetable oil or animal fat for producing lipid-derived biofuels, it is important to explore alternative resources such as microbial lipids. Oleaginous microorganisms are widely recognized as microbial cell factories that can accumulate lipids (from 20% up to 87% of their dry weight) more efficiently than plants and with a short production cycle (Bharathiraja et al., 2017; Sitepu et al., 2014). A variety of oleaginous microorganisms such as microalgae, fungi (yeast), and bacteria have been studied for their ability to accumulate lipids mainly in the form of triglycerides, free fatty acids (FFAs), polar lipids, sterols, and hydrocarbons by utilizing refined/cellulosic sugars. Oleaginous yeasts accumulate lipids via de novo (from sugar substrate under nitrogen limitation) or ex novo (from lipid substrate) (Beopoulos et al., 2009; Carsanba et al., 2018). A potential high value-added utilization of microbial lipids could be a substitute for plant oils and animal fats for producing oleochemicals and lipid-derived biofuels. The lipid production from oleaginous yeasts could prove to be more advantageous in comparison with microalgae or plant oils because the growth rate of oleaginous yeasts is much faster than microalgae since they present duplication times of even lower than 1 h (Koutinas et al., 2014). The seasonal and geographic variations do not affect the cultivation of yeasts as it is in the case of plant oils.

The present review provides an overview of the lipid production from oleaginous yeasts for its applications in the biofuel sector. The production of oleaginous yeast-derived lipids per unit area of bioenergy crops grown has been compared with the productivity of lipid-producing traditional crops such as soybean, palm, and rapeseed. The analogy between the composition of microbial and plant-derived lipids has been demonstrated in terms of the fatty acid profiles. The existing information on the economics and life cycle assessment (LCA) of microbial lipids production and their conversion into biofuels has been compiled. The economically and ecologically stressed unit operations for producing microbial lipid-derived biofuels have been demonstrated highlighting the major economic and environmental bottlenecks in the industrial-scale production of biofuels from oleaginous yeasts. Furthermore, recommendations are provided on the existing research gaps that need to be explored in the future.

PRODUCTIVITY OF MICROBIAL AND PLANT-BASED LIPIDS

The productivity or lipid yield is an essential parameter that affects the cost of production of microbial lipids especially by utilizing the sugar-rich hydrolysates from the bioenergy crops. The theoretical yield of lipid production is 0.32 g/g glucose and 0.34 g/g xylose (Papanikolaou & Aggelis, 2011; Ratledge & Wynn, 2002). The preliminary stoichiometry of the total carbon flux from glucose to triacylglycerol (TAG) was suggested by Ratledge and Wynn (2002). One mole of glucose generates two moles of pyruvate when metabolized via glycolysis and hence around 15 moles of glucose are required to synthesize 1 mole of TAG i.e. 32 g lipid can be produced by 100 g of glucose with an assumption that the glucose is solely used for lipid production (Ratledge & Wynn, 2002). The ability of the oleaginous yeasts to utilize lignocellulosic sugars (preferably glucose and xylose) from bioenergy crops provides an opportunity to compare the potential of these crops with conventional lipid-bearing crops for producing microbial and vegetative lipids respectively. Table 1 represents the maximum theoretical yields of microbial lipids per unit area of the bioenergy crops grown. The maximum theoretical lipid yields calculated for the oleaginous yeasts grown on lignocellulosic sugars surpasses the lipid yields from most of the conventional lipid-producing crops including rapeseed, soybean, sunflower, coconut, and olive (Table 2). The oleaginous yeasts grown on the napier grass, sweet sorghum, and switchgrass hydrolysate could produce lipid yields in the range 3.5–8.2, 3.6–6.0, and 4.6 t/ha respectively which is comparable to or even higher than the lipid yield from palm, that is, 3.0–5.0 t/ha of the palm trees grown. Hence the oleaginous yeasts grown on sugar-rich hydrolysates from bioenergy crops have the potential to compete with the conventional oil-bearing crops for the producing lipid-derived biofuels. However, in the real scenario, the microbial lipid yields would vary depending on the oleaginous yeast species, feedstock type, conditions of cultivation, and the percent conversion of sugars in the hydrolysate of bioenergy crops to lipids in the oleaginous yeast cells. The lipid yield of the theoretical maximum could vary from 25% to 90% for different oleaginous yeasts (Jin et al., 2015; Juanssilfero et al., 2018; Tsigie et al., 2011). The actual lipid yields would also be affected by the method of extraction in the case of both plants and microbes. Even the initial conversion of the lignocellulosic biomass into total sugars in the hydrolysate would vary. Gallego-García et al. (2023) have provided a detailed compilation of the most recent literature on using lignocellulosic feedstocks for lipid production by oleaginous yeasts.

TABLE 1 Lipid yield per unit area from oleaginous yeasts grown on lignocellulosic hydrolysates.

[Table omitted. See PDF]

^aTheoretical lipid yield: 0.33 g microbial lipids per g of total sugars (glucose + xylose).

^bThe productivity of bagasse was calculated with the assumption of 60% juicing efficiency of the sugarcane/Oilcane 1566 stems.

TABLE 2 Lipid yield per unit area from conventional lipid-producing crops.

[Table omitted. See PDF]

COMPOSITIONAL ANALOGY BETWEEN MICROBIAL AND PLANT-BASED LIPIDS

The properties of lipid-derived biofuel are influenced by the composition of the lipids (microbial/vegetative). Wang et al. (2022) have reviewed in detail the intrinsic interactions between lipid feedstocks and lipid-based biofuels, including biodiesel, and renewable equivalents to conventional gasoline, diesel, and jet fuel. The composition of microbial lipids significantly depends upon the growth conditions and varies with different lignocellulosic feedstocks. The oleaginous microorganisms produce different classes of lipids, namely, acyl glycerides, phospholipids, FFAs, glycolipids, sterols, lipoproteins, hydrocarbons, and pigments (Dong et al., 2016). TAGs are known as storage lipids while phospholipids and glycolipids are the main constituents of the cell membrane. TAGs are among the main components of microbial lipids making them chemically similar to vegetative lipids. The existing analogy between the composition of vegetative and microbial lipids is represented in Table 3. Palmitic acid (16:0), oleic acid (C18:1), and linoleic acid (C18:2) are among the common fatty acids in the profiles of microbial and plant-derived lipids. Hence, oleaginous yeasts could be potential alternatives to traditional oil-bearing plants for biofuel production. FFAs are usually formed after the harvesting of microbial biomass due to enzymatic hydrolysis of lipids (Balasubramanian et al., 2013; Chen et al., 2012). It is therefore expected to have high contents of FFAs in the microbial biomass during storage. To produce biodiesel or hydrocarbon-based fuels such as renewable jet fuel, TAGs, and FFAs are the preferred precursors. However, in the case of biodiesel production, FFAs affect the efficiency of the base-catalyzed transesterification reaction by forming soap and consuming the catalyst. The highly unsaturated glycolipids and phospholipids with long carbon chains are not convertible to biodiesel by conventional methods (Arif et al., 2020). Smaller amounts of phospholipids (0.5%–0.6%, w/w) are routinely removed from vegetable oil by degumming (dos Passos et al., 2022; Jiang et al., 2015). However, considering the potentially high content of the microbial polar lipids, it is critical to convert them to biodiesel to achieve better yields and avoid additional refinement costs. Enzymatic transesterification is often preferred over the conventional one in this case (Wu et al., 2017).

TABLE 3 Comparison of the fatty acid profile of lipids from oleaginous yeasts and plant sources.

Lipid source	Lauric acid (C12:0) (%)	Myristic acid (C14:0) (%)	Palmitic acid (C16:0) (%)	Palmitoleic acid (C16:1) (%)	Stearic acid (C18:0) (%)	Oleic acid (C18:1) (%)	Linoleic acid (C18:2) (%)	Linolenic acid (C18:3) (%)	References
Oleaginous yeasts
Rhodosporidium toruloides	-	-	18	3	3	66	-	-	Jones et al. (2019)
Rhodotorula glutinis	-	-	37	1	3	47	8	-	Jones et al. (2019)
Lipomyces starkeyi	-	-	34	6	5	51	3	-	Jones et al., (2019); Rajak et al. (2022)
Crypotococcus albidus	-	-	12	1	3	73	12	-	Rajak et al. (2022)
Rhizopus arrhizus	-	-	18	-	6	22	10	12	Rajak et al. (2022)
Yarrowia lipolytica	-	-	11	6	1	28	51	-	Rajak et al. (2022)
Plant sources
Palm	-	Traces	39–50	-	3–5	38–45	8–12		Montoya et al. (2014); Sargeant et al. (2014)
Rapeseed	-		2–5	-	0–4	51–68	18–25	7–11	Biljana et al. (2015); Guan et al. (2019); Sargeant et al. (2014)
Soybean			10–12	-	3–5	18–26	49–57	6–13	Clemente and Cahoon (2009); Knothe (2008)
Coconut	49	8	8	-	2	6	2	-	Boateng et al. (2016)
Sunflower oil	-	-	5–7		3–6	14–40	48–74	-	Knothe (2008)
Cottonseed oil			22–26		2–3	15–22	47–58	Traces	Knothe (2008)

PRODUCTION OF MICROBIAL LIPIDS AND CONVERSION INTO BIOFUELS

The recovery of yeast lipids or any other intracellular component is still an experimental challenge. The sequence and type of industrial unit operations required for the extraction of microbial cell components depend upon the type of target cell component and its final application. These operations are collectively known as downstream processes and represent a considerable cost of the industrial-scale production and application of microbial lipids. Though the cultivation of the oleaginous microorganisms via fermentation is one of the main unit operations, the production of biofuels from microbial lipids is classified into three broad categories: (1) extraction of lipids followed by transesterification for conversion into biodiesel; (2) direct conversion of microbial cell lipids into biodiesel by in situ transesterification; and (3) hydrothermal liquefaction (HTL) of oleaginous yeast cells into crude bio-oil that is processed into renewable diesel. The schematics for each of these processes with economically and ecologically stressed unit operations have been demonstrated in Figure 1. The cultivation of oleaginous yeast cells via fermentation is one of the major economically and ecologically stressed unit operations. The cost of substrate or feedstock contributes to the economics for the production of oleaginous yeast cell biomass while the GHG emissions make it ecologically stressed. Fermentation leads to achieving the highest lipid yield and productivity when optimum culture conditions (aeration, optimum pH, and temperature) are provided. The type and size of the fermenter to be used for growing oleaginous yeasts and the energy spent for providing the optimum temperature and aeration condition are the major cost-driving factors. According to Rajak et al. (2022), small fermenters with high productivity provide the best combination to achieve high lipid yields with less expenditure of energy and a lower cost of the fermenter, making the overall process cost-efficient. Higher cell biomass productivity could be ascertained by partial biomass recycling, with batch culture, continuous and fed-batch culture (Ykema et al., 1988). High lipid density was obtained with different modes of the fed-batch culture system including dissolved oxygen-stats feeding mode, pulse feeding mode, and online sugar feeding mode (Fei et al., 2016). Fed-batch culture specifically assists in reducing growth inhibition due to high substrate concentration. Furthermore, Lynd et al. (2022) have demonstrated that in the context of fuel production, the cost of aerobic fermentation is impractically high. Previous studies have also suggested that rapid lipid production is crucial for obtaining a sustainable process since aerobic cultivation requires much more energy input than the almost anaerobic process of ethanol production (Brandenburg et al., 2021; Karlsson et al., 2016). Thus, shortening the cultivation time is an important factor to obtain sustainable microbial lipid production (Karlsson et al., 2017).

FIGURE 1. Process schemes to produce lipid-derived biofuels from oleaginous yeasts depicting the major economically (blue) and ecologically (green) stressed unit operations/raw materials. Some of the unit operations are both economically and ecologically stressed (gray).

To enhance the extractability of intracellular microbial lipids, the yeast cell mass obtained after fermentation needs to be conditioned to alter the cell wall permeability. Different conditioning or pretreatment strategies have been developed for the recovery of yeast lipids. These mainly include acid-assisted, alkali-assisted, detergent-assisted, physical, enzymatic, or mechanical methods. The recovery of lipids from wet microbial biomass is often challenging due to the limited lipid accessibility, blocking effects from insoluble biomass residue, and the formation of a stable emulsion. Hence the pretreatment or cell disruption of wet microbial biomass is required to increase lipid accessibility, improve mass transfer, and reduce the formation of emulsions. The pretreatment of microbial cell biomass should be energy efficient and should be easily scalable on an industrial scale. Table 4 shows the recent studies on different pretreatment strategies for the recovery of lipids from oleaginous yeasts. Cell conditioning or pretreatment is a part of the two-step process for biodiesel production. The extracted lipids are subjected to transesterification that converts the triacylglycerides into fatty acid methyl esters. Ultrasound, microwave irradiation, and bead milling have been evaluated as potential methods for lipid recovery by disrupting the cell walls of the oleaginous yeast Yarrowia lipolytica IFP29 (Meullemiestre et al., 2016). The lipid extraction efficiency by these methods have been compared to the pretreatment with freezing/defrosting and cold drying before the conventional solvent extraction process. Extraction by bead milling appears as the most efficient intensified extraction method while cold drying under pressure appears as the best pretreatment method for lipid extraction from yeast, with a yield two times more than conventional maceration. However, the energy consumption and environmental impact studies revealed that bead milling extraction would be the best alternative for lipid recovery compared with a pretreatment by cold drying which appears to be the most energy-consuming technique (Meullemiestre et al., 2016). The Soxhlet extraction method for the recovery of lipids suffers from the major drawbacks of lipid degradation, longer extraction time, and high consumption of energy. Other methods such as the microwave and ultrasound-assisted disruption of oleaginous yeasts are highly efficient on a laboratory scale but might not be that efficient at a large scale mainly due to the high energy input requirement. Bead milling for disrupting microbial cells can be easily scaled up, but its efficiency varies with the yeast cell types. The lipid extraction procedures are ecologically stressed majorly because of the usage of organic solvents. The transesterification process is equally responsible for ecological stress due to the usage of methanol which could lead to different ailments in the human population as well as to the environment. Furthermore, the extraction and the transesterification processes are also economically stressed unit operations due to the involvement of the cost of organic solvents (or alternate green solvents), enzymes and cell disruption technologies. Most of the lipid extraction methods work well in laboratory settings and have not been used extensively at an industrially relevant scale. However, the mechanical methods to disrupt the cell and solvent extraction have been extensively preferred on an industrial scale for the extraction of microbial lipids (Gorte et al., 2020; Saini et al., 2021). In addition, aqueous extraction of corn oil after fermentation in the dry grind ethanol process has been commercialized (Dickey et al., 2009; Moreau et al., 2014). Similar aqueous methods could be devised for the extraction of microbial lipids via effective cell disruption followed by centrifugation resulting in a floating oil layer that could be recovered easily at an industrial scale.

TABLE 4 Common conditioning strategies with the corresponding recovery of lipids from oleaginous yeasts.

[Table omitted. See PDF]

In situ transesterification is another process for biodiesel production, that occurs in a single step of lipid extraction and transesterification (Martinez-Silveira et al., 2019). By excluding the extraction and refining of lipids, this process also lowers the production cost (Ehimen et al., 2010). The process involves the simultaneous extraction of lipids from microbial cells and transesterification of the extracted lipids into biodiesel by utilizing an extracting solvent and acid/base as a catalyst for the transesterification reaction. Methanol is widely used as the extracting solvent during in situ transesterification (Martinez-Silveira et al., 2019) while a few reports have explored binary solvent systems such as methanol-hexane (Chopra et al., 2016). Sulfuric acid is the preferred acid catalyst for in situ transesterification. The reaction temperature for in situ transesterification varies from 40 to 70°C while the incubation time varies from 3 to 20 h for the best yields in different studies (Chopra et al., 2016; Martinez-Silveira et al., 2019). Martinez-Silveira et al. (2019) have reported that the in situ transesterification of the lipids from Rhodotorula graminis S1/S2 led to a yield that was 123% higher than that obtained with a two-step process. During the in situ transesterification of lipids from Pichia guilliermondii, a maximum fatty acid methyl esters yield of 92% (w/w of lipid), was achieved (Chopra et al., 2016). This yield is comparable to that obtained by the ex situ multistep transesterification process which requires approximately 7 h more than the in situ process, thereby resulting in greater productivity. The quality of biodiesel produced by in situ transesterification has passed the standard tests set up by the American Society for Testing and Materials (ASTM) D6751 standards (Haas & Scott, 2007) and European standards (EN) 14214 (Kasim et al., 2010). The in situ transesterification is anticipated to reduce the lipid production cost by eliminating the lipid extraction step.

Hydrothermal liquefaction is another promising technology that converts oleaginous yeasts into liquid biofuels by converting the whole microbial biomass into an energy-dense fuel precursor called bio-crude. Collett et al. (2019) have proposed an integrated route for obtaining renewable diesel via HTL of oleaginous yeast Lipomyces starkeyi and residual lignin obtained from corn stover. HTL represents a promising alternative to the conventional method of lipid extraction for biofuel production (Jena et al., 2015). Along with the production of fuel-grade crude, the HTL of organic slurries also produces an aqueous phase (that partitions from the biocrude via gravity separation) and a solid phase (rich in ash) (Elliott et al., 2015). The aqueous phase resulting from the HTL of organic slurries is often rich in organic acids and phenolics (Watson et al., 2020) along with the presence of toxic heavy metals (Ramirez et al., 2015). The aqueous phase resulting from HTL could be utilized in oleaginous yeast fermentation for the co-production of value-added chemicals such as triacetic acid lactone, itaconic acid, lipids, and citric acid (Cordova et al., 2020). HTL could be preferred over other thermochemical and biochemical processes for the conversion of microbial lipids into biofuels. It promises higher performance than thermochemical processes such as pyrolysis because it can work directly with wet biomass without the need for additional energy required for evaporative drying in pyrolysis. Unlike the biochemical conversion methods, HTL does not focus on pretreatments for microbial cell lysis and lipid extraction. Hence, in the case of HTL, the overall economics are influenced by the improvements in biomass productivity rather than the extractable lipid content. However, the high operating pressure associated with HTL and the scarcity of information on standard product separation procedures are the major disadvantages associated with this process of biofuel production (Jena et al., 2015). The high operating pressures could result in high capital investments and operational issues making HTL as the economically stressed unit operation.

ECONOMICS OF MICROBIAL LIPID PRODUCTION FOR BIOFUEL APPLICATIONS

The commercialization of lipid-derived biofuels from oleaginous microbes depends upon their cost competitiveness with plant-derived lipids. The overall cost of producing biofuels from microbial lipids includes the costs associated with the cultivation of oleaginous microbes for lipid accumulation, recovery of microbial lipids, and their conversion into biofuels. The economics of microbial lipid production is widely controlled by the species of microbe chosen, the number of cells produced, the concentration of lipids accumulated within the cells, and the cost of carbon source required for the cultivation of the oleaginous microbe. Further costs are added by the downstream processing steps including harvesting of cells and lipid recovery along with the costs associated with the conversion of the extracted microbial lipids into biofuels. The previous techno-economic analysis study found that biodiesel production via transesterification of extracted microbial oil proved a more cost-competitive process compared with the direct transesterification of dried yeast cells (Koutinas et al., 2014). In another study, the volume of stirred tank bioreactor (750 m³) was compared to that of open tanks (1260 m³) for the cultivation of oleaginous yeasts to estimate the production cost (Braunwald et al., 2016). In the case of stirred tank bioreactors, the cost of production was USD 2.35 per kg where fermentation, cell harvesting, and drying of cell biomass contributed 87% of the total cost. The cost of production (USD 1.72 per kg) was less in the case of open ponds with a 43% contribution to the cost (Braunwald et al., 2016). Table 5 summarizes the preliminary studies on the economics of the production and extraction of lipids from oleaginous yeast sources. Karamerou et al. (2021) evaluated a wide range of scenarios for their effect on the selling price of microbial lipids. These scenarios included wet cell extraction, growing a thermotolerant species, using a non-sterile process, access to inexpensive electricity, using a species with extracellular production of lipids, and selling the cell as one product comprising lipids, proteins, carbohydrates, etc. The price of lipids was calculated to be USD 1.81 per kg for a production of 8000 tons per year. This price could further be reduced to USD 1.20 per kg upon increasing the production to 48,000 tons of lipid per year demonstrating economy of scale. Using a thermotolerant strain reduced the price from USD 1.20 to USD 1.15 per kg. Access to zero-cost electricity could reduce the price to USD 1.12 per kg. Similarly, using non-sterile conditions, wet extraction of lipids, production of extracellular lipids, and selling the whole yeast cell reduce the price to USD 1.19, 1.16, 0.99, and 0.81 per kg, respectively. Masri et al., 2019 demonstrated the effect of yeast-specific cell wall hydrolases (on-site production) on lipid recovery and estimated the production cost at USD 1.6 per kg of lipid (Masri et al., 2019). The price is still higher than that of conventional palm oil (USD 0.5 per kg) but is comparable to that of eco-certified palm oil (USD 2.1 per kg) (Masri et al., 2019). The cost of production of microbial oil (USD 1.81 per kg) reported by Karamerou et al. (2021) is higher than that of soybean oil (0.70 USD per kg) for the same scale of production (8000 tons of oil/year) (You et al., 2008). However, in some of the abovementioned scenarios, the cost of production of microbial lipids at a particular production scale is lower when compared to that of soybean bean oil. Cheng and Rosentrater (2017) have presented the cost of production of soybean oil to be in the range of USD 5.74–2.69 per kg oil for a production scale of 4000–415,000 tons of oil per year. Sae-ngae et al. (2020) evaluated the techno-economic analysis and CO₂ emissions for bio valorization of abundant agro-industrial wastes for biodiesel by potential oleaginous yeasts. Besides, the internal recycling of nutrients and wastewater effluent for growing microbial cultures could be an attractive strategy to improve both the economic viability and sustainability of bioprocessing for microbial oil production. Yang et al. (2015) reported the application of spent cell mass and lipid fermentation wastewater for microbial oil production from corn stalk hydrolysate by R. toruloides Y4 and showed that up to three cycles could be applied in fully recycling the resources with only slight reduction in the lipid yields. Reports have revealed that culturing oleaginous yeasts and lipid extraction solely for biodiesel production might become economically viable by integrating the process with the co-production of other value-added products in a biorefinery model (Chopra et al., 2022; Gnansounou & Kenthorai Raman, 2016).

TABLE 5 Summary of economics for the production of microbial lipids from oleaginous yeasts.

[Table omitted. See PDF]

LCA OF THE PRODUCTION OF MICROBIAL LIPIDS FOR BIOFUEL APPLICATIONS

Microbial lipids from yeasts are likely to play an important role in the future production of biofuels, bio-based chemicals, or food ingredients. It is therefore essential to understand the environmental implications of recovering microbial oils for industrial applications. LCA is a systematic methodology to assess the potential impacts of the life cycle stages of a product starting from its production to its end of life. Table 6 summarizes the LCA conducted for the production of lipid-derived biofuels from oleaginous yeasts. Chopra et al. reported the LCA of an oleaginous yeast biorefinery combining the direct in-situ transesterification of yeast lipid with the HTL of residual de-oiled biomass (Chopra et al., 2020). According to the combined analysis of the mid-point and end-point indicators, HTL and transesterification have higher impacts when compared to other sub-processes such as cultivation of yeast and harvesting. The authors have suggested that the higher impacts could be attributed to the higher input of energy/electricity for these two processes. In another study, the global warming potential for yeast oil production was assessed to be 3.56 kg CO₂ equivalents to be emitted per kg of yeast oil produced (Masri et al., 2019). The LCA of plant-based oil production indicates the generation of 4.8, 3.0, 3.5, and 7.5 kg CO₂ equivalents per kg of palm oil, rapeseed oil, sunflower oil, and peanut oil, respectively (Schmidt, 2015). The CO₂ emissions for yeast oil production were similar to those obtained for rapeseed and sunflower oil while it was lower than those reported for palm oil and peanut oil. Cortés-Peña et al. (2023) have reported that microbial oil-based biodiesel production in a sugarcane biorefinery leads to half the carbon intensity of biodiesel derived from soybean oil and two–six times greater biodiesel yield per unit of land.

TABLE 6 Summary of LCA for the production of lipid-derived biofuel from oleaginous yeasts.

Microorganism	Mode of cultivation	System boundary	Major unit operations	Impact indicators	Major contributors to GHG emissions	References
Yeast	Batch-Bioreactor	Cradle to gate	Fermentation, harvesting, cell disruption, transesterification, HTL	Human health, resources, ecosystem, GWP	Solvents energy requirement in HTL, fermentation, and cell disruption	Chopra et al. (2022)
Yeast	Shake flask	Cradle to gate	Biomass pretreatment, enzymatic hydrolysis, fermentation, lipid extraction	Human carcinogenic toxicity	Production of enzymes, heat, and organic solvents in lipid extraction and distillation	Barbanera et al. (2021)
Yeast	Cylindrical reactor	Well to tank	Pressure filtration to 70% moisture, homogenization, hexane extraction	Energy use	Agitation and aeration of the bioreactors for lipid accumulation and yeast propagation; Drying the yeast was also energy-demanding	Karlsson et al. (2016)
Yeast	Bioreactor	Well to tank	Centrifugation, hydrothermal liquefaction (300–350°C, 18–20 Mpa)	GHG emissions/energy	Hydrothermal liquefaction process	Summers et al. (2015)

Abbreviations: GHG, greenhouse gas; GWP, global warming potential; HTL, hydrothermal liquefaction.

OLEAGINOUS YEASTS IN A BIOREFINERY

The economics of producing lipid-derived fuels from oleaginous yeasts could be more promising by adopting a biorefinery approach. A biorefinery facility is analogous to a petroleum refinery that integrates the biomass conversion processes and equipment for the valorization of biomass (Cherubini, 2010). The concept of biorefinery embraces a wide range of technologies to fractionate biomass resources into individual components such as carbohydrates, proteins, and triglycerides. These fractions can then be converted into a portfolio of value-added bioproducts such as chemicals and biofuels. Figure 2 represents a consolidated bioprocessing strategy wherein oleaginous yeasts could be incorporated into a lignocellulosic biorefinery to get multiple products. This approach might improve the economics to a great extent. Studies have suggested that the cost of substrate for growing oleaginous microbes contributes to the cost of cultivation which in turn is a major contributor to the overall cost of production of microbial lipids. Lignocellulosic hydrolysate (Shaigani et al., 2021), crude glycerol (Liu et al., 2017), and organic fractions of urban solid waste (Banerjee & Arora, 2021) could be used as potential substrates for growing any kind of microbes. Engineered Y. lipolytica could use xylose-rich agave bagasse hydrolysates as a feedstock for the production of high lipid yields of 0.34 g/g sugars with a titer of 16.5 g/L and a productivity of 1.85 g/L/h (Niehus et al., 2018). Such efficient utilization of xylose by the engineered yeast species would be a step further for the commercial production of microbial lipid-derived fuels in an integrated biorefinery. The suitability of different carbon sources and the cultivation processes for increased lipid accumulation in oleaginous yeasts has been reviewed elsewhere (Poontawee et al., 2023).

FIGURE 2. Sustainable consolidated bioprocessing strategy incorporating oleaginous yeast in a lignocellulosic biorefinery.

Previous studies have demonstrated proof-of-concept for incorporating the oleaginous yeasts in biorefineries based on bioenergy crops such as sorghum (Cheng et al., 2021), oilcane (Deshavath et al., 2024), and miscanthus (Banerjee et al., 2024) for producing microbial lipids. The lipid-accumulating bioenergy crops such as oilcane, energy cane, and oil sorghum could be potential feedstocks to produce microbial lipids along with inherent vegetative lipids. The lignocellulosic components of these lipid-producing crops could be pretreated and hydrolyzed into sugar-rich hydrolysates for growing oleaginous yeasts. The vegetative lipids along with the microbial lipids could potentially be converted into drop-in fuels. Furthermore, in a biorefinery based on transgenic oil-bearing sugarcane, the integrated recovery of both plant and microbial oil through lignocellulosic pretreatment constitutes a process intensification by reducing the number of unit operations. However, the additional utility and capital expenses from a larger pretreatment reactor might result in costs comparable to traditional mechanical oil recovery (Cortés-Peña et al., 2023).

After recovering lipids from microbial cells, the residual cell debris could be used for a variety of applications to evaluate their inherent biorefinery potential. Spent cells have been valued at anywhere between USD 0.5–2.5 per kg and have been demonstrated to increase the revenue from microbial oil production (Koutinas et al., 2014; Parsons et al., 2019; Vieira et al., 2016). It is reported that higher revenue is achieved when spent cells are used as animal feed in comparison to the generation of energy and can make up for other process expenses, such as the cost of raw materials. The oleaginous yeasts belonging to the Xanthophyllomyces genera (Phaffia), Rhodotorula, Rhodosporidium, Sporidiobolus, and Sporobolomyces are also known for their potential for producing pigments such as carotenoids (Kanamoto et al., 2021). These are specifically named red yeasts due to their appearance. An efficient strategy for recovering this value-added pigment could also improve the economics for the production of lipid-derived biofuels from oleaginous yeasts. Furthermore, the valorization of by-products such as crude glycerol generated in the biodiesel industry makes the conversion of microbial lipids into biodiesel more economically attractive (Liu et al., 2022).

CONCLUSION, RECOMMENDATION, AND FUTURE PROSPECTS

The industrial-scale production of lipid-derived biofuels from oleaginous yeasts suffers a major economic bottleneck due to the existing cost competition with plant oils (such as rapeseed, and palm oil). On an economic front, the availability and cost of feedstock, productivity of triglyceride oil, and the process steps for recovery of oil are the major challenges that block the industrial-scale production of microbial oils. Therefore, to make the production of microbial oils economically viable on a commercial scale, the entire process chain and feedstock utilization need to be reorganized. The process chain includes the selection of feedstock, maximizing microbial oil productivity, minimizing the process steps for recovery of oil from microbial cells, and value addition of the by-product streams. The identification and selection of oleaginous yeast strains with a high capacity for lipid accumulation would be the first step. From an economical point of view, the screening of strains that are capable of assimilating low-cost substrates is of high importance. The cost of substrate or carbon source specifically contributes to the final cost of production of microbial lipids and lipid-derived biofuel. The LCA studies reveal that the major ecological bottleneck is attributed to the cultivation of oleaginous yeasts for being the greatest contributor to GHG emissions. Furthermore, the identification of efficient cell disruption strategies and green solvents for lipid extraction is of high importance for cost-effective and ecologically sustainable production of lipid-derived biofuels. The energy-intensive dehydration of microbial biomass for lipid extraction precludes its application in biofuel production. Hence, methods for effective extraction of lipids from wet cell biomass are required for competitive process economics. The application of oleaginous yeasts as potential feedstocks for producing lipid-derived biofuel is expected to be commercially viable when incorporated into biorefineries, utilizing low-cost raw materials for growth, benefitting from shared processing lines and with the integral use of the cell biomass completely leading to zero-waste production. It would be worthwhile to identify and evaluate subsequent product streams rich in proteins and polysaccharides from the microbial cell biomass that could generate additional revenue owing to its potential demand in the industrial sector.

AUTHOR CONTRIBUTIONS

Shivali Banerjee: Conceptualization; data curation; formal analysis; investigation; visualization; writing – original draft. Vijay Singh: Conceptualization; funding acquisition; supervision; visualization; writing – review and editing.

ACKNOWLEDGMENTS

This work was funded by the DOE Center for Advanced Bioenergy and Bioproducts Innovation (U.S. Department of Energy, Office of Science, Biological and Environmental Research Program under Award Number DE-SC0018420). Any opinions, findings, and conclusions or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the views of the U.S. Department of Energy. APC funding was approved for this article.

CONFLICT OF INTEREST STATEMENT

The authors declare that there is no conflict of interest.

DATA AVAILABILITY STATEMENT

No new data was generated.