The cultivation medium profoundly influences microalgal growth and composition, primarily through constituent nutrients and physical factors such as temperature, light, light-dark cycles, and chemical factors including pH, turbulence, salinity, and CO₂ concentrations (Zarrinmehr et al. 2020). Nitrogen and phosphorus, among the nutrients, play a crucial role in microalgae growth and protein synthesis when adequately supplied in the cultivation medium. However, lipid content typically does not thrive in microalgae cultivated in nutrient-replete conditions (Yodsuwan et al. 2017). Thus, limited concentrations of certain nutrients like nitrogen and phosphorus can enhance lipid productivity (Yaakob et al. 2021).

Beyond cultivation-based nutrient optimization, emerging approaches such as nanoparticle application provide additional mechanisms to enhance lipid productivity. Nanoparticles have been integrated into microalgae for bioenergy production, capitalizing on their unique physical and chemical properties. These properties, including their large surface area and strong, persistent, elastic, and electrical characteristics, are leveraged to enhance microalgal growth, lipid production, harvesting efficiency, and catalytic biodiesel production. Specifically, nanoparticles facilitate increased gas-liquid mass transfer rates, thereby elevating CO₂ concentrations at the gas-liquid interface to promote both microalgal growth and lipid content (Zhu et al. 2010). Exposure to various nanoparticle composites significantly affected both the growth rate and lipid productivity of Scenedesmus obliquus. For example, a concentration of 5 mg.L^-1 of carbon nanotubes (CNT) led to a growth rate of 7.5 × 107 cells.mL^-1 and an 8.9% increase in lipid content, while the maximum lipid content of 39.6% was observed with a dosage of 5 mg.L^-1 of α-Fe₂O₃. Conversely, the growth rate declined when MgO nanoparticles were applied, but a concentration of 40 mg.L^-1 of MgO nanoparticles increased lipid content to 18.5% in S. obliquus (He et al. 2017). However, some composites of nanoparticles, such as gold, silver, palladium, selenium, zinc oxide, copper oxide, and ferrous oxide, exhibited toxic properties to various microalgae strains, particularly at high doses. Hence, reducing the doses of these nanoparticles would reduce toxicity and enhance content of lipids in microalgae (Rawat et al. 2022).

Excessive nanoparticle doses result in elevated levels of reactive oxygen species, leading to microalgae cell death, subsequently reducing growth rates and lipid productivity (He et al. 2017). Nanoparticles are employed not only to enhance algal growth and lipid productivity but also to efficiently harvest microalgal yield, thereby increasing lipid content, with silica-coated magnetic nanoparticles demonstrating the ability to separate over 95% of various freshwater and marine microalgal strains, such as Chlamydomonas reinhardtii, Chlorella vulgaris, Phaeodactylum tricornutum, and Nannochloropsis salina, through high flocculation facilitated by adsorption of micron-sized algal cells onto submicron-sized nanoparticles, depending on nanoparticle and medium characteristics (Cerff et al. 2012). Furthermore, the utilization of iron oxide nanoparticles in conjunction with the cationic polyelectrolyte poly(dimethyldiallylammonium chloride); PDDA enabled the efficient harvesting of Chlorella sp. microalgae, resulting in a 99% removal efficiency (Lim et al. 2012). Moreover, under optimal conditions, Fe₃O₄ nanoparticles achieved a 98% efficient removal of Botryococcus braunii and Chlorella ellipsoidea (Xu et al. 2011).

Beside inducing growth rate, lipid productivity, and harvesting, nanoparticles have been utilized to catalyze transesterification reaction to produce biodiesel from microalgae lipids. Transesterification is a chemical reaction in which FA contained in lipid classes such as triglycerides, phospholipids or glycolipids are converted into esters with help of base and/or acid catalyst (Senusi et al. 2024). In this context, nanoparticle composites were used to catalyze the transesterification reaction. As an example, calcium oxide (CaO) was synthesized from eggshell waste and utilized as a catalyst for transesterifying microalgal C. vulgaris biomass, resulting in a 92.03% yield of biodiesel (Pandit and Fulekar 2019). This biodiesel yield is considered quite high compared to another study that utilized the same microalgal strain with an alkaline NaOH catalyst, which produced 77.6% biodiesel (Velasquez-Orta et al. 2012). This proves the significant contribution of nanoparticles in enhancing biodiesel yield synthesized from microalgae lipids. Table 3 shows the applications of various nanoparticles in enhancing lipid productivity, harvesting microalgal biomass, and catalyzing transesterification of microalgae biomass.

In contrast to external methods of nutrient optimization and nanoparticle application, genetic engineering enables direct modification of intracellular metabolic pathways for increasing lipid accumulation. The significant advancements in genetic engineering over the last few decades have spurred researchers to employ genetic tools for enhancing lipid productivity in microalgae biomass. Genetic modifications in microalgae can be achieved through random mutagenesis, physical or chemical adaptive laboratory evolution, and genetic engineering, with the latter being the most precise method due to its ability to accurately insert, substitute, or delete targeted genes and avoid undesired outcomes (Muñoz et al. 2021). With the progress in omics technology and genetic modification tools, various genetic engineering strategies have been devised to increase lipid content in microalgal biomass. These strategies encompass adjustments to fatty acid synthesis metabolism, the Kennedy pathway, polyunsaturated fatty acid metabolism, transcription factors, nicotinamide adenine dinucleotide phosphate (NADPH) generation, central carbon metabolism, lipid catabolism, and other related pathways (Muñoz et al. 2021). Fatty acid synthesis occurs in the chloroplast through a series of conversion processes from acetyl-CoA to malonyl-CoA, then malonyl-ACP, and finally to saturated fatty acids (SFAs), facilitated by various enzymatic catalyses. The pivotal step in this process is the formation of malonyl-CoA, which is catalyzed by acetyl-CoA carboxylase (ACC). Therefore, overexpressing the ACC gene plays a significant role in increasing lipid content in microalgae strains (Li-Beisson et al. 2019). Moreover, genes such as acetyl-CoA carboxylase 1 (ACC1), glycerol-3-phosphate dehydrogenase 1 (GPD1), glycerol kinase (GUT1), acetyl-CoA synthetase (ACS), and Chlamydomonas reinhardtii fatty acid desaturase 2 (CrFAB2) have been identified to significantly contribute to lipid enhancement through the manipulation of fatty acid synthesis metabolism, as detailed in Table 4. In the Kennedy pathway, glycerolipid synthesis begins with glycerol-3-phosphate, catalyzed by glycerol 3-phosphate acyltransferase (GPAT), and undergoes a series of conversion processes ultimately leading to the formation of triacylglycerols (TAGs), which are abundant lipids in microalgae with potential applications in the biofuel industry (Yu et al. 2011).

Table 4 presents examples of how transcription factors contribute to lipid enhancement in microalgae. Photosynthesis in microalgae is initiated by light energy, where electrons coupled with H+ from water (H₂O) transfer from stroma to lumen, facilitating ATP formation. Additionally, glyceraldehyde 3-phosphate (GAP) is synthesized from atmospheric CO₂, which is then converted into sugars and lipids through various biological and chemical processes within the microalgae structure. Consequently, modifying photosynthetic capacity and enhancing reducing equivalent supplies can lead to lipid augmentation (Subramanian et al. 2013). Overexpressing Ferredoxin 1 (PETF) and 5 (FDX5) genes has been demonstrated to enhance the light reactions involved in lipid synthesis in microalgae strains (Huang et al. 2015). Moreover, the synthesis of lipids necessitates nicotinamide adenine dinucleotide phosphate (NADPH). Consequently, augmenting these factors results in an increase in lipid formation (Cornish-Bowden 2014). Central carbon metabolism is crucial for directing carbon flux and regulating acetyl-CoA and G3P, which can be utilized for TAG synthesis or converted into dihydroxyacetone phosphate (DHAP), thereby influencing glycolysis or gluconeogenesis (Subramanian et al. 2013; Han et al. 2016). Ultimately, the lipid catabolism strategy relies on triacylglycerols (TAGs) stored within multifunctional cytosolic lipid droplets (LDs). While the formation of LDs remains somewhat obscure, they primarily consist of TAGs enclosed by phospholipids. Consequently, certain microalgae have been genetically engineered to augment lipid content using this approach (Muñoz et al. 2021).

Overall, lipid enhancement methods in microalgae feedstock demonstrate a clear integration between biomass and lipid productivity. Nutrient deprivation methods, nitrogen and phosphorus limitation, are simple and less costly. However, they often result in reduced biomass yield, which may negatively affect the total lipid productivity since the produced feedstock is low. Conversely, nanoparticle application approaches offer multifunctional merits such as improved growth, harvesting efficiency, and catalytic conversion. The toxicity, cost, and environmental sustainability remain a great concern to large-scale implementation of nanoparticle application. Genetic engineering provides the most targeted and potentially high-yield techniques, enabling precise manipulation of metabolic pathways, yet its industrial application is limited by regulatory challenges, genetic stability, and scalability issues. Importantly, most studies focus primarily on lipid accumulation without sufficiently evaluating how these upstream strategies influence downstream biodiesel conversion efficiency, fuel properties, and techno-economic feasibility. Therefore, future research should emphasize integrated approaches that balance lipid productivity, biomass yield, and downstream processing performance to ensure economically viable biodiesel production.

Recovering lipids from microalgae is vital for their use in biodiesel industry, but the thick cell wall surrounding microalgal cells represents a challenge in accessing and extracting the lipidic compounds. Therefore, breaking down the rigidity of the cell wall is necessary to maximize the extraction of microalgal lipid content. The process of lysing the cell wall precedes lipid extraction, although they are often carried out simultaneously as a single operation (Lee et al. 2021). Figure 2 depicts various mechanical, chemical, and physical approaches employed to disrupt the microalgal cell wall.

Mechanical disruption involves applying external force to microalgae to rupture the cell wall. There are multiple mechanical techniques that can be used to disrupt the rigid microalgal cell wall. High-pressure homogenization technique forces microalgal cells to go through a high pressurized and narrow valve that leads to break the microalgal cell wall. In contrast, rotor–stator homogenization breaks the rigid microalgal cell wall by the shearing action between a constant outer stator and a fast-spinning inner roto. Another technique depends on bead milling, in which agitating beads collide with the microalgae cells, leading to the crushing of the cell walls. Grinding with mortar and pestle uses two hard surfaces to crush the microalgal cell wall. Ultrasonication technique depends on high frequency waves of sound to make cavitation and rupture the cell wall of microalgae. Cavitation that ruptures the microalgal cell wall can be resulted from the rapid change in pressure, this technique is called hydrodynamic cavitation. The final mechanical disruption method for the microalgal cell wall is screw expeller pressing, in which dry microalgal cells are positioned in a cavity and compressed (Vasistha et al. 2021).

Chemical disruption utilizes acids, alkalis, enzymes, and other agents to lyse the cell wall without force. Alkalis or acids are used to solubilize the microalgal cell wall through saponification. The proteins contained within the microalgal cell membrane can also be solubilized by detergent or surfactant molecules. Lastly, the rigid microalgal cell wall can be digested chemically using enzymes that promote hydrolysis reaction (Lee et al. 2021; Vasistha et al. 2021).

Physical disruption methods such as thermolysis, osmosis, electrolysis, and radiation are also used to cleave the rigid walls of microalgal cells. The technique of repeated freeze-thaw disrupts microalgal cell wall through repeated freezing and thawing process. During osmotic shock technique, microalgal cell receives amounts of water causing internal pressure and bursting the cell. Moreover, the cell wall can be lysed by gas bubbles escaping from the cell as a result of a sudden release of pressure. Electroporation has the effect of cell disruption when it is applied in high electric fields which called pulsed electric field. In addition, the oscillating electric field initiated by microwaves results in frictional forces and heat, causing water to move rapidly inside the microalgal cell, leading to the rupture of the cell wall. Finally, heat is also considered a physical cell disruption technique, where the microalgal cell wall can be completely disrupted at 121°C for a duration of 30 minutes. This technique is called thermolysis (Lee et al. 2021).

Beside the effectiveness of prior cell disruption techniques, the yields of extraction of lipids from microalgae is influenced by factors such as the microalgae structure, lipid composition, pretreatment, and the efficiency of the extraction technique, with considerations for both polar and nonpolar lipids, which must be addressed alongside technical and economic considerations (Zhou et al. 2022). The lipid extraction techniques from microalgae, as illustrated in Figure 3, are broadly categorized into solvent-assisted extraction, enzyme-assisted extraction, electric field-assisted extraction, ionic liquids extraction, and instrument-assisted extraction. Table 5 resumes approaches of lipid extraction from various microalgal strains employing these techniques.

Initially, conventional transesterification for processing microalgae as a biodiesel feedstock involves several steps including collection, dehydration, lipid extraction, and transesterification. The process may include pretreatment or esterification depending on the free fatty acids (FFA) contained in the sample of microalgae oil. During transesterification reaction, ester group is exchanged with an alcohol group (generally methanol) to form FAMEs. This process is promoted by either acid or base catalyst. In addition to biodiesel, glycerol is a by-product resulting from the conventional transesterification. However, the high moisture content in some microalgae biomass, which could be as high as 90%, leads to additional expenses for thorough dehydration. Moreover, the lipid extraction process often results in the loss of valuable microalgal oil components, complicating traditional transesterification processes (Pandey et al. 2024).

In response to the challenges associated with the conventional transesterification, in situ transesterification (IST) emerged as a solution, allowing for the direct utilization of freshly harvested microalgae for biodiesel production in a single step. Generally, IST process refers to the direct procedures of converting raw microalgae into FAME. Since the first attempts at IST were in the 2000s, this approach was considered a modern and recent technique for biodiesel production from microalgae feedstock. The IST was classified into wet IST and dry IST. The wet IST is the process of utilizing freshly harvested microalgae for biodiesel production without dehydration and lipid extraction. Although wet IST shows promise in reducing energy consumption and costs by eliminating the need for dehydration and lipid extraction, challenges such as high-water content, the presence of microalgal proteins, and FFAs hindering catalyst activity have led to low efficiency in early experiments. This prompted the exploration of techniques like dry IST, which involves dehydrating the microalgae feedstock before subjecting it to the IST process. Despite advancements and promising outcomes from both wet and dry IST approaches, further efforts are needed to improve efficiency and reduce costs in biodiesel production to meet industry standards (Kim et al. 2019). Table 6 explores part of previous studies that implemented both wet IST and dry IST to synthesize biodiesel from microalgae feedstock. In addition, Figure 4 illustrates the integrated tracks of conventional transesterification, wet IST, and dry IST for the production microalgal biodiesel.

The synthesis of microalgae biodiesel highlights a transition from conventional multi-step processes toward more integrated and efficient method of in situ transesterification. While conventional transesterification remains well-established and reliable, its dependence on energy-intensive drying and extraction steps constrains its economic feasibility. conversely, in situ transesterification simplifies the process by compiling extraction and conversion, thereby decreasing processing time, solvent usage, and costs. However, challenges related to moisture content, catalyst inhibition, and process optimization still hinder its large-scale implementation. Importantly, the success of both approaches is strongly dependent on upstream factors of lipid content, composition, and extraction. Hence, optimizing biodiesel yield needs a holistic integration of lipid enhancement, recovery efficiency, and conversion processes to achieve maximum economic outcomes.