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Microalgal lipids hold significant potential for the production of biodiesel and dietary supplements. To enhance their cost-effectiveness and commercial competitiveness, it is imperative to improve microalgal lipid productivity. Metabolic engineering that targets the key enzymes of the fatty acid synthesis pathway, along with transcription factor engineering, are effective strategies for improving lipid productivity in microalgae. This review provides a summary of the advancements made in the past 5 years in engineering the fatty acid biosynthetic pathway in eukaryotic microalgae. Furthermore, this review offers insights into transcriptional regulatory mechanisms and transcription factor engineering aimed at enhancing lipid production in eukaryotic microalgae. Finally, the review discusses the challenges and future perspectives associated with utilizing microalgae for the efficient production of lipids.

In this work, microbial lipid production from non-detoxified Eucalyptus bark hydrolysate (EBH) with oleaginous xylose-utilizing Ashbya gossypii strains was explored. The best producing strain from a set of engineered strains was identified in synthetic media mimicking the composition of the non-detoxified EBH (SM), the lipid profile was characterized, and yeast extract and corn steep liquor (CSL) were pinpointed as supplements enabling a good balance between lipid accumulation, biomass production, and autolysis by A. gossypii. The potential of the engineered A. gossypii A877 strain to produce lipids was further validated and optimized with minimally processed inhibitor-containing hydrolysate and high sugar concentration, and scaled up in a 2 L bioreactor. Lipid production from non-detoxified EBH supplemented with CSL reached a lipid titer of 1.42 g/L, paving the way for sustainable single-cell oil production within the concept of circular economy and placing lipids as an alternative by-product within microbial biorefineries.

Oleic acid is increasingly required in many industries, causing the indiscriminate extension of land for the cultivation of certain agricultural products to extract their oil. The current contribution aimed to cultivate Candida wangnamkhiaoensis (CW) for the production of lipids and determine the profile of fatty acids in these lipids. The lipid yield was compared in the yeast when using glucose or glycerol as the substrate, in both cases being over 24%. The main fatty acids in the oil derived from CW were oleic, palmitic, stearic, and linoleic acid. The fatty acid composition of the oil from CW was very similar to that of avocado oil and resembled that of olive oil and palm oil. The advantages of cultivating CW include its relatively high percentage of oleic acid and the balance of other fatty acids, its capacity to generate lipids in a short time (48–72 h), the controlled environment of production (versus the variability of the cultivation of agricultural products), and the relatively limited surface area required. CW shows potential as an alternative and economical source of oleic acid for the food, drug, cosmetics, lubricant, and biofuel industries, and does not require the alteration of large extensions of land.

Low biomass yield and nutrient removal efficiency are problems challenging the employment of microorganisms for wastewater remediation. Starch processing effluent (SPE) was used as a fermentation substrate to co-culture Chlorella vulgaris and Rhodotorula glutinis for biofuel feedstock production. Co-culture options were compared, and the optimal conditions were identified. The result shows that microalgae and yeast should be inoculated simultaneously at the beginning of SPE-based fermentation to achieve high biomass yield and the optimal inoculation ratio, light intensity, and temperature should be 2:1, 150 μmol/m2/s, and 25 °C, respectively. Under the optimal conditions, the lipid yield of microorganisms was 1.81 g/L and the carbon–conversion ratio reached 82.53% while lipid yield and the carbon–conversion ratio in a monoculture fell in the range of 0.79–0.81 g/L and 55.93–62.61%, respectively. Therefore, compared to the monoculture model, the co-fermentation of Chlorella vulgaris and Rhodotorula glutinis in starch processing effluent could convert nutrients to single-cell oil in a more efficient way. It should be noted that with the reduced concentration of residual organic carbon in effluent and the increased carbon–conversion ratio, co-fermentation of microalgae and yeast can be regarded as a promising and applicable strategy for starch processing effluent remediation and low-cost biofuel feedstock production.

Lipid extraction from microbial and microalgae biomass requires the separation of oil-rich cells from the production media. This downstream procedure represents a major bottleneck in biodiesel production, increasing the cost of the final product. Flocculation is a rapid and cheap system for removing solid particles from a suspension. This natural characteristic is displayed by some microorganisms due to the presence of lectin-like proteins (called flocculins/adhesins) in the cell wall. In this work, we showed, for the first time, that the heterologous expression of the adhesin Cfl1p endows the oleaginous species Cutaneotrichosporon oleaginosus with the capacity of cell flocculation. We used Helm’s test to demonstrate that the acquisition of this trait allows for reducing the time required for the separation of lipid-rich cells from liquid culture by centrifugation without altering the productivity. This improves the lipid production process remarkably by providing a more efficient downstream.

The interest in microorganisms to produce microbial lipids at large-scale processes has increased during the last decades. Rhodosporidium toruloides-1588 could be an efficient option for its ability to simultaneously utilize five- and six-carbon sugars. Nevertheless, one of the most important characteristics that any strain needs to be considered or used at an industrial scale is its capacity to grow in substrates with high sugar concentrations. In this study, the effect of high sugar concentrations and the effect of ammonium sulfate were tested on R. toruloides-1588 and its capacity to grow and accumulate lipids using undetoxified wood hydrolysates. Batch fermentations showed a catabolic repression effect on R. toruloides-1588 growth at sugar concentrations of 120 g/L. The maximum lipid accumulation was 8.2 g/L with palmitic, stearic, oleic, linoleic, and lignoceric acids as predominant fatty acids in the produced lipids. Furthermore, R. toruloides-1588 was able to utilize up to 80% of the total xylose content. Additionally, this study is the first to report the effect of using high xylose concentrations on the growth, sugar utilization, and lipid accumulation by R. toruloides-1588.

As an interesting alternative microbial platform for the sustainable synthesis of oleochemical building blocks and biofuels, oleaginous yeasts are increasing in both quantity and diversity. In this study, oleaginous yeast species from northern Thailand were discovered to add to the topology. A total of 127 yeast strains were isolated from 22 forest soil samples collected from mountainous areas. They were identified by an analysis of the D1/D2 domain of the large subunit rRNA (LSU rRNA) gene sequences to be 13 species. The most frequently isolated species were Lipomyces tetrasporus and Lipomyces starkeyi. Based on the cellular lipid content determination, 78 strains of ten yeast species, and two potential new yeast that which accumulated over 20% of dry biomass, were found to be oleaginous yeast strains. Among the oleaginous species detected, Papiliotrema terrestris and Papiliotrema flavescens have never been reported as oleaginous yeast before. In addition, none of the species in the genera Piskurozyma and Hannaella were found to be oleaginous yeast. L. tetrasporus SWU-NGP 2-5 accumulated the highest lipid content of 74.26% dry biomass, whereas Lipomyces mesembrius SWU-NGP 14-6 revealed the highest lipid quantity at 5.20 ± 0.03 g L−1. The fatty acid profiles of the selected oleaginous yeasts varied depending on the strain and suitability for biodiesel production.

Environmental pollution, greenhouse gas emissions, depletion of fossil fuels, and a growing population have sparked a search for new and renewable energy sources such as biodiesel. The use of waste or residues as substrates for microbial growth can favor the implementation of a biorefinery concept with reduced environmental footprint. Cyanobacteria constitute microorganisms with enhanced ability to use industrial effluents, wastewaters, forest residues for growth, and concomitant production of added-value compounds. In this study, a recently isolated cyanobacterium strain of Pseudanabaena sp. was cultivated on hydrolysates from pretreated forest biomass (silver birch and Norway spruce), and the production of biodiesel-grade lipids was assessed. Optimizing carbon source concentration and the (C/N) carbon-to-nitrogen ratio resulted in 66.45% w/w lipid content when microalgae were grown on glucose, compared to 62.95% and 63.79% w/w when grown on spruce and birch hydrolysate, respectively. Importantly, the lipid profile was suitable for the production of high-quality biodiesel. The present study demonstrates how this new cyanobacterial strain could be used as a biofactory, converting residual resources into green biofuel.

This paper studied the bifurcation phenomena that can occur in a continuous bioreactor for lipid-rich biomass production using Rhodococcus opacus that feeds on refinery wastewater. An unstructured model of the bioreactor was developed based on experimentally validated studies reported in the literature. The analysis of the unsteady-state model was carried out both analytically and through numerical simulations. It was shown that the inhibition effect of the biomass growth rate can lead to the occurrence of static bistability in the model. Periodic behavior was also found for some range of model parameters. The effect of model parameters on the productivity of the bioreactor was also investigated. The analysis carried out in this paper allowed for the detection of unsafe operating regions in the bioreactor which, would help in the attenuation of these operational problems in the early stage of process design.

Adding organic compounds to wastewater can improve the carbon/nitrogen ratio and benefit microalgae growth. We studied microalgal growth, nutrient removal and lipid accumulation of Chlorella pyrenoidosa cultured in a mixture of swine wastewater (SW) and furfural wastewater (FW). The mix ratio of SW:DFW (diluted furfural wastewater) had a significant effect on microalgae growth. As the mix ratio of SW:DFW decreased from 1:0.5 to 1:19, the maximum microalgal biomass increased, while the specific growth rate initially increased and then decreased. The efficiency of nutrient removal also depended on the mix ratio of wastewater. The highest chemical oxygen demand (COD) removal efficiency (57.30%) occurred at the mix ratio of SW:DFW = 1:3. The highest removal efficiencies of total phosphorous (TP) reached 61.93% when the mix ratio of SW:DFW was 1:9. Wastewater at the mix ratio of SW:DFW = 1:19 had a maximum lipid productivity of 49.48 mg L−1 d−1, which was 4.9 times higher than that at a mix ratio of SW:DFW = 1:0.5. These results showed that C. pyrenoidosa can be used to remove nutrients from mixed wastewater sources and simultaneously produce algal lipids.

With the aim of developing bioprocesses for waste valorization and a reduced water footprint, we optimized a two-step fermentation process that employs the oleaginous yeast Cutaneotrichosporon oleaginosus for the production of oil from liquid cheese whey permeate. For the first step, the addition of urea as a cost-effective nitrogen source allowed an increase in yeast biomass production. In the second step, a syrup from candied fruit processing, another food waste supplied as carbon feeding, triggered lipid accumulation. Consequently, yeast lipids were produced at a final concentration and productivity of 38 g/L and 0.57 g/L/h respectively, which are among the highest reported values. Through this strategy, based on the valorization of liquid food wastes (WP and mango syrup) and by recovering not only nutritional compounds but also the water necessary for yeast growth and lipid production, we addressed one of the main goals of the circular economy. In addition, we set up an accurate and fast-flow cytometer method to quantify the lipid content, avoiding the extraction step and the use of solvents. This can represent an analytical improvement to screening lipids in different yeast strains and to monitoring the process at the single-cell level.

In this study, magnetic Fe3O4 nanoparticles (NPs) were used as an effective enhancer to increase the biomass and total lipid production of Chlorella sp. UJ-3. It was found that the biomass of algal cells increased significantly when they were exposed to low concentrations of Fe3O4 NPs (20 mg/L), while the best total lipid content of algal cells was achieved when they were exposed to high concentrations of Fe3O4 NPs (100 mg/L). Therefore, we established a strategy to promote the growth and lipid accumulation of microalgae by initially exposing the algal cells to low concentrations of Fe3O4 NPs and then treating them with an increased concentration of Fe3O4 NPs after 12 days of culture. For this strategy, the biomass and total lipid production of algal cells increased by 50% and 108.7%, respectively, compared to the untreated control. The increase in lipid production and change in the fatty acid composition of Chlorella cells were found to help them to cope with the increased number of reactive oxygen species produced due to oxidative stress in alga cells after the addition of Fe3O4 NPs. This study provided a highly efficient way to improve the lipid production of microalgae using nanoparticles.

Microbial lipid production with cost effectiveness is a prerequisite for the oleochemical sector. In this work, genome-wide transcriptional responses on the utilization of xylose and glucose in oleaginous Aspergillus oryzae were studied with relation to growth and lipid phenotypic traits. Comparative analysis of the active growth (t1) and lipid-accumulating (t2) stages showed that the C5 cultures efficiently consumed carbon sources for biomass and lipid production comparable to the C6 cultures. By pairwise comparison, 599 and 917 differentially expressed genes (DEGs) were identified in the t1 and t2 groups, respectively, in which the consensus DEGs were categorized into polysaccharide-degrading enzymes, membrane transports, and cellular processes. A discrimination in transcriptional responses of DEGs set was also found in various metabolic genes, mostly in carbohydrate, amino acid, lipid, cofactors, and vitamin metabolisms. Although central carbohydrate metabolism was shared among the C5 and C6 cultures, the metabolic functions in acetyl-CoA and NADPH generation, and biosynthesis of terpenoid backbone, fatty acid, sterol, and amino acids were allocated for leveraging biomass and lipid production through at least transcriptional control. This study revealed robust metabolic networks in the oleaginicity of A. oryzae governing glucose/xylose flux toward lipid biosynthesis that provides meaningful hints for further process developments of microbial lipid production using cellulosic sugar feedstocks.

Optimization of the microalgae culture conditions could significantly reduce the production costs of microalgae-derived biodiesel. In the current study, a new process of adding different forms using the multiple small-dose method was employed. The effects of different forms of nitrogen (NaNO3, NH4Cl, and CH4N2O) and their concentrations (0.1, 0.5, 1, and 2 mg L−1) on the growth and lipid production of Scenedesmus obliquus were studied. Algae density and lipid production increased with increasing nitrogen concentration for all different forms of nitrogen except NH4Cl. The Scenedesmus obliquus growth was promoted by adding NaNO3 and CH4N2O, but was inhibited by adding NH4Cl. Adding 2 mg N L−1 of CH4N2O daily yielded the highest cell density (1.7 × 107 cells mL−1) and lipid production (242.4 mg L−1). These conditions can thus maintain the biomass of Scenedesmus obliquus, increase its lipid accumulation, and decrease the costs of biodiesel production.

Lipid production is an important indicator for assessing microalgal species for biodiesel production. In this work, the effects of medium composition on lipid production by Scenedesmus sp. were investigated using the response surface methodology. The results of a Plackett–Burman design experiment revealed that NaHCO3, NaH2PO4·2H2O and NaNO3 were three factors significantly influencing lipid production, which were further optimized by a Box–Behnken design. The optimal medium was found to contain 3.07 g L−1 NaHCO3, 15.49 mg L−1 NaH2PO4·2H2O and 803.21 mg L−1 NaNO3. Using the optimal conditions previously determined, the lipid production (304.02 mg·L−1) increased 54.64% more than that using the initial medium, which agreed well with the predicted value 309.50 mg L−1. Additionally, lipid analysis found that palmitic acid (C16:0) and oleic acid (C18:1) dominantly constituted the algal fatty acids (about 60% of the total fatty acids) and a much higher content of neutral lipid accounted for 82.32% of total lipids, which strongly proved that Scenedesmus sp. is a very promising feedstock for biodiesel production.

Microalgal lipids are a source of valuable nutritional ingredients in biotechnological industries, and are precursors to biodiesel production. Here, the effects of salt-induced stresses, including NaCl, KCl, and LiCl stresses, on the production of lipid in green microalga Chlamydomonas reinhardtii (137c) were investigated. NaCl stress dramatically increased saturated fatty acids (SFAs), which accounted for 70.2% of the fatty acid methyl ester (FAMEs) under stress. In contrary, KCl stress led to a slight increase in SFAs (47.05%) with the remaining being polyunsaturated fatty acids (PUFAs) (45.77%). RT-PCR analysis revealed that the genes involved in FA biosynthesis, such as PDH2, ACCase, MAT and KAS2, were up-regulated by NaCl-induced stress. Conversely, the genes responsible for the Kennedy pathway were suppressed. The alteration of FA homeostasis was further assessed by overexpressing MAT, the enzyme responsible for the production of malonyl-ACP, a key building block for FA biosynthesis, in the cyanobacterium Synechococcus elongatus PCC 7942. Intracellular FA composition was affected, with a predominant synthesis of SFAs in transformed cells. Owing to the diversity and relative abundance of SFAs, monounsaturated fatty acid (MUFAs) and PUFAs enable the feasibility of using microorganisms as a source of microalgal lipids or valuable nutritional ingredients; salt-induced stress and expression of MAT are useful in providing precursors for enhanced lipid production.

The microalga Nannochloropsis gaditana is a natural producer of triacylglycerol (TAG) and the omega-3 fatty acid eicosapentaenoic acid (EPA). TAG accumulation is induced by nitrogen starvation. The biomass-specific photon supply rate used had an effect on EPA and TAG accumulation during nitrogen starvation as well as on the localization of EPA accumulation. Clear differences in TAG yield on light were found for different biomass-specific photon supply rates and light regimes during nitrogen starvation. De novo EPA synthesis or the translocation of EPA between lipid fractions might be limiting for EPA accumulation in TAG. Further studies are needed to fully understand EPA accumulation in TAG during nitrogen starvation. To elucidate the function of EPA in TAG nitrogen recovery, experiments are suggested. The overexpression of genes involved in de novo EPA synthesis and translocation is proposed to elucidate the exact metabolic routes involved in these processes during nitrogen starvation. This work addresses future opportunities to increase EPA accumulation.

Water surface-floating microalgae have great potential for biofuel applications due to the ease of the harvesting process, which is one of the most problematic steps in conventional microalgal biofuel production. We have collected promising water surface-floating microalgae and characterized their capacity for biomass and lipid production. In this study, we performed chemical mutagenesis of two water surface-floating microalgae to elevate productivity. Floating microalgal strains AVFF007 and FFG039 (tentatively identified as Botryosphaerella sp. and Chlorococcum sp., respectively) were exposed to ethyl methane sulfonate (EMS) or 1-methyl-3-nitro-1-nitrosoguanidine (MNNG), and pale green mutants (PMs) were obtained. The most promising FFG039 PM formed robust biofilms on the surface of the culture medium, similar to those formed by wild type strains, and it exhibited 1.7-fold and 1.9-fold higher biomass and lipid productivities than those of the wild type. This study indicates that the chemical mutation strategy improves the lipid productivity of water surface-floating microalgae without inhibiting biofilm formation and floating ability.

Lipid extraction from microbial and microalgae biomass requires the separation of oil-rich cells from the production media. This downstream procedure represents a major bottleneck in biodiesel production, increasing the cost of the final product. Flocculation is a rapid and cheap system for removing solid particles from a suspension. This natural characteristic is displayed by some microorganisms due to the presence of lectin-like proteins (called flocculins/adhesins) in the cell wall. In this work, we showed, for the first time, that the heterologous expression of the adhesin Cfl1p endows the oleaginous species Cutaneotrichosporon oleaginosus with the capacity of cell flocculation. We used Helm's test to demonstrate that the acquisition of this trait allows for reducing the time required for the separation of lipid-rich cells from liquid culture by centrifugation without altering the productivity. This improves the lipid production process remarkably by providing a more efficient downstream.

As an interesting alternative microbial platform for the sustainable synthesis of oleochemical building blocks and biofuels, oleaginous yeasts are increasing in both quantity and diversity. In this study, oleaginous yeast species from northern Thailand were discovered to add to the topology. A total of 127 yeast strains were isolated from 22 forest soil samples collected from mountainous areas. They were identified by an analysis of the D1/D2 domain of the large subunit rRNA (LSU rRNA) gene sequences to be 13 species. The most frequently isolated species were Lipomyces tetrasporus and Lipomyces starkeyi . Based on the cellular lipid content determination, 78 strains of ten yeast species, and two potential new yeast that which accumulated over 20% of dry biomass, were found to be oleaginous yeast strains. Among the oleaginous species detected, Papiliotrema terrestris and Papiliotrema flavescens have never been reported as oleaginous yeast before. In addition, none of the species in the genera Piskurozyma and Hannaella were found to be oleaginous yeast. L. tetrasporus SWU-NGP 2-5 accumulated the highest lipid content of 74.26% dry biomass, whereas Lipomyces mesembrius SWU-NGP 14-6 revealed the highest lipid quantity at 5.20 ± 0.03 g L -1 . The fatty acid profiles of the selected oleaginous yeasts varied depending on the strain and suitability for biodiesel production.

Environmental pollution, greenhouse gas emissions, depletion of fossil fuels, and a growing population have sparked a search for new and renewable energy sources such as biodiesel. The use of waste or residues as substrates for microbial growth can favor the implementation of a biorefinery concept with reduced environmental footprint. Cyanobacteria constitute microorganisms with enhanced ability to use industrial effluents, wastewaters, forest residues for growth, and concomitant production of added-value compounds. In this study, a recently isolated cyanobacterium strain of Pseudanabaena sp. was cultivated on hydrolysates from pretreated forest biomass (silver birch and Norway spruce), and the production of biodiesel-grade lipids was assessed. Optimizing carbon source concentration and the (C/N) carbon-to-nitrogen ratio resulted in 66.45% w / w lipid content when microalgae were grown on glucose, compared to 62.95% and 63.79% w / w when grown on spruce and birch hydrolysate, respectively. Importantly, the lipid profile was suitable for the production of high-quality biodiesel. The present study demonstrates how this new cyanobacterial strain could be used as a biofactory, converting residual resources into green biofuel.

In this study, magnetic Fe 3 O 4 nanoparticles (NPs) were used as an effective enhancer to increase the biomass and total lipid production of Chlorella sp. UJ-3. It was found that the biomass of algal cells increased significantly when they were exposed to low concentrations of Fe 3 O 4 NPs (20 mg/L), while the best total lipid content of algal cells was achieved when they were exposed to high concentrations of Fe 3 O 4 NPs (100 mg/L). Therefore, we established a strategy to promote the growth and lipid accumulation of microalgae by initially exposing the algal cells to low concentrations of Fe 3 O 4 NPs and then treating them with an increased concentration of Fe 3 O 4 NPs after 12 days of culture. For this strategy, the biomass and total lipid production of algal cells increased by 50% and 108.7%, respectively, compared to the untreated control. The increase in lipid production and change in the fatty acid composition of Chlorella cells were found to help them to cope with the increased number of reactive oxygen species produced due to oxidative stress in alga cells after the addition of Fe 3 O 4 NPs. This study provided a highly efficient way to improve the lipid production of microalgae using nanoparticles.