Engineering Fatty Acid Biosynthesis in Microalgae: Recent Progress and Perspectives.

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.

Role of microalgae-bacterial consortium in wastewater treatment: A review.

In the global effort to reduce CO2 emissions, the concurrent enhancement of pollutant degradation and reductions in fossil fuel consumption are pivotal aspects of microalgae-mediated wastewater treatment. Clarifying the degradation mechanisms of bacteria and microalgae during pollutant treatment, as well as regulatory biolipid production, could enhance process sustainability. The synergistic and inhibitory relationships between microalgae and bacteria are introduced in this paper. The different stimulators that can regulate microalgal biolipid accumulation are also reviewed. Wastewater treatment technologies that utilize microalgae and bacteria in laboratories and open ponds are described to outline their application in treating heavy metal-containing wastewater, animal husbandry wastewater, pharmaceutical wastewater, and textile dye wastewater. Finally, the major requirements to scale up the cascade utilization of biomass and energy recovery are summarized to improve the development of biological wastewater treatment.

Concentration-dependent dual roles of proanthocyanidins on oxidative stress and docosahexaenoic acid production in Schizochytrium sp. ATCC 20888.

Antioxidant addition is an effective strategy to achieve docosahexaenoic acid (DHA) overproduction in oleaginous microorganisms. Nevertheless, antioxidants like phenolic compounds sometimes exert pro-oxidant activity. In this work, effects of proanthocyanidins (PAs) on fermentation performance and oxidative stress in Schizochytrium sp. were investigated. Low PAs addition (5 mg/L) reduced reactive oxygen species and enhanced lipogenic enzymes activities and NADPH, resulting in significant increase in lipid (20.3 g/L) by 33.6 % and DHA yield (9.8 g/L) by 53.4 %. In contrast, high PAs addition (500 mg/L) exerted pro-oxidant effects, aggravated oxidative damage and lipid peroxidation, leading to sharp decrease in biomass (21.3 g/L) by 35.1 %, lipid (8.2 g/L) by 46.0 %, and DHA (2.9 g/L) by 54.8 %. Therefore, the antioxidant concentration is especially crucial in DHA production. This study is the first to report concentration-dependant dual roles of PAs in oxidative stress and DHA production in Schizochytrium sp., providing new insights into microbial DHA production.

Enhanced microalgal biomass and lipid production with simultaneous effective removal of Cd using algae-bacteria-activated carbon consortium added with organic carbon source.

Recently, using microalgae to remediate heavy metal polluted water has been attained a huge attention. However, heavy metals are generally toxic to microalgae and consequently decrease biomass accumulation. To address this issue, the feasibility of adding exogenous glucose, employing algae-bacteria system and algae-bacteria-activated carbon consortium to enhance microalgae growth were evaluated. The result showed that Cd2+ removal efficiency was negatively correlated with microalgal specific growth rate. The exogenous glucose alleviated the heavy metal toxicity to algal cells and thus increased the microalgae growth rate. Among the different treatments, the algae-bacteria-activated carbon combination had the highest biomass concentration (1.15 g L-1) and lipid yield (334.97 mg L-1), which were respectively 3.03 times of biomass (0.38 g L-1) and 4.92 times of lipid yield (68.08 mg L-1) in the single microalgae treatment system. Additionally, this algae-bacteria-activated carbon consortium remained a high Cd2+ removal efficiency (91.61%). In all, the present study developed an approach that had a great potential in simultaneous heavy metal wastewater treatment and microalgal lipid production.

Microalgae cultivation with recycled harvesting water achieved economic and sustainable production of biomass and lipid: Feasibility assessment and inhibitory factors analysis.

This study was conducted to achieve economic and sustainable production of biomass and lipids from Chlorella sorokiniana by recirculating cultivation with recycled harvesting water, to identify the major inhibitory factors in recirculating culture, and to analyze accordingly economic benefits. The results showed that recirculating microalgae cultivation (RMC) could obtain 0.20-0.32 g/L biomass and lipid content increased by 23.1 %-38.5 %. Correlation analysis showed that the extracellular polysaccharide (PSext), chemical oxygen demand (COD) and chromaticity of recirculating water inhibited photosynthesis and induced oxidative stress, thus inhibiting the growth of C. sorokiniana. In addition, the economic benefits analysis found that circulating the medium twice could save about 30 % of production cost, which is the most economical RMC solution. In conclusion, this study verified the feasibility and economy of RMC, and provided a better understanding of inhibitory factors identification in culture.

Advanced treatment of antibiotic-polluted wastewater by a consortium composed of bacteria and mixed cyanobacteria.

This study constructed a cyanobacteria-bacteria consortium using a mixture of non-toxic cyanobacteria (Synechococcus sp. and Chroococcus sp.) immobilized in calcium alginate and native bacteria in wastewater. The consortium was used for the advanced treatment of sulfamethoxazole-polluted wastewater and the production of cyanobacterial lipid. Mixed cyanobacteria increased the abundances of denitrifying bacteria and phosphorus-accumulating bacteria as well as stimulated various functional enzymes in the wastewater bacterial community, which efficiently removed 70.01-71.86% of TN, 91.45-97.04% of TP and 70.72-76.85% of COD from the wastewater. The removal efficiency of 55.29-69.90% for sulfamethoxazole was mainly attributed to the upregulation of genes encoding oxidases, reductases, oxidoreductases and transferases in two cyanobacterial species as well as the increased abundances of Stenotrophomonas, Sediminibacterium, Arenimonas, Novosphingobium, Flavobacterium and Hydrogenophaga in wastewater bacterial community. Transcriptomic responses proved that mixed cyanobacteria presented an elevated lipid productivity of 33.90 mg/L/day as an adaptive stress response to sulfamethoxazole. Sediminibacterium, Flavobacterium and Exiguobacterium in the wastewater bacterial community may also promote cyanobacterial lipid synthesis through symbiosis. Results of this study proved that the mixed cyanobacteria-bacteria consortium was a promising approach for advanced wastewater treatment coupled to cyanobacterial lipid production.

Neuro-fuzzy modelling of a continuous stirred tank bioreactor with ceramic membrane technology for treating petroleum refinery effluent: a case study from Assam, India.

A continuous stirred tank bioreactor (CSTB) with cell recycling combined with ceramic membrane technology and inoculated with Rhodococcus opacus PD630 was employed to treat petroleum refinery wastewater for simultaneous chemical oxygen demand (COD) removal and lipid production from the retentate obtained during wastewater treatment. In the present study, the COD removal efficiency (CODRE) (%) and lipid concentration (g/L) were predicted using two artificial intelligence models, i.e., an artificial neural network (ANN) and a neuro-fuzzy neural network (NF-NN) with a network topology of 6-25-2 being the best for NF-NN. The results revealed the superiority of NF-NN over ANN in terms of determination coefficient (R2), root mean square error (RMSE), and mean absolute percentage error (MAPE). Three learning algorithms were tested with NF-NN; among them, the Bayesian regularization backpropagation (BR-BP) outperformed others. The sensitivity analysis revealed that, if solid retention time and biomass concentrations were maintained between 35 and 75 h and 3.0 g/L and 3.5 g/L, respectively, high CODRE (93%) and lipid concentration (2.8 g/L) could be obtained consistently.

Selecting for a high lipid accumulating microalgae culture by dual growth limitation in a continuous bioreactor.

A dual-growth-limited continuous operated bioreactor (chemostat) was used to enhance lipid accumulation in an enrichment culture of microalgae. The light intensity and nitrogen concentration where both limiting factors resulting in high lipid accumulation in the mixed culture. Both conditions of light and nitrogen excess and deficiency were tested. Strategies to selectively enrich for a phototrophic lipid-storing community, based on the use of different nitrogen sources (ammonium vs. nitrate) and vitamin B supplementation in the growth medium, were evaluated. The dual limitation of both nitrogen and light enhanced the accumulation of storage compounds. Ammoniacal nitrogen was the preferred nitrogen source. Vitamin B supplementation led to a doubling of the lipid productivity. The availability of vitamins played a key role in selecting an efficient lipid-storing community, primarily consisting of Trebouxiophyceae (with an 82 % relative abundance among eukaryotic microorganisms). The obtained lipid volumetric productivity (387 mg L-1 d-1) was among the highest reported in literature for microalgae bioreactors. Lipid production by the microalgae enrichment surpassed the efficiencies reported for continuous microalgae pure cultures, highlighting the benefits of mixed-culture photo-biotechnologies for fuels and food ingredients in the circular economy.

A Coculture of Photoautotrophs and Hydrolytic Heterotrophs Enables Efficient Upcycling of Starch from Wastewater toward Biomass-Derived Products: Synergistic Interactions Impacting Metabolism of the Consortium.

Even with particular interest in sustainable development, due to the limited types of bioavailable carbon sources that could support heterotrophic/mixotrophic growth, microalgae-derived products still suffer from inconsistent yield and high costs. This study demonstrates a successful cocultivation of the photoautotroph Chlorella vulgaris with a hydrolytic-enzyme-abundant heterotroph, Saccharomycopsis fibuligera, enabling efficient starch upcycling from water/wastewater toward enhancing microalgae-dominant biomass and lipid production. The enzymatic activities of S. fibuligera contributed to the hydrolysis of starch into glucose, generating a 7-fold higher biomass through mixotrophic/heterotrophic growth of C. vulgaris. Further, scanning transmission electron microscopy (STEM) and quantitative analysis suggested a significantly induced accumulation of lipids in C. vulgaris. Results of meta-transcriptomics revealed the critical regulatory role of illumination in interaction shifting. Gene expression for glycolysis and lipid biosynthesis of C. vulgaris were highly activated during dark periods. Meanwhile, during illumination periods, genes coding for glucoamylase and the sulfur-related activities in S. fibuligera were significantly upregulated, leading to induced starch hydrolysis and potential increased competition for sulfur utilization, respectively. This study indicates that hydrolytic organisms could collaborate to make starch bioavailable for nonhydrolytic microalgae, thus broadening the substrate spectrum and making starch a novel biotechnological feedstock for microalgae-derived products, e.g., biofuels or single-cell protein.

Orange Peel Waste as Feedstock for the Production of Glycerol-Free Biodiesel by the Microalgae Nannochloropsis oculata.

The bioconversion of agri-food waste into high-value products is gaining growing interest worldwide. Orange peel waste (OPW) is the main by-product of orange juice production and contains high levels of moisture and carbohydrates. In this study, the orange waste extract (OWE) obtained through acid hydrolysis of OPW was used as a substrate in the cultivation of the marine microalgae Nannochloropsis oculata. Photoheterotrophic (PH) and Photoautotrophic (PA) cultivations were performed in OWE medium and f/2 medium (obtained by supplementing OWE with macro- and micronutrients of f/2 medium), respectively, for 14 days. The biomass yields in PA and PH cultures were 390 mg L-1 and 450 mg L-1, while oil yields were 15% and 28%, respectively. The fatty acid (FA) profiles of PA cultures were mostly represented by saturated (43%) and monounsaturated (46%) FAs, whereas polyunsaturated FAs accounted for about 10% of the FAs. In PH cultures, FA profiles changed remarkably, with a strong increase in monounsaturated FAs (77.49%) and reduced levels of saturated (19.79%) and polyunsaturated (2.72%) FAs. Lipids obtained from PH cultures were simultaneously extracted and converted into glycerol-free biodiesel using an innovative microwave-assisted one-pot tandem protocol. FA methyl esters were then analyzed, and the absence of glycerol was confirmed. The FA profile was highly suitable for biodiesel production and the microwave-assisted one-pot tandem protocol was more effective than traditional extraction techniques. In conclusion, N. oculata used OWE photoheterotrophically, resulting in increased biomass and oil yield. Additionally, a more efficient procedure for simultaneous oil extraction and conversion into glycerol-free biodiesel is proposed.

A novel fungal-algal coupling system for slaughterhouse wastewater treatment and lipid production.

In this study, a novel fungal-algal coupling system was established for slaughterhouse wastewater treatment with Chlorella sp. DT025 and a new fungus, Penicillium sp. AHP141. With the optimization of cultivation conditions for the fungal-algal coupling system, the harvest efficiency of Chlorella sp. DT025 reached 99.79%. The mechanism of microalgae harvest of the fungal-algal system was revealed to be related to the morphological characteristics, surface charge, and the secretion of humic acid-like compounds and tryptophan on the surface of the fungi cells. For the original slaughterhouse wastewater treatment, the fungal-algal coupling system had a better removal efficiency of COD, TN, and TP than both monoculture systems. In the high-concentration artificial slaughterhouse wastewater, COD removal of the fungal-algal system reached more than 5350 mg/L. The lipid production of the fungal-algal coupling system in the high-concentration artificial slaughterhouse wastewater treatment was improved by 343.33% to 1.33 g/L compared to the microalgae monoculture treatment.

Responses of microalgae under different physiological phases to struvite as a buffering nutrient source for biomass and lipid production.

Microalgae cultivation for biodiesel production is promising, but the high demand for nutrients, such as nitrogen and phosphorus, remains a limiting factor. This study investigated effects of struvite, a low-cost nutrient source, on microalgae production under different physiological phases. Changes in element concentrations were determined to characterize the controllable nutrient release properties of struvite. Results showed that nutrient elements could be effectively supplemented by struvite. However, responses of microalgae under different growth stages to struvite varied obviously, achieving the highest biomass (0.53 g/L) and the lowest (0.32 g/L). Moreover, the microalgal lipid production was obviously increased by adding struvite during the growth phase, providing the first evidence that struvite could serve as an alternative buffering nutrient source to culture microalgae. The integration of microalgae cultivation with struvite as a buffering nutrient source provides a novel strategy for high ammonia nitrogen wastewater treatment with microalgae for biodiesel production.

Krüppel-like factor 4 (KLF4) facilitates lipid production in immortalized human sebocytes via regulating the expression of SREBP1.

BACKGROUND: Acne is associated with the excessive production of sebum, a complex mixture of lipids, in the sebaceous glands. The transcription factor Krüppel-like factor 4 (KLF4) plays an important role in skin morphogenesis, but its role in sebum production by sebocytes is not well known. PURPOSE: In this study, we investigated the possible action mechanism of KLF4 during calcium-induced lipogenesis in immortalized human sebocytes. METHODS: Sebocytes were treated with calcium, and lipid production was confirmed by thin-layer chromatography (TLC) and Oil Red O staining. To investigate the effect of KLF4, sebocytes were transduced with the KLF4-overexpressing adenovirus, and then lipid production was evaluated. RESULTS: Calcium treatment resulted in increased sebum production in terms of squalene synthesis in sebocytes. In addition, calcium increased the expression of lipogenic regulators such as sterol-regulatory element binding protein 1 (SREBP1), sterol-regulatory element binding protein 2 (SREBP2), and stearoyl-CoA desaturase (SCD). Similarly, the expression of KLF4 was increased by calcium in sebocytes. To investigate the effect of KLF4, we overexpressed KLF4 in sebocytes using recombinant adenovirus. As a result, KLF4 overexpression increased the expression of SREBP1, SREBP2, and SCD. Parallel to this result, lipid production was also increased by KLF4 overexpression. Chromatin immunoprecipitation revealed the binding of KLF4 to the SREBP1 promoter, indicating that KLF4 may directly regulate the expression of lipogenic regulators. CONCLUSION: These results suggest that KLF4 is a novel regulator of lipid production in sebocytes.

Effect of Different Carbons on Lipid Production and SNF1 Transcription in Mucor Circinelloides.

Sucrose non-fermenting 1 (SNF1) protein kinase plays an important role in the utilization of selective carbon sources and regulation of lipid metabolism. In order to further explore the function of SNF1 in regulating lipid accumulation by responding nutritional signals from non-glucose carbon sources, in the present study, the lipid production and SNF1 transcriptional levels of Mucor circinelloides were analyzed and compared on different carbon sources. The results indicated that M. circinelloides could effectively utilize some secondary metabolic carbon sources of monosaccharides and disaccharides for growth and lipids production, such as fructose, maltose and galactose. Snf-ß subunit was associated with the regulation of lipid metabolism in response to nutritional signals from different carbon sources. This is the first report on the transcriptional analysis of SNF1 subunits on different carbons metabolism in oleaginous filamentous fungi. This research has suggested that genetic engineering of SNF1 subunits will alter the lipid production of M. circinelloides from alternative carbon sources. Supplementary Information: The online version contains supplementary material available at 10.1007/s12088-023-01070-z.

Expression of malic enzyme reveals subcellular carbon partitioning for storage reserve production in soybeans.

Central metabolism produces amino and fatty acids for protein and lipids that establish seed value. Biosynthesis of storage reserves occurs in multiple organelles that exchange central intermediates including two essential metabolites, malate, and pyruvate that are linked by malic enzyme. Malic enzyme can be active in multiple subcellular compartments, partitioning carbon and reducing equivalents for anabolic and catabolic requirements. Prior studies based on isotopic labeling and steady-state metabolic flux analyses indicated malic enzyme provides carbon for fatty acid biosynthesis in plants, though genetic evidence confirming this role is lacking. We hypothesized that increasing malic enzyme flux would alter carbon partitioning and result in increased lipid levels in soybeans. Homozygous transgenic soybean plants expressing Arabidopsis malic enzyme alleles, targeting the translational products to plastid or outside the plastid during seed development, were verified by transcript and enzyme activity analyses, organelle proteomics, and transient expression assays. Protein, oil, central metabolites, cofactors, and acyl-acyl carrier protein (ACPs) levels were quantified overdevelopment. Amino and fatty acid levels were altered resulting in an increase in lipids by 0.5-2% of seed biomass (i.e. 2-9% change in oil). Subcellular targeting of a single gene product in central metabolism impacts carbon and reducing equivalent partitioning for seed storage reserves in soybeans.

Algal-bacterial aerobic granular sludge for real municipal wastewater treatment: Performance, microbial community change and feasibility of lipid recovery.

Despite various research works on algal-bacterial aerobic granular sludge for wastewater treatment and resource recovery processes, limited information is available on its application in real wastewater treatment in terms of performance, microbial community variation and resource recovery. This study investigated the performance of algal-bacterial aerobic granular sludge on real low-strength wastewater treatment in addition to the characterization of microbial community and fatty acid compositions for biodiesel production. The results demonstrated 71% COD, 77% NH4+-N and 31% phosphate removal efficiencies, respectively. In addition, all the water parameters successfully met the effluent standard A, imposed by the Department of Environment (DOE) Malaysia. Core microbiome analyses revealed important microbial groups (i.e., Haliangium ochraceum, Burkholderiales and Chitinophagaceae) in bacterial community. Meanwhile the photosynthetic microorganisms, such as Oxyphotobacteria and Trebouxiophyceae dominated the algal-bacterial aerobic granular sludge, suggesting their important roles in granulation and wastewater treatment. Up to 12.51 mg/gSS lipid content was recovered from the granules. In addition, fatty acids composition showed high percetages of C16:0 and C18:0, demonstrating high feasibility to be used for biodiesel production application indicated by the cetane number, iodine value and oxidation stability properties.

CO2 capture and lipid production performance of microalgae in the S-shaped photobioreactor under different culture modes.

An S-shaped photobioreactor was designed by adding grooves and baffles in the traditional photobioreactor to improve the culture efficiency of microalgae. After that, the parameters of the characterization of the S-shaped photobioreactor, such as the mixing time, gas holdup, and gas-liquid mass transfer coefficient, were determined. The biomass, lipid production rate, and average CO2 capture rate of microalgae were then analyzed under different culture modes. Finally, the feasibility of using digested piggery wastewater combined with simulated flue gas was explored as a culture mode for the microalgae and the lipid properties of the microalgae were analyzed. The results revealed that, at a flow rate of 0.08 vvm, the mixing time was reduced by 8.5 s, the gas hold-up increased by 44.6% and the gas-liquid mass transfer ability was also improved. Improvements were also observed in the biomass values, lipid production rate, and average CO2 capture rate of the microalgae under different culture conditions, with respective values reaching 0.23 g·(L·d)-1, 70.28 mg·(L·d)-1, and 0.43 g·(L·d)-1 under the mixotrophic mode. Additionally, digested piggery wastewater combined with the simulated microalgae flue gas culture was determined to be feasible. The biomass, lipid production rate, and the average CO2 capture rate of microalgae, the values of which were 0.22 g·(L·d)-1, 52.55 mg·(L·d)-1, and 0.41 g·(L·d)-1, respectively. Lipid was observed to have the potential to produce high-quality biofuel.

Nutrient removal and lipid production by the co-cultivation of Chlorella vulgaris and Scenedesmus dimorphus in landfill leachate diluted with recycled harvesting water.

Applying microalgae for landfill leachate (LL) treatment is promising. However, LL usually needs to be diluted with much fresh water, aggravating water shortage. In this study, mono- and co-culturing microalgae (Chlorella vulgaris and Scenedesmus dimorphus) were used to treat LL diluted with recycled harvesting water, to investigate nutrient removal and lipid production. The results showed that microalgae in co-culture treatment had more biomass and stronger superoxide dismutase activity, which might be related to humic acids contained in recycled harvesting water, according to dissolved organic matters (DOMs) analysis. In addition, the lipid content and yield of co-cultured microalgae reached 27.60 % and 66.87 mg·L-1, respectively, higher than those of mono-culture, proving the potential of co-culture for the improvement of lipid production. This study provided a freshwater-saving dilution method for LL treatment with recycled harvesting water as well as a strategy for the increase of biomass and lipid accumulation by microalgae co-cultivation.

Simultaneous chromium removal and lipid accumulation by microalgae under acidic and low temperature conditions for promising biodiesel production.

Microalgae have become the hotspot of recent researches as heavy metals (HMs) adsorbent and biodiesel production feedstock. In this study, the cell growth, lipid production and Cr6+ removal of Parachlorella kessleri R-3 at pH 3.5 and 15 °C were investigated. It was found that low concentration of Cr6+ (0.5 to 2 mg/L) promoted the algal growth, whereas Cr6+ higher than 5 mg/L inhibited the growth of P. kessleri R-3. Biomass concentration (2.40 g/L) and lipid productivity (131.79 mg/L d-1) reached the highest at 2 mg/L Cr6+, and lipid content (61.03 %) reached the highest at 5 mg/L Cr6+. The maximum Cr6+ removal efficiency of 91 % was obtained at 0.5 mg/L Cr6+ treatment. Furthermore, fatty acid composition analysis showed that strain R-3 had a high C16-18 content of 74.88-89.21 %. This study provides new insight into the treatment of HMs and lipid production in cold regions.