Hydrothermal liquefaction of microalgae is a promising route for biocrude production; however, accurately modelling the complex nonlinear relationships among biomass composition, operating conditions, and biocrude properties remains challenging. Accordingly, an integrated artificial neural network–response surface methodology–Box–Behnken design (ANN–RSM–BBD) framework was developed to predict and optimize biocrude yield, heteroatom content, and higher heating value. The model was trained using an extensive dataset of 880 experimental observations encompassing biochemical composition, including lipids, proteins, carbohydrates, and ash, as well as elemental composition, including C, H, O, N, and S, under varied operating conditions. The integrated ANN–RSM framework was applied to predict biocrude yield (YBc), nitrogen, oxygen, and sulfur contents, and higher heating value (HHV). The ANN model demonstrated superior predictive accuracy, with R2 values exceeding 0.98 and RMSE values below 5%, compared with standalone RSM models with R2 values of approximately 0.94, confirming its ability to capture complex nonlinear and multiparameter interactions. Optimization results indicated that lipid-rich strains, including Chlorella vulgaris and Nannochloropsis salina, achieved maximum YBc values exceeding 45 wt% and HHV values above 35 MJ kg−1 under moderate HTL conditions of 330 °C, 20 MPa, and 30–60 min residence time. Furthermore, YBc reached 100%, while HHV reached 80 MJ kg−1, depending on algal strain, biochemical composition, and HTL operating conditions. The proposed Neural Engineering framework provides an efficient, data-driven approach for predicting and enhancing biocrude yield and quality through the integration of AI-based modelling and experimental design.
Low-molecular-weight fucoidan (LMWF) is highly desirable for food and pharmaceutical applications, yet conventional degradation methods often compromise its bioactive sulfate content. In this study, we employed a TEMPO/NaClO/NaBr system to degrade fucoidan under mild alkaline conditions and systematically investigated the degradation kinetics, structural changes, and anticoagulant activity of the resulting LMWF products. The degradation process was monitored by NaOH consumption and molecular weight distribution analysis. The optimal reaction time was determined to be 0.5 h, during which the molecular weight decreased from 337.4 kDa to 24.3 kDa, yielding LMWF with a relatively narrow molecular weight distribution (PDI = 1.08). Structural characterization by monosaccharide composition analysis, FT-IR, and 1H NMR suggested that TEMPO-mediated oxidation favored oxidation at C6 primary hydroxyl groups, as indicated by changes in galactose and galacturonic acid contents, while no significant loss of total sulfate content was observed. In contrast, the non-selective NaClO/NaBr system caused substantial losses of fucose (18.97%) and total sulfate content (6.13%). Notably, the TEMPO-derived LMWF retained potent anticoagulant activity in the APTT assay while exerting a minimal effect on the PT pathway, suggesting a potentially lower bleeding risk. This study provides a promising strategy for the controlled preparation of LMWF with minimal loss of total sulfate content and a potentially favorable safety profile.
Germplasm of kelp (e. g. Saccharina japonica) is preserved as gametophyte clones under low light and low temperature. However, long-term storage causes a gradual decline in gametophyte vigor, specifically, the loss of reproductive capacity, threatening effective germplasm utilization. Using gametophytes preserved from 1981 to 2020, we systematically evaluated vigor changes and characterized the decline from morphological, physiological, biochemical, and epigenetic perspectives. Gametogenesis and development rates decreased significantly with storage duration. Long-preserved gametophytes exhibited elongated rod-shaped cells, thickened cell walls, and increased plastids. Soluble protein content, catalase activity (CAT), and total antioxidant capacity (T-AOC) were negatively correlated with storage duration (P < 0.01), whereas H₂O₂ and malondialdehyde (MAD) levels were positively correlated (P < 0.01), indicating intensifying oxidative stress. Global 5-mC methylation declined sharply with storage time (r = −0.952, P < 0.01), and multiple methylation-related genes showed dynamic expression during gametophyte development: the methyltransferase gene SJ02917 was upregulated on day 6 (the transition period from vegetative growth to gametogenesis), SJ00150 peaked on day 9 (the oogonia formation stage), whereas the demethylase gene SJ04314 showed the highest expression level on day 0 (the initial developmental stage). Based on these findings, we propose an integrated hypothesis: long-term storage disrupts ROS homeostasis, which is associated with global DNA hypomethylation. We speculate that this epigenetic alteration may affect the expression of cell cycle regulators, thereby modulating the transition from mitosis to cell differentiation. However, this hypothesis currently lacks direct causal evidence and requires future validation. Soluble protein, CAT, T-AOC, H₂O₂, MDA, and 5-mC% emerged as key vigor indicators. This study provides both a mechanistic framework for understanding vigor decline and a practical basis for monitoring germplasm health during long-term preservation.
Algae are important sources, but environmental fluctuations severely restrict their growth. To efficiently utilize algal resources, it is necessary to investigate transcriptional regulatory mechanisms under different conditions. Recently, long non-coding RNAs (lncRNAs) have been recognized as critical regulatory elements affecting gene transcription. In algae, thousands of lncRNAs have been identified, but their effects in metabolic pathways remain poorly characterized. Since investigating the regulatory relationships between lncRNAs and protein-coding genes may help to discover lncRNA functions, a resource for exploring lncRNA transcriptional activity and functional analysis is needed. Previously, AlgaePath integrated numerous high-throughput gene expression datasets, enabling users to identify differentially expressed genes under various conditions and to explore the metabolic pathways in which they are involved. Here, we present AlgaePath 2.0, a substantially enhanced platform that integrates large-scale transcriptomic data with multi-level functional analysis. AlgaePath 2.0 incorporates 1395 RNA-seq samples from Chlamydomonas reinhardtii across 537 experimental conditions, providing expression profiles for 22,367 genes, including 2639 lncRNAs. It features dynamic gene correlation network reconstruction, promoter analysis tools, enrichment analysis for 132 pathways and 3725 Gene Ontology terms, and an interactive module for user-uploaded gene expression analysis. By integrating transcriptome profiling, lncRNA annotation, regulatory network analysis, and functional enrichment into a single platform, AlgaePath 2.0 provides a comprehensive resource for exploring candidate regulatory relationships and generating hypotheses regarding transcriptional regulation in algal metabolic pathways. The updated database is freely available at https://algaepath.itps.ncku.edu.tw/.
Microalgal biorefineries are considered an extremely promising approach for achieving a circular and carbon-neutral bioeconomy by synergistically combining CO2 sequestration with wastewater utilization. The present research highlights an integrated experimental-computational framework to maximize carbon capture, utilization, and storage, along with bioremediation and the synthesis of precursors for biomass/biofuels production in the pre-acclimatized Scenedesmus obliquus. Continuous chemostat cultivations with dilution rates from 0.005 h−1 to 0.040 h−1 were operated using seawater-drain-wastewater containing externally-added heavy metals and bicarbonates. Measured extracellular fluxes, quantified with HPLC, GC–MS, and CHNS/O analyses, were used as experimental constraints in a novel genetic-algorithm metabolic-flux-analysis approach, implemented in MATLAB®. Employing a stoichiometrically balanced network of 185 reactions, the computational workflow resulted in precise measurement of intracellular carbon partitioning. The carbon was directed towards overflow metabolism at the low dilution rate, resulting in acetate (2.588C−mmol/gbiomass/h). At a high dilution rate (0.040 h−1), the co-utilization of carbon leads to the highest biomass (1.689C−mmol/gbiomass/h), a remarkable carbon capture efficiency of 98.5%, while simultaneously maximizing lipid productivity (181.15 mg/L/h). This concurrently sustained the co-production of high-value pigments, yielding intracellular productivity of 0.453 mg/L/h for ‘chlorophylla’ and 4.803 mg/L/h for phycocyanin. The steady-state at 0.025 h−1 achieved the highest heavy metal removal efficiency (reaching 99.04%, 99.17%, and 99.01% for Cu2+, Cr3+, and Cd2+, respectively). Through identifying key targets for metabolic engineering to reduce byproduct secretion while simultaneously maximizing biorefinery systems, this research presents an innovative, scalable solution that directly supports the climate-water-energy nexus and United Nations Sustainable Development Goals 6, 7, 12, and 13.
The transition toward high-density microalgae cultivation has become essential for improving biomass productivity and spatial efficiency in microalgae biorefinery. However, harvesting for high-density biomass has not been explored intensively, and even considered as a bottleneck since the performance declines as the culture density increases. To address this issue, a new harvesting strategy, AOP-FFP, a combination of flocculation-flotation process (FFP) with advanced oxidation process (AOP) was proposed here. At a biomass concentration of 2.0 g L−1, the AOP-FFP system alleviated the inhibitory influence of algal organic matter (AOM) and increased the final harvesting efficiency by 11.1 ± 2.1% compared to non-AOP process (FFP only). Since excessive UV/H2O2 in AOP could damage cell surfaces, the treatment intensity was optimized to maintain cell integrity, correlated with the flocculation performances. The effect of optimized AOP treatment in alleviating flocculation inhibition became more noticeable with increasing polysaccharide concentration, indicating that the combined process effectively prohibits the negative influence of polysaccharide-rich AOM in high-density cultures. Furthermore, AOP-FFP achieved harvesting efficiency comparable to that of FFP despite using 25% less flocculant, reducing biomass harvesting costs by approximately $0.3 kg−1. These results demonstrate that the AOP-FFP system is quite effective for harvesting high-density microalgae biomass. Each step, AOP and FFP is a scalable and energy-efficient process, and the combined harvesting process, proposed here, could contribute to the industrial-scale biorefinery of microalgae biomass.
This study evaluated the dietary supplementation of Hy-Line Brown® laying hens with a microalgal blend composed of Microchloropsis gaditana and Muriellopsis sp. strain MCH-35 at inclusion levels of 1% and 3% (microalgal mix) and 10% (M. gaditana), aiming to improve the nutritional and functional quality of eggs. The formulated pellets were analyzed for proximate composition, fatty acid profile, carotenoid content, and antioxidant capacity, while the feeding trials were conducted over eight weeks. Microalgal inclusion enhanced the nutritional profile of the feed by increasing protein, lipid, and mineral contents while reducing crude fiber. M. gaditana contributed high levels of eicosapentaenoic acid (EPA), whereas Muriellopsis sp. exhibited higher lutein and total carotenoid contents, significantly increasing antioxidant capacity. In egg yolks, supplementation produced a progressive, dose-dependent increase in pigmentation, with the most intense coloration observed in the 3% and 10% treatments. Additionally, a significant increase in docosahexaenoic acid (DHA) and a reduction in linoleic acid (n-6) were recorded, improving the n-6/n-3 ratio from 24:1 (control) to 7:1 (3%) and 6:1 (10% M. gaditana). Total carotenoid and lutein contents in yolks increased by 2.3- and 2.7-fold, respectively, without affecting egg physical parameters. Biochemical profiles of the hens confirmed the absence of hepatic or metabolic stress, validating the safety of supplementation. Microalgae-enriched pellets offer an innovative and sustainable feed alternative that enables the production of functional eggs enriched with omega-3 fatty acids and carotenoids, enhancing their nutritional quality while adding both economic and environmental value to the poultry sector.
The growing prevalence of Cd(II) and Cu(II) contamination in aquatic systems underscores the need for efficient remediation strategies; however, the low surface area and insufficient active sites of algal biochar restrict its adsorption performance. In this work, freshwater algae were used as a precursor to prepare iron-modified algal biochar (FeBC) through the synergistic strategy of KOH activation and Fe(NO₃)₃·9H₂O impregnation. The adsorption performance and mechanisms of FeBC for Cd(II) and Cu(II) removal were systematically investigated. Compared with pristine algal biochar, FeBC exhibited significantly enhanced adsorption performance. Under identical experimental conditions, the removal efficiency of Cd(II) (100 mg/L) increased from 25.5% to 100%, with adsorption capacity increasing from 7.08 to 27.78 mg/g. Similarly, Cu(II) (30 mg/L) removal efficiency increased from 36.3% to 100%, with adsorption capacity increasing from 2.48 to 6.82 mg/g. SEM, BET, XRD, FTIR, and XPS analyses revealed significant structural and surface modifications of FeBC after KOH activation and iron impregnation. The specific surface area increased from 2.30 m2/g to 180.88 and 206.74 m2/g, accompanied by the formation of Fe–O/Fe–OH active sites and oxygen-containing functional groups. The adsorption kinetics and isotherms were better described by the pseudo-second-order and Langmuir models, respectively, indicating the contribution of chemisorption and monolayer adsorption. XPS analysis confirmed that surface complexation, ion exchange, and redox reactions contributed to Cd(II) and Cu(II) removal. After three adsorption–desorption cycles, FeBC retained 71.85% and 41.15% of its initial adsorption capacity for Cd(II) and Cu(II), respectively, demonstrating good stability and reusability. This synergistic KOH activation‑iron impregnation strategy overcame the limitations of algal biochar and provided an effective approach for biomass valorization and heavy metal remediation.
The present study showed that Aurantiochytrium sp. SZ13 could simultaneously accumulate docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), and odd-chain fatty acids (OcFAs) with distinct accumulation profiles, which reached the highest to 34.4% (96 h), 5.92% (168 h) and 20.48% (192 h) of total fatty acids (TFA), respectively. Time-resolved transcriptomics linked DHA accumulation to PUFA synthase pathway expression, EPA accumulation to elongase (ELOVL5/7 and HSD17B12) and desaturase (DES5) expression, whereas OcFAs accumulation appeared to be more closely associated with propionyl-CoA supply than with transcriptional changes in fatty acid biosynthetic genes. Furthermore, carbonic anhydrase (CA), O-linked N-acetylglucosamine transferase (OGT), and sugar transporter (SLC50A) were proposed to be key regulatory genes. Lastly, cytokinin at a concentration of 0.05 mg L−1 increased the yields of DHA and OcFAs by 36.8% and 72.6%, respectively, while gibberellin at 5 mg L−1 enhanced EPA yield by 73.8%. This study provided valuable insights into the regulatory mechanisms and suggested the potential of phytohormone-mediated regulation for functional fatty acid production.
The intertidal red alga Pyropia haitanensis, with its exceptional salt tolerance, serves as an ideal model for elucidating plant salt adaptation mechanisms. However, the regulatory mechanisms at the post-translational level, particularly the dynamic protein phosphorylation events underlying its stress response, remain poorly understood. This study employed integrated quantitative proteomics and phosphoproteomics to systematically investigate the dynamic response of P. haitanensis to hypersaline stress (110‰) across different time points (0 min, 15 min, 1 h, 12 h). A total of 16,404 phosphorylation sites on 2904 proteins were identified, revealing a regulatory pattern that shifts from early dephosphorylation-dominant to late phosphorylation-dominant states. The early stress response (15 min - 1 h) was characterized by extensive protein dephosphorylation, promoting rapid energy conservation through the suppression of processes such as carbon metabolism, photosynthesis, and nitrogen assimilation. In contrast, prolonged stress (12h) shifted towards phosphorylation dominated regulation, activating various reconstruction and adaptation pathways, including SNARE-mediated vesicle trafficking, spliceosome assembly, nucleotide excision repair, and inositol phosphate metabolism. Crucially, the vast majority of differentially phosphorylated proteins exhibited no significant changes in abundance (90.82%), indicating that phosphorylation acts as a core rapid response strategy operating independently of transcriptional and translational regulation. This study elucidates the dynamic phosphoregulatory network underpinning the salt tolerance of P. haitanensis at the post-translational level, providing a valuable theoretical foundation and potential candidate targets for enhancing salt tolerance in crops.
Microalgae exhibit high metabolic versatility and the ability to synthesize valuable biomolecules such as extracellular polymeric substances (EPS), positioning them as promising bio-based reagents for sustainable bioreagents in mineral processing. Although extensively studied in wastewater treatment and metal bioremediation, their application in mining systems remains relatively unexplored. This review examines the biological and physicochemical properties that make microalgae suitable for improving solid–liquid separation, recycled water quality, and tailings management.Microalgal EPS, rich in anionic polysaccharides and functional groups such as carboxyl, phosphate, and hydroxyl moieties, can promote particle aggregation through mechanisms including charge neutralization, polymer bridging and cation–induced flocculation. These properties enable the formation of dense organic–mineral flocs that enhance sedimentation and water clarification, offering a biodegradable alternative to synthetic flocculants. In addition, the reactive surface chemistry of microalgae and their EPS enables interactions with metal-bearing particles and dissolved ions, contributing to particle aggregation, surface modification, and improved water quality in mineral processing systems.Their tolerance to variable pH, salinity and metal concentrations allow microalgae to operate in challenging mining environments. Controlled cultivation strategies can further enhance EPS productivity and tailor bioflocculant properties to specific mineral suspensions. Despite these advantages, important challenges remain regarding species selection, large–scale cultivation, EPS extraction and pilot-scale validation. Overall, microalgae and their EPS represent a promising platform for development of environmentally compatible reagents aligned with sustainable mining practices.
Phaeodactylum tricornutum is a promising diatom for the co-production of biofuel precursors and high-value carotenoids. In this study, an integrated cultivation strategy combining optimized nutrient supply (nitrogen, iron, and silicon) with light shift (red light followed by blue-green light) was developed to enhance the simultaneous production of biomass, lipids, and fucoxanthin in P. tricornutum. Compared with the conventional F/2 medium, nitrogen- and iron-rich (+N + 186.4 μM Fe) culture significantly increased fucoxanthin yield (20.98 mg/L) by 117.8% and fucoxanthin content (16.87 mg/g) by 43.6%, while lipid content (35.87 DW%) increased by 98.7%. Crucially, a 2.18-fold elevation of the high-value coproduct fucoxanthin production was maintained, supporting the co-production of biofuel and high-value products. Multi-omics analyses, further supported by RT-qPCR of representative genes, revealed that the improved productivity was associated with coordinated activation of the photosynthetic system and the central carbon metabolism, which increased energy generation, precursor supply, and carbon flux toward both biomass formation and the parallel biosynthesis of lipids and carotenoids. These results demonstrate an effective nutrient-light regulation strategy for improving the productivity of P. tricornutum and provide mechanistic support for its application in microalgal biorefinery.
