Biotechnology for Biofuels最新文献

Background

Oleaginous microorganisms are promising lipid producers that accumulate an abundance of lipids from different carbon sources. However, the cost of the carbon source in the culture medium is a significant component of the total substrate cost. In this study, lignocellulose from corncob hydrolysate (CBH) was used instead of glucose as a low-cost medium for Schizochytrium fermentation.

Results

Eicosapentaenoic acid (EPA) content was 7.31%, after 110 h of fermentation, when the total sugar concentration of CBH was 80 g/L, which was greater than that of pure glucose medium. Replacing 40% of freshwater with fermentation wastewater (FW) resulted in biomass, lipid titer, and EPA titer of 42.16 g/L, 23.05 g/L, and 1.72 g/L, respectively. Compared with the initial CBH medium, the lipid and EPA titers in the 7.5-L bioreactor employing the FW recycling strategy using CBH as a carbon source increased by 12.10% and 9.26%, respectively.

Conclusions

Corncob hydrolysate can be used as a potential low-cost and effective carbon source for EPA production by Schizochytrium sp. The recycling of FW provides a reference for reducing freshwater consumption and environmental pollution and realizing green and economic recycling fermentation.

This review critically examines the entire value chain of microalgal biorefineries, with the central aim of elucidating the key technological, economic, and environmental enablers and barriers that govern their transition from pilot-scale demonstrations to commercially viable, circular-economy applications. A systematic literature search was conducted across five major scientific databases using predefined Boolean strings: “algal biorefineries,” “microalgae biofuel,” “techno-economic analysis,” “life-cycle assessment,” and “bioproduct recovery.” Inclusion criteria encompassed peer-reviewed studies and authoritative policy documents published between January 2007 and March 2025 that provided empirical data on upstream cultivation, midstream processing, and downstream conversion, as well as techno-economic assessments (TEA) and life-cycle analyses (LCA). Exclusion criteria included non-English commentaries, purely theoretical models without experimental validation, and studies that focused exclusively on single-product streams. Unlike previous reviews that address isolated segments of the algal biorefinery pipeline, this work delivers a novel, integrative framework that synthesizes recent advances across cultivation modes, genetic and metabolic engineering, AI‐enabled optimization, and IoT‐driven monitoring. This review critically evaluates the trade-offs between CAPEX and OPEX, energy penalties associated with harvesting and drying, and inconsistencies in LCA to identify, where performance improvements yield the most significant economic and environmental returns. Finally, this review proposes a targeted research roadmap, spanning multivariate strain engineering, hybrid cultivation architectures, low‐energy harvesting technologies, cascade‐compatible fractionation platforms, standardized LCA/TEA protocols, and supportive policy mechanisms, that charts a clear path toward overcoming current bottlenecks. This comprehensive, evidence‐based synthesis aims to inform both academic research and industrial strategy, thereby advancing the field of sustainable algal biorefineries.

Population growth throughout the world has led to increased pollution and overconsumption of fossil resources. Microalgae are increasingly recognized as sustainable biofactories for producing lipids and astaxanthin, two commercially significant metabolites with wide-ranging applications in biofuel, pharmaceutical, cosmetic, and nutraceutical industries. Enhancing the yields of these compounds remains a major challenge due to growth–productivity trade-offs and limited understanding of regulatory mechanisms. This review aims to bridge that gap by providing a comprehensive and comparative analysis of traditional and modern strategies employed to enhance lipid and astaxanthin production in microalgae. We critically evaluate stress-based methods (e.g., salinity, light, nutrient limitation), phytohormone treatments, cultivation system optimization, and genome editing technologies, including CRISPR/Cas9. Special emphasis is given to gene-level responses and pathway-level regulation involved in these enhancements. This review article highlights the novel synchronization between astaxanthin and fatty acid biosynthesis under various stress conditions which emphasizes the role of diacylglycerol acyltransferase (DGAT) enzymes to enhance astaxanthin accumulation. Editing technologies with base suggest a novel strategy to reduce off-target effects and enhance metabolic efficiency related to lipid and astaxanthin biosynthesis.

Graphical Abstract

Enzymes capable of catalysing the production of hydrocarbons hold promise for sustainable fuel synthesis. However, the native activities of these enzymes are often insufficient for their exploitation in industrial bioprocesses. Enzyme engineering approaches including directed evolution (DE) can be used to improve the properties of enzymes to meet desirable standards for their industrial application. In this review, we summarise DE methods for engineering hydrocarbon-producing enzymes, including both screening- and selection procedures. The efficacy of DE depends on several factors, including sensitive and accurate detection of enzyme activity, the throughput of screening or selection steps, and the scale of diversity generation. Although DE is a well-established approach, its application in engineering hydrocarbon-producing enzymes has not been widely demonstrated. This can be attributed to the physiochemical properties of the target molecules, such as aliphatic hydrocarbons, which can be insoluble, gaseous, and chemically inert. Detection of these molecules in vivo presents several unique challenges, as does dynamically coupling their abundance to cell fitness. We conclude with a discussion on future directions and potential advancements in this field.

Biomass is greatly influenced by geographic location, soil composition, environment, and climate, making the efficient and accurate identification of growing areas highly significant. This study proposes a classification model for tobacco growing areas based on time series features from thermogravimetric analysis (TGA). This study combines Convolutional Neural Networks (CNN) with Long Short-Term Memory (LSTM) model to process the derivative thermogravimetric (DTG) data, aiming to uncover the inherent time series properties and the continuous and dynamic relationship between temperatures for classifying tobacco growing areas. By analyzing 375 tobacco samples from ten different provinces, CNN is employed to extract local features, while LSTM captures long-term dependencies in the DTG data. The dataset used in this study has a limited sample size, a wide variety of classes, and an imbalance in the number of samples across these classes. Despite these challenges, the model achieves 86.4% accuracy on the test set, significantly surpassing the performance of the traditional Support Vector Machine model, which only achieves 68.2% accuracy. Additionally, the model reveals key temperature ranges crucial for growing area classification associated with the pyrolysis temperature ranges of volatile components, hemicellulose, cellulose, lignin, and CaCO₃ in the tobacco. This model lays the groundwork for the future use of geographical labels to accurately represent tobacco’s style and quality, enabling more precise differentiation and improved quality control.

Background

Industrial lignocellulosic side streams are considered an attractive carbon source for the cultivation of biotechnologically important fungi, although the presence of toxic pretreatment by-products is a major challenge yet to be overcome. Aspergillus oryzae is a filamentous fungus with a large secretion capacity, high tolerance for toxins, and a wide substrate range, making it a promising candidate for side stream utilization. In the present study, the cellular mechanisms of tolerance against furfural, 5-hydroxymethylfurfural (HMF), levulinic acid, ferulic acid, and vanillin were studied at the transcriptome level.

Results

A. oryzae RIB40 was grown in the presence of different inhibitors commonly found in lignocellulosic side streams, and RNA sequencing was utilized to investigate the transcriptomic changes in response to the inhibitors. Analysis of the transcriptomic response in all conditions indicates that a large fraction of differentially expressed genes responded to the inhibitor-induced formation of reactive oxygen species (ROS). Apart from levulinic acid, all inhibitors showed strong initial suppression of metabolic pathways related to cell cycle, ribosome functions, protein folding, and sorting in the endoplasmic reticulum. Genes associated with cellular detoxification, namely, NAD(P)H-dependent oxidoreductases and efflux transporters, such as the ATP-Binding Cassette (ABC) transporters and major facilitator superfamily (MFS) transporters, showed strong upregulation upon exposure to the inhibitors.

Conclusions

The results obtained provide important insights into the stress response of A. oryzae to the xenobiotic compounds and their cellular detoxification. Aldehydic inhibitors, especially HMF, caused a strong and severe stress response in A. oryzae RIB40. Additionally, we identified several highly upregulated uncharacterized genes upon exposure to the inhibitors. These genes serve as promising targets for strain engineering to build robust industrial strains capable of utilizing lignocellulosic side streams as feedstock.

Advances in tissue printing and wound healing necessitate a continuous global supply of collagen. Microbial systems are highly desirable to meet these demands as recombinant collagenous proteins can be guaranteed as free from animal viruses. The filamentous cell factory Aspergillus niger has been instrumental for decades in the production of organic acids, enzymes and proteins, yet this fungus has not been explored for recombinant collagen production. In this study, we conducted extensive genetic engineering and fermentation optimization to provide proof of principle that A. niger can produce hydroxylated collagen. We used a modular cloning system to generate a suite of cassettes encoding numerous N-terminal secretion signals, native collagen genes and, additionally, various prolyl-4-hydroxylases (P4H) for protein hydroxylation. Collagen transcription was supported by both luciferase reporter and eGFP tagged approaches. Peptide sequencing from culture supernatant confirmed A. niger produced partially hydroxylated collagen. We then conducted a range of media optimizations and RNA sequencing to, respectively, increase collagen production and identify proteases which we hypothesized were detrimental to recombinant protein titers. Thus, we deleted an endopeptidase encoding gene, protA, which was likely responsible for degrading secreting collagen. Ultimately, we were able to generate an isolate capable of producing hydroxylated collagen at titers of 5 mgL⁻¹ in shake flask models of fermentation. This study thus proves A. niger is a promising heterologous system to address the demand for virus-free collagen.

Background

The red alga Palmaria palmata is a rich source of sugar compounds, particularly mixed-linkage xylans present in the cell walls of the algae. In contrast to their terrestrial lignocellulosic counterparts, these xylans are more easily accessible. They can be hydrolyzed enzymatically into valuable xylooligosaccharides (XOS), known for their prebiotic, antioxidant, and immunomodulatory properties.

Results

This study introduces a simplified, one-step enzymatic process utilizing the endo-1,4-β-xylanase FO15_GH10 that directly hydrolyzes P. palmata biomass to produce XOS, eliminating the need for prior xylan extraction and improving efficiency. The exact structure of the resulting XOS was determined using NMR and MS/MS techniques. In addition, the xylosidase FO17_GH43 can be added to break down all residual 1,4-linked XOS. As a result, only 1,3- and mixed-linkage XOS (degree of polymerization (DP) 2–4) remains under simultaneous increase of the xylose obtained. Using FO15_GH10 alone, it was possible to produce approximately 17.6 (± 0.16) % (176 mg) XOS from 1 g of powdered biomass while combining both enzymes resulted in 22.6 (± 0.51) % (226 mg) XOS. Further optimization upon upscaling offers the possibility of achieving even greater improvements.

Conclusion

In summary, our one-step enzymatic approach offers an efficient and sustainable method for producing XOS directly from P. palmata biomass. This streamlined process overcomes the need for resource-consuming extraction processes. The further characterization of the obtained XOS and the potential to gain solely 1,3- and mixed-linkage XOS is paving the way for future studies on their functional properties.

3-Methyl-1-butanol (3MB) is a promising renewable solvent, drop-in fuel, and precursor for various industrial products, including flavors, fragrances, and surfactants. Due to the myriad of intertwined biosynthetic pathways that share metabolic precursors, conventional metabolic engineering strategies to overproduce 3MB in yeast have typically resulted in yields that are far too low for economic viability. However, because 3MB is naturally produced by yeast, 100 million liter of 3MB are already produced annually as a byproduct of bioethanol fermentations. Despite its significant commercial value, this 3MB fraction is currently discarded due to its low relative concentration within the fusel alcohol mixture. Here, we present a novel strategy to produce 3MB along with the conventional bioethanol fermentation, leveraging the existing bioethanol industry by valorizing the discarded fusel alcohol byproduct stream. We first identified a robust industrially relevant chassis strain and explored different strategies to alleviate the valine and leucine feedback inhibition within the 3MB pathway, showing that mutating the leucine-inhibition site of Leu4p increased 3MB yield by 2.9-fold. Finally, we tested in silico-predicted gene deletion targets to reduce the byproduct acetate. Our final engineered strain achieved a 4.4-fold increase in 3MB yield compared to the wild type (1.5 mg/g sugars), average productivity of 5 mg/Lh, and a 3MB proportion increase from 42 to 71% within the fusel alcohol mix, while ethanol production remained comparable to the Ethanol Red® industrial reference. Our study thus opens a new route for co-producing 3MB and ethanol from sugarcane molasses in Saccharomyces cerevisiae, laying the groundwork toward an economically viable and sustainable approach for 3MB production alongside existing bioethanol production.

Graphical Abstract

Background

Cyanobacteria are promising platforms for metabolic engineering to convert carbon dioxide into valuable fuels and chemicals, addressing both energy demands and global climate change. Among various fuels and chemicals, isobutanol (IB) and 3-methyl-1-butanol (3M1B) have gained increasing attention due to their superior physical properties, such as high energy density, low water solubility, and low hygroscopicity. Heterologously expressing α-ketoisovalerate decarboxylase (Kivd^S286T) in the unicellular cyanobacterium Synechocystis sp. PCC 6803 (Synechocystis) enables microbial production of IB and 3M1B through the 2-keto acid pathway, with Kivd^S286T identified as a key bottleneck limiting production efficiency.

Results

To address this limitation, a high-throughput screening method based on the consumption of the substrate 2-ketoisovalerate was successfully established. This screen was coupled with random mutagenesis, via error-prone PCR, of Kivd^S286T. Out of the 1600 variants, 1B12, featuring dual substitutions K419E and T186S, exhibited a 55% increase in IB production and a 50% increase in 3M1B production in Synechocystis on the fourth day of cultivation. The crystal structure of Kivd^S286T was determined as a tetramer with a resolution of 2.8 Å to provide a framework for analyzing the structural basis for the enhanced butanol production conferred by the K419E and T186S substitutions.

Conclusions

A novel Kivd variant, 1B12, was successfully generated via directed evolution, with enhanced catalytic activity for microbial IB and 3M1B biosynthesis. To our knowledge, this study represents the first successful application of directed evolution on the rate-limiting enzyme of a specific metabolic pathway to enhance biochemical production in cyanobacteria.