Biotechnology for Biofuels最新文献

Background

Oleaginous yeast are prodigious producers of oleochemicals, offering alternative and secure sources for applications in foodstuff, skincare, biofuels, and bioplastics. Nitrogen starvation is the primary strategy used to induce oil accumulation in oleaginous yeast as part of a global stress response. While research has demonstrated that post-translational modifications (PTMs), including phosphorylation and protein cysteine thiol oxidation (redox PTMs), are involved in signaling pathways that regulate stress responses in metazoa and algae, their role in oleaginous yeast remain understudied and unexplored.

Results

Towards linking the yeast oleaginous phenotype to protein function, we integrated lipidomics, redox proteomics, and phosphoproteomics to investigate Rhodotorula toruloides under nitrogen-rich and starved conditions over time. Our lipidomics results unearthed interactions involving sphingolipids and cardiolipins with ER stress and mitophagy. Our redox and phosphoproteomics data highlighted the roles of the AMPK, TOR, and calcium signaling pathways in regulation of lipogenesis, autophagy, and oxidative stress response. As a first, we also demonstrated that lipogenic enzymes including fatty acid synthase are modified as a consequence of shifts in cellular redox states due to nutrient availability.

Conclusions

We conclude that lipid accumulation is largely a consequence of carbon rerouting and autophagy governed by changes to PTMs, and not increases in the abundance of enzymes involved in central carbon metabolism and fatty acid biosynthesis. Our systems-level approach sets the stage for acquiring multidimensional data sets for protein structural modeling and predicting the functional relevance of PTMs using Artificial Intelligence/Machine Learning (AI/ML). Coupled to those bioinformatics approaches, the putative PTM switches that we delineate will enable advanced metabolic engineering strategies to decouple lipid accumulation from nitrogen limitation.

Microbial fuel cells (MFCs) have emerged as a promising technology to convert biomass and organic waste into electricity, offering an eco-friendly and sustainable alternative to fossil fuels. Recent innovations in nanotechnology have significantly enhanced the performance and efficiency of MFCs by improving electron transfer rates, expanding surface areas, and optimizing the properties of anode and cathode materials. This review provides a detailed assessment of the fundamental and functional components of MFCs. These components include the anode, which facilitates the oxidation of organic matter, and the cathode, where the reduction of oxygen or other electron acceptors occurs. Another critical component is the proton exchange membrane (PEM), which allows the transfer of protons from the anode to the cathode while preventing oxygen from diffusing into the anode chamber. In addition to discussing these key elements, the article explores the role of various microorganisms involved in MFCs. These microorganisms, which include both naturally occurring species and genetically engineered strains, play a vital role in facilitating extracellular electron transfer (EET), a process that enables the conversion of chemical energy stored in organic compounds into electrical energy. We analyze different biomass pretreatment strategies, such as physical, chemical, and biological approaches, that enhance the breakdown of lignocellulosic biomass to improve energy output. Furthermore, the review highlights optimization techniques for improving biomass-powered MFC performance, such as electrode modification, pH control, and organic loading rate management. The application potential of MFCs is extensively discussed, covering bioremediation, wastewater treatment, biosensors, and power generation, with a particular focus on MFC-based biosensors for environmental monitoring and medical diagnostics. Despite their immense potential, challenges such as low power output, biofouling, and high operational costs hinder large-scale commercialization. To address these issues, we propose innovative strategies, including the integration of nanomaterials, electroactive microorganisms, and advanced membrane designs, to enhance the efficiency and reliability of MFCs. We conclude that nanotechnology-enabled MFCs, combined with engineered microbes and optimized system designs, hold immense potential for revolutionizing sustainable energy generation and biosensing applications, paving the way for a cleaner and more efficient future.

Background

Waste valorisation refers to processes of reusing or recycling waste materials to create valuable products. In the Rum distillery industry, the primary waste byproducts include bagasse, a solid waste made up of sugar cane residue and vinasse, a thick and acidic liquid. Although vinasse has been repurposed in agricultural fields, it has also contributed to both soil and ocean pollution. Despite several potential solutions having been suggested, an effective and environmentally safe use for vinasse has yet to be found.

Results

The valorisation of vinasse for biofuel production was explored by assessing its potential as a growth medium for lipid production by non-conventional yeasts. The oleaginous yeast strain Yarrowia lipolytica, known for its lipid production capabilities, was initially tested on vinasse but required further adaptation and optimization. To circumvent this, we isolated a novel yeast strain from old vinasse waste, named V1, which demonstrated strong growth potential. The growth conditions of V1, including temperature and acidity, were characterized, and its potential for bioengineering was evaluated. This strain exhibited resistance to highly acidic pH levels and higher temperatures when cultivated on YPV, an artificial laboratory medium designed to mimic the acidity and glycerol content of vinasse. Whole genome sequencing (WGS) identified V1 as Pichia kudriavzevii. We demonstrated that V1 could be transformed with Yarrowia lipolytica vectors using the classical yeast heat shock protocol, thus enabling potential genetic engineering. Finally, lipid content in V1 was analysed in different conditions, confirming the strain's potential for biofuel production.

Conclusions

Pichia kudriavzevii is not a traditional yeast, but its ability to adapt and grow under extreme pH and higher temperature conditions makes it a promising candidate for rum industry waste management applications. This strain could potentially be utilised to convert vinasse and other food waste products into valuable biofuels. Although further research is required to engineer and optimize this novel strain for vinasse cultivation, our findings highlight its great potential as a micro-factory in rum-producing regions and high locations, where agricultural waste is in need of valorisation solutions.

Background

Zeaxanthin is a yellow xanthophyll naturally found in plants and algae, where it plays a crucial role in light absorption and photoprotection. In mammals, ingestion of zeaxanthin through the diet is essential as it accumulates in the retina where it absorbs excessive blue light to protect photoreceptors from photooxidative stress. Chlamydomonas reinhardtii is an established model organism for pigment biosynthesis and bioengineering. Previous studies developed double knockout mutants (dzl) using CRISPR-Cas9 to eliminate ZEP and LCYE genes, achieving zeaxanthin production up to 6.84 mg/L with medium optimization. However, these approaches have not explored additional enzyme overexpression strategies combined with advanced cultivation techniques, leaving significant potential for enhanced zeaxanthin biosynthesis unexplored.

Results

In this study, we strategically enhanced zeaxanthin biosynthesis in C. reinhardtii by genome editing to knockout competing pathways coupled with overexpression of rate limiting enzymes and optimization of cultivation for efficient biomass accumulation. We employed the knockout of lycopene epsilon cyclase (LCYE; dL mutant), which resulted in a 2.83-fold increase in zeaxanthin levels. Additionally, knocking out zeaxanthin epoxidase (ZEP, dLZ mutant) redirected metabolic flux towards zeaxanthin biosynthesis, further enhancing its accumulation by 14.07-fold. Overexpression of β-carotene hydroxylase (CHYB, dLZ_C strains) enabled efficient hydroxylation of β-carotene and increasing zeaxanthin concentration further by1.80-fold without compromising growth. In addition, elevated acetate concentrations supported mixotrophic growth and resulted in a zeaxanthin yield of 21.68 ± 0.90 mg/L, threefold higher compared to previously reported values and a culminated 190-fold increase compared to the parental strain (UVM4) grown in standard medium.

Conclusion

Our study developed a zeaxanthin-producing mutant strain by integrating gene modification, gene overexpression, and culture optimization. Among various green microalgae, the engineered strain dLZ_C demonstrated notable zeaxanthin productivity, reaching 6.70 mg/L/day over a period of 3 days, suggesting its potential as a candidate for industrial production. Its improved efficiency may offer advantages for large-scale applications in microalgal-based zeaxanthin production. Additionally, these findings indicate that Chlamydomonas reinhardtii could serve as a viable and sustainable platform for biotechnological applications in the health, nutrition, and biotechnology sectors.

Background

Palmitoleic acid, a valuable functional fatty acid, is notably scarce in traditional oil crops, with the exception of certain wild plants such as macadamia nuts and sea buckthorn. Recently, the lipid from Saccharomyces cerevisiae was found to contain approximately 50% palmitoleic acid. Consequently, S. cerevisiae has the potential to sustainably produce palmitoleic acid through fermentation, provided that the issue of promoting its lipid content is addressed.

Results

In this work, based on the previously isolated oleaginous wild strain of S. cerevisiae, the mutagenesis by zeocin combined with ARTP was carried out to generate S. cerevisiae mutants, and then the high lipid content mutants were isolated using the flow-mode Raman-activated cell sorting (FlowRACS) technique, which allowed for the high-throughput selection of these mutants in a label-free and non-invasive manner. The mutant MU2R48 was finally obtained and its lipid content was 40.26%, 30.85% higher than the original type. Transcriptome and targeted metabolome analysis revealed a coordinated interaction of fatty acid precursor biosynthesis, the pentose phosphate pathway, ethanol degradation, and amino acid metabolism, synergistically channeling carbon flux from acetyl-CoA and NADPH into lipid biosynthesis. Additionally, key transcriptional regulators within the lipid metabolism network were implicated in this enhanced lipid accumulation.

Conclusion

In this study, a mutant strain of Saccharomyces cerevisiae MU2R48 with 40.26% lipid content was successfully generated through zeocin-ARTP mutagenesis combined with Raman-activated cell sorting. Multi-omics analysis revealed that the enhanced lipid accumulation was driven by coordinated up-regulation of precursor biosynthesis, carbon flux redirection, and key transcriptional regulators, with increased acetyl-CoA and NADPH production fluxes likely serving as the pivotal determinants.

Food loss and waste (FLW) generated by unsustainable linear food systems are major contributors to greenhouse gas (GHG) emissions. Although microalgal lipid production has advanced significantly for applications such as biofuels and high-value polyunsaturated fatty acids (PUFAs), the use of FLW as an alternative feedstock to cultivate lipid-rich microalgal biomass within a circular bioeconomy remains insufficiently explored. This review critically evaluates the feasibility of converting FLW into nutrient-rich media for microalgae cultivation, with particular focus on its effects on biomass productivity and lipid accumulation. Pre-treatment methods for food waste are essential to enhance nutrient recovery, especially of carbon sources, and significantly influence subsequent microalgae cultivation. These methods affect the bioavailability of key nutrients, particularly the carbon-to-nitrogen-to-phosphorus (C/N/P) ratio, which regulates metabolic pathways involved in lipid biosynthesis. Despite encouraging laboratory-scale outcomes, large-scale implementation remains constrained by feedstock heterogeneity, high energy demands during harvesting and lipid extraction, and regulatory challenges. To overcome these barriers and facilitate scale-up, this review highlights integrative strategies such as metabolic engineering, automated cultivation systems, and a closed-loop microalgae-based biorefinery. Moreover, life cycle assessment (LCA) is emphasized as a tool to assess environmental performance and inform policy decisions, supporting alignment with Sustainable Development Goals (SDG 12 and SDG 13).

Graphical Abstract

Background

As outlined by the Intergovernmental Panel on Climate Change, we need to approach global net zero CO₂ emissions by approximately 2050 to prevent warming beyond 1.5 °C and the associated environmental tipping points. Future microbial electrosynthesis (MES) systems could decrease net CO₂ emissions by capturing it from industrial sources. MES is a process where electroactive microorganisms convert the carbon from CO₂ and reduction power from a cathode into reduced organic compounds. However, no MES system has attained an efficiency compatible with a financially feasible scale-up. To improve MES efficiency, we need to consider the energetic constraints of extracellular electron uptake (EEU) from an electrode to cytoplasmic electron carriers like NAD⁺. In many microbes, EEU to the cytoplasm must pass through the respiratory quinone pool (Q-pool). However, electron transfer from the Q-pool to cytoplasmic NAD⁺ is thermodynamically unfavorable. Here, we model the thermodynamic barrier for Q-pool dependent EEU using the well-characterized bidirectional electron transfer pathway of Shewanella oneidensis, which has NADH dehydrogenases that are energetically coupled to proton-motive force (PMF), sodium-motive force (SMF), or uncoupled. We also tested our hypothesis that Q-pool dependent EEU to NAD⁺ is ion-motive force (IMF)-limited in S. oneidensis expressing butanediol dehydrogenase (Bdh), a heterologous NADH-dependent enzyme. We assessed membrane potential changes in S. oneidensis + Bdh on a cathode at the single-cell level pre to post injection with acetoin, the substrate of Bdh.

Results

We modeled the Gibbs free energy change for electron transfer from respiratory quinones to NADH under conditions reflecting changes in membrane potential, pH, reactant to product ratio, and energetically coupled IMF. Of the 40 conditions modeled for each method of energetic coupling (PMF, SMF, and uncoupled), none were thermodynamically favorable without PMF or SMF. We also found that membrane potential decreased upon initiation of EEU to NAD⁺ for S. oneidensis on a cathode.

Conclusions

Our results suggest that Q-pool-dependent EEU is both IMF-dependent and is IMF-limited in a proof-of-concept system. Because microbes that rely on Q-pool-dependent EEU are among the most genetically tractable and metabolically flexible options for MES systems, it is important that we account for this thermodynamic bottleneck in future MES platform designs.

Advancing biomethane production from anaerobic digestion (AD) is essential for building a more reliable and resilient bioenergy system. However, incomplete conversion of lignocellulose-rich agricultural waste remains a key limitation, often leaving energy-dense residues in the digestate by-product. In this study, we introduce a novel application of chelator-mediated Fenton (CMF) post-treatment to recover untapped biomethane potential from these recalcitrant residues, representing a significant departure from conventional pre-treatment strategies. By systematically varying pH, iron-chelator concentration, and hydrogen peroxide dosage, we identified reaction conditions (pH 6–8, 5 mM Fe²⁺-dihydroxybenzene, 3–4 wt.% H₂O₂) that enhanced lignocellulose deconstruction and increased dissolved organic carbon (DOC) availability for methanogenesis. CMF post-treatment led to up to a tenfold increase in biomethane potential compared to untreated controls. Microbial community analysis revealed enrichment of cellulolytic species, suggesting enhanced hydrolytic activity as a driver of improved conversion. Application of the CMF post-treatment method to isolated poplar lignin further demonstrated its versatility for diverse lignocellulosic substrates. These findings position CMF post-treatment as a promising strategy to enhance AD efficiency and valorize digestate.

Graphical Abstract

Rhodotorula glutinis is an important oleaginous yeast that can synthesize various valuable compounds, including carotenoids, lipids, and exopolysaccharides. The effect of combined heat stress and glucose starvation on carotenoid biosynthesis in R. glutinis was investigated in this study. Carotenoid production in R. glutinis was promoted by heat stress, and this effect was further enhanced when glucose starvation was applied to the strain. The results of multiomics analysis revealed that the effects of heat stress and glucose starvation on promoting carotenoid biosynthesis appeared to be additive, with the combined stress leading to a further increase in reactive oxygen species (ROS) levels and a reduction in enzymatic antioxidant capacity, while carotenoid biosynthesis was prioritized simultaneously. The key responses of R. glutinis to combined stress include the regulation of the cell cycle and energy metabolism, maintenance of membrane integrity, an increase in ROS scavenging capacity, and non-enzymatic antioxidant activity. Additionally, several candidate genes and metabolites associated with the combined stress response were identified. To summarize, we provided new insights into optimizing fermentation processes for increased carotenoid production in Rhodotorula glutinis and established a molecular basis for further genetic engineering to increase carotenoid yield.