Biotechnology for Biofuels最新文献

Background

Anaerobic digestion (AD) or acidogenic fermentation (AF) of biomass can generate either biogas fuel or C₂ ‒ C₈ volatile fatty acids (VFAs) as feedstocks for synthesis of other petrochemical products. Typical AD feedstocks require large amounts of land that could otherwise be used for food production. Unlike these traditional bioenergy crops, plants using the crassulacean acid metabolism pathway (CAM), such as cacti and succulents, may be cultivated on degraded or semi-arid land that cannot support conventional agriculture. This could allow significant biorefinery feedstock to be sourced with minimal impact on existing agriculture or biodiversity. Several economically important CAM crops (e.g. pineapple, agave, prickly pear) are cultivated globally, with waste biomass that could be valorised as a biorefinery feedstock.

Results

Here, we investigate the fermentation kinetics of this novel feedstock class (CAM plants) against traditional bioenergy crops with two contrasting inocula: AD sludge and rumen fluid. Fermentations were performed under the influence of a methanogenesis inhibitor (bromoethane sulfonate) to isolate the acidogenic fermentation processes. CAM and non-CAM substrates in this study demonstrated distinct degradation kinetics (yields and degradation rates). We demonstrate that regardless of the inoculum type, CAM crops show higher hydrolysis rates for VFA production. Moreover, yields of VFAs from three CAM crops (0.41 ± 0.01 – 0.48 ± 0.02 g/g_vs) were higher than for the three non-CAM crops (0.21 ± 0.01 – 0.38 ± 0.01 g/g_vs) when AD sludge was used as the inoculum. This superior performance appeared to correlate with a higher abundance of soluble material and lower structural carbohydrate content in CAM biomass.

Conclusions

At industrial scale, the observed kinetic advantages of VFA production from CAM-plant feedstocks could translate into process enhancements that would greatly improve the cost-competitiveness of anaerobic biorefinery. Assuming comparable biomass productivities of CAM and non-CAM crops, this high yield could allow higher VFA production per unit of cultivated land, improving the environmental credentials of CAM biorefinery.

Graphical abstract

Unspecific peroxygenases (UPOs) are versatile enzymes capable of oxidizing a broad range of substrates, using hydrogen peroxide as the sole co-substrate. In this study, UPOs were evaluated for their potential in the selective oxyfunctionalization of the phenolic lignin monomer 4-propylguaiacol (4-PG) to generate versatile scaffolds for the synthesis of high-value compounds. In addition to the desired peroxygenase reaction, the phenolic group of 4-PG is susceptible to undesirable one-electron oxidation (peroxidase activity). Assessment of the activity of 19 UPOs from phylogenetically diverse clades toward 4-PG revealed that several UPOs could serve as potential biocatalysts for the functionalization of 4-PG, with some enzymes showing both promising conversion yields (>50%) and regioselectivity for the peroxygenase reaction. Pronounced differences in peroxygenase:peroxidase activity ratios and regioselectivity were observed. Comparative analysis—supported by experimental activity profiles and structural data—suggest that a more constrained active-site topology contributes to the peroxygenase activity. UPOs from a clade within the Ascomycota phylum with high peroxygenase activity possess a unique aliphatic pocket in their catalytic centers. Our study provides valuable insights into the structure–function relationships underpinning enhanced peroxygenase activity of UPOs and provides a functional mapping of a broad UPO-sequence space for 4-PG, highlighting these enzymes as promising catalysts for the selective oxyfunctionalization of a phenolic lignin monomer.

Background

A deployable, continuous enzymatic hydrolysis (CEH) process can address cost and commercialization risks associated with second-generation (Gen2) biorefinery sugar/lignin/ethanol production while contributing to energy supply and security. Developments in commercial enzymatic hydrolysis formulations targeting Gen2 pretreated biomass such as deacetylated mechanically refined (DMR) biomass necessitate a reassessment of the existing hybrid simultaneous saccharification and fermentation (SSF) approach. Notably, the practice of "finishing hydrolysis" in SSF has become problematic with the introduction of oxidative enzymes, such as lytic polysaccharide monooxygenases (LPMOs), into commercial cellulase formulations as these require specific redox conditions and cofactor. Moreover, continuous SSF has not been demonstrated at commercial scale, limiting deployment and the associated economic benefits to farmers, producers, and support industries.

Results

Continuous enzymatic hydrolysis (CEH) was demonstrated at bench scale to enable optimal saccharification performance of deacetylated mechanically refined (DMR) pretreated biomass. Diafiltration was demonstrated to retain pretreated biomass solids and enzymes for continuous reaction while removing solubilized product sugars in situ. A significant breakthrough afforded by the CEH process is its ability to achieve equivalent endpoint conversions with approximately 50% lower enzyme loading. Yields of glucose and xylose were increased ~ 15% and ~ 4%, respectively, over batch hydrolysis. Unlike SSF using yeast or Zymomonas, CEH allows precise optimization of pH, temperature, oxygen tension, LPMO mediator concentration, and removal of end-product inhibitors.

Conclusions

Advanced CEH holds promise as a transformational, process-intensified, and cost-effective method for producing soluble clarified biomass sugars and insoluble lignin-rich streams. Enhancing saccharification performance, optimizing operating parameters, and employing membrane filtration will help overcome existing challenges and enable the efficient production of valuable biomaterials from lignocellulosic biomass.

Plant specialised metabolism generates a vast array of compounds with significant potential across agriculture, medicine, cosmetics, and the food industry. A key challenge lies in optimising their production in the plant, as these compounds are often present in trace amounts in a complex metabolic cocktail. Given their high economic value, extensive efforts have been made to elucidate their biosynthetic pathways and pinpoint key regulatory and enzymatic targets. This knowledge has been applied for metabolic engineering to enhance the carbon flux towards metabolites of interest, thereby broadening the utility of plants as a source of high-value compounds. This review examines different metabolic engineering strategies employed today using the phenylpropanoid pathway as a case study and highlights the potential of integrating plant and microbial research to drive cross-disciplinary innovation.

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.