Biotechnology for Biofuels最新文献

Background

Green microalgae, such as Monoraphidium minutum 26B-AM, have garnered significant commercial interest due to their high biomass production and lipid yield, providing promising candidates for various bioprocessing applications. However, the economic viability of large-scale algal cultivation in open raceway ponds is limited by biocontamination and environmental stressors, necessitating deeper understanding of the molecular mechanisms that underpin resilience and productivity in these systems. We hypothesized that the molecular signature associated with the cellular responses of M. minutum to environmental stressors will reveal critical information for the timely prediction of resilience and productivity in algal cultures within open pond systems.

Results

To test this hypothesis, we conducted a longitudinal multi-omic study, integrating transcriptomics, proteomics, metabolomics, and phenomics, to monitor the acclimation, growth dynamics, and pathogen responses of algal cultures in two 1000 L raceway ponds, before and after the introduction of a pathogen as a stressor. We identified a number of molecular patterns that correlate with changes in the algal environment, and we can track these changes within the ponds per time. Furthermore, we identify scale-up and infection-specific molecular pathways through integrated multi-omics, showing that most patterns are unique to each studied stressor/transition.

Conclusions

Ultimately, this study demonstrates the utility of multi-omics observations at scale, revealing unique signatures and laying the groundwork for developing molecular detection techniques and predictive models that can improve the sustainability and efficiency of large-scale algae biomass production.

Gas fermentation offers a sustainable alternative for valorizing climate-active gases and industrial off-gases. Currently, these gases require energy-intensive purification steps before they can be used in chemical processes such as Fischer–Tropsch synthesis. In gas fermentation, anaerobic bacteria produce acetate from industrial off-gases. Compared to chemical processes, the anaerobic bacteria offer greater tolerance to varying gas concentrations and impurities. One major product of these anaerobic valorization processes is acetate, which can be used as a co-substrate in a variety of biological processes. This study evaluates Corynebacterium glutamicum and Ustilago maydis in benchtop cultivations using 10–20% (v/v) sterile-filtered acetate-rich supernatants from Acetobacterium woodii fermentation to produce L-lysine and triglycerides. Partial substitution of glucose with these supernatants supported robust growth and required no additional purification beyond sterile filtration. C. glutamicum achieved a L-lysine concentration of 3.5 ± 0.27 g∙L⁻¹ and exhibited a diauxic growth pattern on glucose and acetate. In U. maydis, supernatant addition shortened the lag phase by approximately 2 h but reduced triglyceride yields modestly due to higher nitrogen availability. Optimizing the nitrogen-to-carbon ratio in benchtop fermentations resulted in a triglyceride concentration of 12.75 ± 1.17 g∙L⁻¹, demonstrating the feasibility of this approach. Collectively, the results demonstrate a viable method for replacing a portion of refined glucose with acetate-rich supernatants, thereby enabling a cost-efficient integration of anaerobic gas valorization with aerobic biomanufacturing.

Graphical Abstract

This study introduces Escherichia coli MC3, demonstrating unique dual functionality in coupling lignin depolymerization with biohydrogen production from untreated food waste, thereby advancing sustainable biorefineries without energy-intensive pretreatments. MC3 demonstrated a lignin degradation rate of 50.3 ± 3.2% and an Azure B decolorization rate of 34.8 ± 2.1% over a 7-day period, as validated by UV–visible spectrophotometry. High-performance liquid chromatography identified ferulic acid (2.355 mg/L) and vanillin (1.018 mg/L) as primary byproducts of lignin degradation, thereby confirming its ligninolytic potential. Enzymatic assays revealed significant hydrolytic activity, with hydrolysis zones measuring 25.4 ± 1 mm (proteases), 28.1 ± 2 mm (cellulases), 20.5 ± 3 mm (xylanases), and 6.8 ± 1 mm (amylases). Whole-genome sequencing of the E. coli strain MC3 revealed a total of 4798 coding sequences. Among these, key genes identified include carbohydrate-active enzymes (CAZymes: AA2, AA3, CE1), hydrolytic enzymes (GH2, GH13, GH8), multicopper oxidases (CueO, CopA), and biohydrogen-related enzymes (NikA, FbpC). These findings highlight the potential of MC3 for lignin degradation and hydrogen production. In batch fermentation assays, the strain demonstrated a hydrogen yield ranging from 0.292 to 1.004 mol H2 per mol of substrate across various substrates, including xylose, glucose, carboxymethyl cellulose, starch, and food waste, with the highest yield obtained from food waste. The E. coli strain MC3 produced the highest cumulative hydrogen yield of 175 ± 10 mL H₂/g VS from food waste. This study confirms the dual function of E. coli strain MC3 for conversion of lignin into vanillin and biomass into biohydrogen, suggesting the excluding energy-intensive pretreatments, substantial potential for sustainable biorefineries.

Bioenergy with carbon capture, utilization, and storage (BECCUS) is a competent technology with the potential to address global climate change challenges. However, its deployment faces significant hurdles across technological, economic, and policy domains. The production of biofuels including ethanol, methane, butanol, and biogas is accompanied by the release of carbon dioxide (CO₂). This CO₂ can be incorporated into organic molecules through various biochemical routes as part of the metabolic mechanisms of carbon absorption. The efficiency of these carbon assimilation pathways can be improved through ongoing developments in metabolic engineering, which can increase the production of valuable bioproducts, improve carbon sequestration, and support efforts to mitigate climate change. The present review recognizes critical avenues for advancing BECCUS, emphasizing market mechanisms, technological innovations, and cross-sector integration in both developed and developing countries such as India. The review recommends policy modifications aimed at establishing a transparent framework related to carbon pricing, emission trading systems, and proper certification mechanisms for biogenic carbon utilization. These modifications, coupled with the integration of renewable energy systems, would not only stimulate BECCUS adoption, but also foster its economic feasibility and sustainability. Additionally, promising technologies such as chemical looping and microalgae-based carbon capture should be technologically scaled up to ensure industrial-level applications. The integration of BECCUS with other sectors is also critical to optimize the impact of this technology on climate change mitigation. Therefore, the present review highlights the need for a robust policy framework, technology-driven innovation, and cross-sector research collaboration to resolve the challenges associated with BECCUS, boost its adoption, and ensure its economic feasibility and environmental sustainability. Moreover, providing regulatory support, augmenting market competitiveness, and aligning research on BECCUS play a transformative role in attaining the goals of the Paris Agreement and promoting environmental sustainability.

Graphical Abstract

Isobutanol holds significant potential as a next-generation biofuel and platform chemical, offering a viable alternative to petroleum across various industries, including pharmaceuticals, and fine chemicals. Biosynthesis has emerged as a highly advantageous approach for isobutanol production, surpassing conventional chemical synthesis methods in terms of environmental sustainability, safety, and ecological compatibility. Consequently, the field of isobutanol biosynthesis has garnered increasing interest. This review provides an overview of recent breakthroughs in isobutanol biosynthesis and explores strategies aimed at improving its production yield from both biological and engineering perspectives. On the biological front, the exploration and optimization of enzymatic pathways, metabolic engineering techniques to enhance precursor availability and flux, and cellular engineering approaches to improve strain tolerance and production efficiency are meticulously analysed. Additionally, engineering aspects such as in situ product removal methods and biomass fermentation are introduced, as they significantly contribute to enhancing yields and economy. Despite the immense promise of isobutanol biosynthesis, several challenges require attention. The modification of enzymatic and metabolic pathways is crucial for increasing production efficiency and yield. The adoption of high-throughput screening techniques becomes indispensable for identifying and selecting optimal strains and enzymes with improved characteristics. Furthermore, the cytotoxic nature and volatility of isobutanol pose additional hurdles, leading to losses during the production process. By effectively addressing these challenges and advancing our understanding of isobutanol biosynthesis, we can pave the way for more efficient and sustainable production methods. This review aims to provide valuable insights and inspiration to researchers in the field, with the ultimate objective of accelerating the development and application of isobutanol biosynthesis.

Spontaneous strain degeneration, defined as the loss of an essential biological function during prolonged usage, is frequently observed in microorganisms and poses a significant challenge to the biotechnology industry. In Trichoderma reesei, a filamentous fungus widely used for large-scale cellulase production, spontaneous loss of cellulase productivity has been reported. However, studies on this phenomenon have focused solely on industrial strains derived from the Rut-C30 lineage. This study analyzes strain degeneration in a different industrial lineage of T. reesei, RL-P37, and its hypercellulase-producing descendant, GEN-3A. We found that RL-P37 and GEN-3A are also affected by the degeneration phenomenon, with the highly productive GEN-3A showing greater susceptibility. The degenerated phenotype was characterized by reduced cellulase productivity, altered growth behavior, and distinct morphological changes. In particular, cellulase hyperproduction was associated with bulbous, highly branched hyphae, while these morphological traits were lost in degenerated isolates. Our study establishes a framework for characterizing strain degeneration in T. reesei, highlights the trade-off between productivity and stability, and identifies distinctive morphological signatures linked to cellulase hyperproduction and degeneration, which may serve as early phenotypic indicators for industrial strain monitoring.

Background

Chromochloris zofingiensis is recognized as a promising resource for the production of carotenoids. However, current knowledge regarding its capacity for zeaxanthin accumulation remains limited, which restricts its potential for commercial application. The development of high-yielding strains and an understanding of the underlying metabolic and regulatory mechanisms are critical to advancing large-scale zeaxanthin production.

Results

In this study, a novel zeaxanthin-enriched mutant CZ-Z12 was obtained using ethyl methyl sulfonate (EMS). CZ-Z12 displayed a rapid growth rate when cultivated heterotrophically. On Day 6, zeaxanthin content and total fatty acid (TFA) content reached 0.229% and 28.6% of dry weight (DW), representing a 4.1 and 1.9-fold increase compared to wild-type (WT), respectively. Differentially expressed genes (DEGs) were significantly enriched in pathways of ABC transporters, endogenous hormones signal transduction, nucleotide excision repair, glycolysis, purine and pyrimidine metabolism, glutathione metabolism, fatty acid and carotenoid biosynthesis. Genes encoding growth-related proteins, such as vegetative storage protein 2 (VSP2), deoxycytidine triphosphate pyrophosphatase 1 (DCTPP1), and fructose-bisphosphate aldolase (FBA), were up-regulated by 1.4, 1.7, and 2.5-fold, respectively. The key genes encoding enzymes in zeaxanthin biosynthesis, including glutathione reductase (GR), phytoene desaturase (PDS), and β-carotene hydroxylase (CHYb), were up-regulated by 3.1, 2.4, and 2.6-fold, respectively.

Conclusion

These findings indicate that CZ-Z12 is an efficient strain for zeaxanthin production and highlight the potential for EMS mutagenesis for metabolic enhancement in heterotrophic C. zofingiensis. By modulating gene expression and key metabolic pathways, this study provides new insights and a practical strategy for improving carotenoid biosynthesis in microalgae, thereby supporting the feasible of large-scale, sustainable zeaxanthin production.

Graphical Abstract

Leveraging alternative electron acceptors to support anaerobic metabolism in industrially relevant microorganisms holds substantial biotechnological potential, especially when coupled to anodic electro-fermentation, which provides a non-depleting electron sink to the microorganisms. Bacillus subtilis is a widely used industrial workhorse for biochemical production, valued for its genetic tractability and environmental stress tolerance. However, the anaerobic, anodic metabolism of B. subtilis has been constrained by limited redox flexibility. Here, deletion of ldh (lactate dehydrogenase) to restrict fermentative NAD⁺ regeneration enabled engineered B. subtilis to survive anaerobically via anodic respiration, partially oxidising glucose while steering the metabolism toward 2,3-butanediol. The anodic metabolism showed enhanced extracellular electron transfer mediated by ferricyanide, with the highest current density of 0.77 mA/cm² reached within 2 h. Carbon flux was directed predominantly to 2,3-butanediol (0.49 ± 0.07 mol_product/mol_glucose) under incomplete glucose oxidation and without pH control. In addition, pH control further improved anodic electro-fermentation performance. At pH 6.5, 66% of the added glucose was consumed, and 2,3-butanediol carbon selectivity rose to 77.1 ± 0.6%, whereas at pH 7.5, cells consumed 89% of the glucose with 73.4 ± 0.7% 2,3-butanediol carbon selectivity. To our knowledge, this is the first investigation of anodic electro-fermentation in B. subtilis that integrates metabolic engineering with pH optimisation strategies, demonstrating a new route to produce high-purity biochemicals from renewable resources.

Background

2,5-Furandicarboxylic acid (FDCA) is a bio-derived compound which serves as a building block of polyethylene furandicarboxylate (PEF) for the synthesis of bio-plastics. 5-Hydroxymethylfurfural oxidase (HMFO) is a promising biocatalyst for the oxidation of 5-hydroxymethylfurfural (HMF) to FDCA through three oxidation steps. The substrate could be fructose and other C6 sugars present in lignocellulosic biomass, providing an eco-friendly and sustainable route for the production of FDCA, an essential monomer for biopolymer development.

Result

The newly identified HMFO enzyme from Pseudomonas poae (PSPO) was heterologously expressed in E. coli, and the recombinant protein was purified and characterized. The catalytic function of PSPO was authenticated for the oxidation of HMF to FDCA. PSPO oxidized HMF, resulting in a 99% yield of FDCA within 24 h. The secondary structure composition of PSPO was analyzed. The optimal enzymatic activity of the recombinant protein was observed at pH 8.0 and temperature of 30℃. PSPO enzyme stability against H₂O₂ was evaluated, and the enzyme was observed to retain ˃90% of residual activity at lower concentrations of H₂O₂ (2 to 6 mM). The kinetic parameters of PSPO for the substrates HMF, FFCA, DFF, and vanillyl alcohol were also determined. The K_m values for HMF, FFCA, DFF, and vanillyl alcohol were 2.252 mM, 5.551 mM, 2.503 mM, and 1.589 mM, respectively.

Conclusion

The identified and characterized PSPO was found to have a potential for the accelerated enzymatic oxidation of HMF to FDCA. Through molecular dynamic (MD) simulations of the catalytic pathway and enzyme mechanisms, alongside assessing the impact of substrate concentration, PSPO turns out to be a potential candidate for enhancing reaction efficiency and maximizing yield.

Background

Mandelic acid (MA) is a high-value chiral platform molecule with broad applications in pharmaceutical synthesis, cosmetic formulations, and polymer production. Conventional chemical synthesis is limited by harsh reaction conditions, poor enantioselectivity, and environmental concerns. Microbial biosynthesis offers a sustainable and stereoselective alternative; however, its industrial application is constrained by low titres, suboptimal productivity, and inefficient downstream recovery. This study reports an engineered microbial chassis that enables enhanced biosynthesis of MA enantiomers with integrated downstream compatibility.

Results

The biosynthetic potential of Escherichia coli was harnessed through targeted metabolic engineering and pathway optimisation for the biosynthesis of (R)- and (S)-MA. Batch fermentations in rich medium produced 1.6 g/L (R)-MA and 1.8 g/L (S)-MA. Transitioning to fed-batch cultivation in defined minimal medium, under non-optimised conditions, increased titres to 2.9 g/L; ee = 99% for (R)-MA and 5.7 g/L; ee = 93% for (S)-MA, representing the highest reported in vivo titres of MA enantiomers achieved in E. coli to date. A two-step downstream process comprising solvent extraction and crystallisation enabled the recovery of MA at high purity (> 99.0%), with recovery efficiencies of 84% for (S)-MA and 77% for (R)-MA. To validate the functional utility of bio-based MA, SAMMA, a sulfuric acid condensation polymer with documented antiviral and contraceptive properties, was synthesised from both bio-based and commercial MA. Additionally, mandelide, a monomer precursor for the biodegradable polystyrene analogue polymandelide (PM), was synthesised to illustrate the platform’s relevance to sustainable polymer applications.

Conclusions

This study establishes a robust proof of concept for a microbial platform enabling enantioselective MA biosynthesis from renewable carbon sources. Through the integration of metabolic engineering, downstream process development and application-driven validation, this platform lays the foundation for a scalable and industrially relevant bioproduction strategy. Aligned with the principles of green chemistry and the circular bioeconomy, this approach offers a sustainable and environmentally responsible route to high-value chiral chemicals.