The transition toward a sustainable bioeconomy requires efficient strategies for converting renewable biomass and industrial side streams into value-added chemicals and fuels. Fusel oil is an important coproduct of bioethanol fermentation and levulinic acid (LA), a key platform molecule derived from lignocellulosic biomass, can be upgraded into alkyl levulinates, which are promising green solvents, specialty chemicals, and fuel-related applications. In this study, a solvent-free enzymatic approach was developed for the synthesis of alkyl levulinates via lipase-catalyzed esterification of levulinic acid with bioethanol-derived fusel oil and its major alcohol components, including 2-methyl-1-butanol and 3-methyl-1-butanol, using immobilized Candida antarctica lipase B (Novozym® 435). Under optimized conditions (50 °C, LA:alcohol molar ratio 1:10, enzyme loading 10 wt
Formate is a promising, sustainable feedstock for microbial conversion into value-added products via the Wood–Ljungdahl pathway. Based on the identification of three distinct formate dehydrogenases in Clostridium sp. AWRP, this study investigated the potential of this acetogen for formate-based growth. Although the wild-type strain exhibited an extended lag phase due to formate toxicity, adaptive laboratory evolution yielded the adapted strain F30T, which demonstrated a significantly enhanced growth rate and tolerance to formate concentrations up to 300 mM. Whole-genome and transcriptomic analyses of the F30T strain revealed a sophisticated, multi-layered strategy for mitigating organic acid stress and optimizing energy conservation. Adaptive laboratory evolution resulted in six key non-synonymous mutations, including those in ackA and adhE1, and triggered the differential expression of 948 genes out of 4199 genes under the formate-supplemented condition. Specifically, F30T achieved intracellular pH buffering and supplemental ATP generation by upregulating the arginine deiminase pathway, histidine biosynthesis, and ethanolamine utilization. To maintain growth near the thermodynamic limit, the strain implemented a stringent energy-saving program by downregulating the F₀F₁-type ATP synthase and the methyl-branch of the Wood–Ljungdahl pathway, while simultaneously enhancing stress resilience through molecular chaperone upregulation. In a pH-stat fed-batch system using formic acid, the adapted F30T strain showed a higher biomass yield while achieving a similar acetate yield from formate as the wild-type strain. This study demonstrated that adaptive laboratory evolution is a highly effective strategy for enhancing the formate utilization and stress tolerance of Clostridium sp. AWRP. Through integrated genomic and transcriptomic analyses, the molecular basis of the F30T strain’s improved performance was elucidated, identifying key mutations and coordinated metabolic and regulatory mechanisms that maintain cellular homeostasis and maximize energy efficiency. These findings provide valuable molecular insights to aid the targeted design and optimization of formate-utilizing acetogens for use in microbial cell factories aimed at sustainable C1-based bioproduction.
Cellulase enzyme secretion by cells of 2G S. cerevisiae yeast is considered a promising strategy to reduce the prohibitive cost of commercial cellulase cocktails in lignocellulosic bioethanol production. Here, we have developed Cellusec® strains by engineering enzyme secretion capacity into an industrial lignocellulosic bioethanol strain and demonstrated its potential in simultaneous saccharification and fermentation (SSF) experiments of real-world feedstocks. A single copy each of CBHI, CBHII and EG encoding genes, and three copies of a BGL encoding gene were inserted into the strain, followed by overexpression of HAC1i. Importantly, all intermediate strains were evaluated throughout the engineering process, allowing identification of performance trade-offs associated with successive genetic modifications. We show that cell-free supernatant from Cellusec® strains reduced exogenous cellulase cocktail requirements by up to 53
Industrial wastewater contains a variety of persistent pollutants that are difficult to treat and pose significant risks to ecosystems. One such waste stream, hydrothermal liquefaction (HTL) aqueous phase (AP), is a nutrient-rich yet toxic byproduct generated during the conversion of biomass to biocrude oil. APs are chemically heterogeneous, posing substantial challenges for detoxification, nutrient reclamation, and commercial application of HTL technology. Fungi exhibit a wide breadth of metabolic capabilities that respond dynamically to their environment, making them promising candidates for degrading or transforming recalcitrant compounds from waste streams. Understanding the genetic basis of fungal tolerance in such environments is critical to fully realize their bioremediation capacity. This work leverages transcriptomic analyses to identify gene families broadly associated with fungal stress tolerance in Trametes versicolor challenged by three distinct APs. Upregulated genes encoding NAD(P)-binding proteins, alpha/beta-hydrolases, aldo/keto reductases, and cytochrome P450s were identified across all conditions, along with concomitant changes in biochemical composition of the APs. These results build greater understanding of fungal responses to HTL-AP exposure and identify gene candidates for future functional validation, a crucial first step toward the development of stress-tolerant strains capable of reclaiming nutrients from diverse and hostile wastewater environments.
The valorization of hempseed oil pomace (HP) using two techniques, solid-state fermentation (SSF) and hydrothermal carbonization (HTC), to produce value-added products was investigated. HP was first pretreated with Thermomyces lanuginosus under SSF for 10 days to produce two enzymes (lipase and xylanase) and fermented HP (a nutritionally enriched, mycelium-based product). The structural and chemical properties of the biomass were characterized, and lipase and xylanase activities were monitored daily throughout the fermentation process. Samples collected after 4, 7, and 10 days of fermentation were further subjected to HTC to produce hydrochar (HC) and a liquid fraction (LF), and to study the effect of SSF pretreatment on the properties of HTC products. Structural analysis of fermented HP using NMR, SEM–EDX and FTIR indicated substantial structural modification and partial degradation of the lignocellulosic matrix following SSF pretreatment. High activities of lipase (up to 0.29 U/mL) and xylanase (up to 79.34 U/mL) were obtained during fermentation, while fermented HP exhibited improved nutritional properties, particularly being enriched with free amino acids, among which glutamic and aspartic acids were the most abundant, each exceeding 5000 µg/g. HC from fermented HP exhibited great energetic and fuel properties (calorific values between 28.9 and 29.7 MJ/kg), comparable to HC from non-fermented HP. SSF treatment affected energy yield, volatile matter, and carbon and ash contents, resulting in increased fixed carbon content and an improved fuel ratio. LF obtained from HTC of fermented HP showed reduced toxicity and increased organic acids, total nitrogen and potassium content. Both HC and LF showed potential for fertilizer-oriented applications, although LF would require appropriate dilution or post-treatment before practical use. To the best of the authors’ knowledge, the application of T. lanuginosus for the integrated production of lipase and xylanase, together with the subsequent generation of hydrochar and biofertilizer from hempseed oil pomace or other oilseed pomaces, has not previously been reported.
Green hydrogen (H2) is a promising alternative to carbon-based fuels. However, delivering economically viable large-scale renewable H2 production remains challenging. Green microalgae, such as Chlamydomonas reinhardtii, offer a sustainable route to H2 production by coupling water oxidation to the photosynthetic electron transport chain. As a result, considerable effort is being devoted to improving microalgal H2 yields through engineering of metabolic and photosynthetic pathways. Although microalgal H2 production has been studied for over two decades, progress towards application remains constrained by the lack of rapid, reproducible and quantitative phenotypic screening tools. Conventional photobioreactor-based assays are low-throughput, labour-intensive and poorly reproducible, limiting comparative assessment across strains and conditions. We developed a cost-effective, plate-based, semi-quantitative, high-throughput assay for screening microalgal H2 production based on an H2-responsive Rhodobacter capsulatus biosensor that reports H2 via GFP fluorescence. The platform enables simultaneous assessment of H2-producing microalgae across multiple strains and conditions in 96-well plates, with a total workflow time of 6 h from setup to data acquisition. Benchmarking the microplate assay against six well-characterised Chlamydomonas reinhardtii strains demonstrated strong concordance with photobioreactor data, while exhibiting good technical precision and biological reproducibility. Chemical-inhibitor treatments revealed pathway-specific contributions to H2 evolution and highlighted the constraining role of PSII-derived O2 under illuminated conditions. Importantly, the assay supports assessment of H2 production under photoautotrophic conditions, which remain challenging to establish in standard photobioreactor workflows. This assay directly addresses the limited throughput and poor reproducibility that currently constrain phenotypic screening in microalgal H2 research. While not a substitute for larger-scale, long-term photobioreactor-based quantitative studies, it provides a rapid, robust and small-scale upstream screening tool to prioritise strains, mutants, and experimental conditions for downstream analysis, thereby accelerating early-stage phenotypic evaluation in microalgal H2-production workflows. In this role, adoption of this assay provides a basis for standardising H2-phenotyping workflows and supporting the development of next-generation microalgal H2 production processes.
The catalytic transformation of biomass to high value sustainable platform chemicals presents an auspicious avenue for developing a renewable bio-economy. The conversion of C5 and C6 from xylose and glucose involves two-step processes of tandem hydrogenative hydrogenolysis reaction. Recently, covalent organic frameworks (COFs) and COF-based hybrid architectonic have gathered considerable research interest for the direct synthesis of fine chemicals from biomass via dihydroxylation owing to their functionalized active sites and well-ordered porous structures. The present review includes the fundamentals of catalytic hydrogenolysis of 5-hydroxymethylfurfural (5‑HMF) to 2,5-dimethylfuran (2,5-DMF) and furfural production. It includes the synthesis, characterization and properties of COFs-based architectonics for catalytic hydrogenolysis. Numerous studies are compared on the basis of their conversion efficiency, catalytic performance, recyclability, and reusability for the potential conversion of 5‑HMF to 2,5-DMF. The mechanistic insights, relationship between the structural features, and reaction pathways are elaborated in detail. Finally, the limitations and future perspectives are covered that will be helpful for “plug-and-play” during the cascade processes including the upgradation of downstream process in combination with continuous-flow reactors, linking with in-situ spectroscopy with DFT and machine learning, etc. for sustainable platform chemical production.
Tobacco biomass constitutes a substantial yet underexploited lignocellulosic resource with considerable potential for bioconversion. However, its efficient enzymatic utilization is constrained by pronounced structural heterogeneity and the presence of diverse non-structural inhibitory components. This review provides a comprehensive overview of tobacco lignocellulose, emphasizing how tissue-specific variations in cellulose, hemicellulose, lignin, and other cell wall components influence enzyme accessibility and catalytic efficiency. Particular attention is given to tobacco-derived inhibitory compounds, including alkaloids, polyphenols, and pigments, which impair enzymatic hydrolysis through multiple mechanisms. Unlike previous reviews on lignocellulose hydrolysis, this review further summarizes recent advances in inhibitor transformation, enzyme engineering to enhance inhibitor tolerance and degradation activity, and enzymatic system optimization. Looking forward, the integration of advanced metagenomic screening and artificial intelligence-driven design is expected to accelerate the development of more robust lignocellulolytic enzymes with enhanced resistance to inhibitory compounds. Collectively, these insights provide a mechanistic basis for efficient and sustainable utilization of tobacco biomass.
Lipase-catalyzed biodiesel preparation in non-aqueous media has the advantages of reduced energy consumption, simplified recovery of fatty acid methyl esters (FAMEs), and minimized environment pollution, etc. To discover a novel thermophilic lipase, achieve its high-level expression, and develop it as an efficient liquid biocatalyst for FAMEs production via methanolysis of plant oils in non-aqueous media, a lipase gene from the thermophilic fungus Rasamsonia emersonii was cloned and efficiently expressed through synergistic strategies including computational codon optimization and increased gene dosage in the host genome. The resulting liquid lipase preparation was directly employed as a biocatalyst for FAMEs production. Under optimized reaction conditions, a conversion rate of approximately 95
The conversion of renewable biological resources into value-added products is essential for supporting the transition to a sustainable bioeconomy. This study explores the use of waste apples—a by-product generated in large quantities along the fresh apple supply chain—as feedstock for the production of two industrially relevant platform chemicals: lactic acid (LA) and succinic acid (SA). A screening identified Heyndrickxia coagulans A203 and Actinobacillus succinogenes DSM 22257 as the most suitable strains for LA and SA production from apple mash, the sugar-rich fraction obtained by pressing waste apples (excluding the solid peel and seed residues). A simplified procedure, omitting costly centrifugation and sterilization steps between enzymatic liquefaction and fermentation, was successfully scaled up, resulting in 73.8 g L−1 LA with a yield of 0.91 g g−1 and 36.8 g L−1 SA with a yield of 0.69 g g−1. The results demonstrate the potential of waste apples as a low-cost, effective feedstock for the biotechnological production of high-value platform chemicals, contributing to the valorization of agri-food waste within a sustainable and circular bioeconomy.
Consolidated bioprocessing (CBP) integrates enzyme production, biomass hydrolysis, and fermentation into a single process for lignocellulosic ethanol production. However, its coupled nonlinear dynamics and limited intermediate-state measurements make dynamic modeling difficult. This study developed a time-series-informed framework for trajectory-level validation of CBP ethanol production using secondary literature-derived ethanol trajectories. After series-level curation, the benchmark retained 23 trajectories and 216 ethanol observations from 26 eligible series, corresponding to 18 training series and 5 held-out test series, with a median of 10 sampled timepoints per trajectory and a median time span of 120.0 h . A weighted, non-retrospective timepoint model was first developed as an empirical benchmark across heterogeneous operating conditions. The selected ET_log model achieved a grouped out-of-fold RMSE of 3.990 g L^-1 , MAE of 1.869 g L^-1 , R^2 of 0.924, and WAPE of 0.205 across all retained observations. On the held-out test split, it achieved an RMSE of 4.631 g L^-1 , MAE of 2.553 g L^-1 , R^2 of 0.923, and WAPE of 0.195. A phase-structured grey-box model was then fitted to the same ethanol trajectories to represent overlapping enzyme-associated, hydrolytic, and fermentative stages. The fitted parameter patterns showed near-unity median multiplicative scale factors, with broader upper-tail variation within the admissible parameter range. The transition-time medians were 38.190 h and 63.397 h for t_1 and t_2 , respectively. Across 22 matched series, the data-driven benchmark outperformed the grey-box model in predictive accuracy, with median trajectory RMSE values of 0.959 g L^-1 and 3.525 g L^-1 , respectively, and median endpoint absolute errors of 0.541 g L^-1 and 4.170 g L^-1 , respectively. The grey-box model, therefore, served primarily as an interpretable dynamic scaffold rather than as the strongest predictor. In an observer-oriented feasibility analysis using ethanol-only UKF measurement mode, the scaled product state was reconstructed accurately, with an online RMSE of 2.75× 10^-4 and R^2=0.9995 . The enzyme-like state also showed good recovery, whereas the sugar state was less reliable under variance-based assessment. Overall, the results show that secondary CBP time-series data can support grouped dynamic benchmarking, trajectory-informed grey-box analysis, and preliminary soft-sensor-oriented interpretation while highlighting the need for denser multi-state experimental data to improve identifiability and observer validation.
Lignin is the most abundant renewable aromatic resource on Earth, yet its efficient conversion into value-added chemicals through biological funneling remains a key challenge. Sphingobium lignivorans SYK-6 exhibits broad metabolic capacity for lignin-derived aromatic compounds, ranging from monomers to dimers, making it a suitable host for biological funneling. Production systems for the polymer building block 2-pyrone-4,6-dicarboxylic acid (PDC) from lignin-derived aromatic compounds have been constructed using SYK-6. In this study, we developed a PDC-responsive biosensor to facilitate high-throughput screening of improved PDC-producing strains. PDC-assimilating bacteria were isolated from environmental samples, and their genomes were analyzed to obtain candidate PDC-responsive transcriptional regulatory systems. Among these, the LysR-type transcriptional regulator PdcR and its target promoter PPDC from Cupriavidus sp. 8B were identified and characterized as a PDC-responsive system, in which PdcR is suggested to act as both a repressor and an activator. A reporter plasmid pCup1 carrying these components was introduced into a PDC hydrolase gene-deficient strain (ΔligI) of SYK-6 to construct the PDC biosensor ΔligI(pCup1). The biosensor was specific for PDC, with no response observed for its upstream metabolites. When vanillic acid or protocatechuic acid was supplied as a substrate, concentration-dependent fluorescence responses were observed over a range of 0.1–5 mM, demonstrating that PDC production from lignin-derived aromatic compounds can be monitored. Fluorescence increased synchronously with PDC accumulation, suggesting real-time monitoring of PDC production. Furthermore, the biosensor responded to PDC produced from aromatic compounds in softwood black liquor extract. ΔligI(pCup1) enables real-time, specific detection of PDC produced from lignin-derived aromatic compounds, including those in black liquor extract derived from lignocellulosic biomass, demonstrating applicability to screening for lignin valorization. Integration of this biosensor with diverse strain engineering approaches, including random mutagenesis, adaptive laboratory evolution, and metagenomics-based enzyme discovery, would provide a platform for efficient selection of SYK-6 strains with enhanced PDC production capability.
Sorghum stalk (Sorghum bicolor) represents an abundant and underutilized lignocellulosic biomass with significant potential for the generation of value-added bioproducts within biorefinery systems. In the present study, sorghum stalk (SS) biomass was valorized for the production of xylooligosaccharides (XOS) through alkaline extraction of xylan, followed by enzymatic hydrolysis. SS was treated with alkali to extract high-purity xylan, where increasing NaOH concentrations significantly (p < 0.05) enhanced xylan recovery. Fourier-transform infrared spectroscopy indicated that xylan extracted using 3 M NaOH contained minimal lignin and represented a higher recovery (87.17 ± 0.32
Petroleum hydrocarbon contamination creates specialized ecological niches that present a source for mining microorganisms with exceptional metabolic flexibility and stress resilience. In this study, we performed a comprehensive genome-level analysis of Bacillus cereus strain GRJBSBT-1, isolated from petroleum refinery sludge, to elucidate the molecular coordination between ethanol production and stress tolerance, two important traits associated with microbial adaptation to ethanol-rich environments. Whole-genome sequencing revealed a 5.41-Mb circular genome with 35.3
Lignocellulosic biomass is a promising feedstock for sustainable bioethanol production due to its abundance, renewability, and high carbohydrate content. Among these feedstocks, sugarcane bagasse (SCB), a fibrous byproduct of sugar and ethanol mills, is particularly abundant in Brazil. However, the complex and recalcitrant structure of lignocellulose hinders its biological conversion into biofuels. There are different enzymatic architectures to overcome this challenge. Fungal enzyme systems rely on non-complexed cellulase components that act independently and are often used in combination with yeast fermentation in a simultaneous saccharification and fermentation (SSF) configuration. By contrast, thermophilic bacteria such as Clostridium thermocellum employ complexed cellulosome systems capable of highly efficient biomass deconstruction, which enables their application in Consolidated Bioprocessing (CBP). Although previous studies have compared these systems on various lignocellulosic feedstocks, no direct comparison exists for unpretreated sugarcane bagasse. This study aimed to compare carbohydrate solubilization between CBP with a coculture of thermophilic bacteria and SSF with a fungal enzyme–yeast system for the conversion of unpretreated SCB, and to assess the effect of particle size. CBP consistently achieved higher carbohydrate solubilization than SSF across all particle sizes, with improvements ranging from 1.9- to 3.7-fold. CBP was also less affected by particle size variation, showing a drop of 0.52-fold in solubilization for particles sized 0.2–4.00 mm, compared to a 2.2-fold for SSF. A strong linear correlation between C6 and C5 sugar solubilization was observed for both strategies. Despite differences in solubilization, residual sugar concentrations were similar between systems. Neither increased enzyme loading nor improved conditions significantly improved SSF performance, indicating intrinsic limitations in the enzymatic hydrolysis of unpretreated SCB. This study provides the first direct comparison of unpretreated SCB deconstruction mediated by a thermophilic bacterial culture to a fungal cellulase preparation in the presence of yeast. Higher carbohydrate solubilization is seen for the bacterial system regardless of particle size. These findings highlight the potential of CBP with thermophilic bacteria as an alternative to conventional strategies. The results also reinforce the importance of standardized metrics, such as fractional carbohydrate solubilization, for cross-study comparison.
One of the most prominent mechanisms for plant cell wall deconstruction in nature, and widely employed in industry, relies on the coordinated action of hydrolytic and oxidative enzymes. However, how redox networks sustain synergistic biomass deconstruction remains incompletely understood, particularly in the industrial workhorse Trichoderma reesei. This fungus lacks a cellobiose dehydrogenase (CDH), a pivotal redox partner for lytic polysaccharide monooxygenases (LPMOs) in many fungal systems. Here, we investigated the oxidative machinery of T. reesei and the contribution of key redox-active enzymes to lignocellulose deconstruction. We demonstrate that the oxidative capacity of the T. reesei secretome is largely driven by a single enzyme, TrLPMO9A, the most abundant oxidoreductase in the secretome. Proteomic analyses also revealed a lower abundance of other redox-active enzymes, including TrLPMO9B and AA5 oxidase. Although deletion of TrLPMO9B and TrAA5 had a less pronounced impact on saccharification efficiency compared with TrLPMO9A, the secretome remodeling triggered by their deletion, along with the associated decrease in saccharification performance, indicates that these redox enzymes play distinct, non-redundant roles. They likely play a system-level role within a cooperative redox network that fuels oxidative cellulose deconstruction, potentially extending beyond direct catalysis to processes associated with redox balance or protein secretion. Finally, we challenged the CDH-lacking paradigm by heterologously expressing a CDH in T. reesei. In vivo reconstitution of this redox duet increased biomass saccharification by 13–19
Acetogenic bacteria are promising platforms for converting gaseous and liquid one-carbon (C1) substrates such as carbon monoxide, carbon dioxide (CO2), formate and methanol into fuels and chemicals, but their application is limited by sparse genetic tools and poor control over metabolic fluxes. Here, we developed a modular plasmid assembly platform and a suite of characterized native, heterologous and methanol-inducible promoters for the model acetogen Acetobacterium woodii, enabling rapid and tunable control of gene expression together with CRISPR-based genome editing. Using an anaerobe-compatible fluorescent reporter (pFAST), promoter strength was quantified, from medium to high expression levels. Subsequently, a minimal promoter replacement at the native adhE (bifunctional aldehyde-alcohol dehydrogenase) locus along with deletion of the neighboring LysR-type regulator was done employing the newly characterized promoters, demonstrating that a single promoter replacement in the genome was sufficient to shift A. woodii from a primarily acetogenic toward a partly ethanologenic phenotype. The engineered strain was able to produce ethanol not only from fructose but also from the C1 substrates methanol + CO2 and formate + syngas. Together with the promoter replacement strategy, these findings underline that ethanol formation in A. woodii is constrained both by energy conservation and by endogenous transcriptional control. Relieving energetic bottlenecks through the choice of energetically favorable C1 substrates and decoupling adhE expression from its native promoter suffices to enable ethanol production from methanol + CO2 and formate + syngas, directly linking the Wood–Ljungdahl pathway to ethanol formation. These results illustrate how small, targeted genome edits can significantly reshape carbon flux in acetogens and positions A. woodii as a viable chassis for C1-based bioproduction.
Sustainable aviation fuels (SAFs) are considered a projected alternative to widely used petroleum-based fuels for reducing the environmental impact of aviation, including the potential mitigation of greenhouse gas emissions and the reduction of particulate matter formation. While numerous studies have addressed feedstocks and production technologies of SAFs, comparatively limited attention has been given to experimental investigations associated with the second stage of the ASTM D4054 fuel certification process, which includes fuel atomization, ignition, combustion characteristics, and emission behavior under realistic engine operating conditions. This review provides a comprehensive analysis of recent experimental studies devoted to these processes. Particular attention is given to the influence of physicochemical fuel properties, including volatility, viscosity, cetane number, and chemical composition, on atomization quality, ignition delay, flame stability, and combustion efficiency. The relationship between fuel molecular structure and soot formation tendencies is also examined. The analysis shows that the lower density, viscosity, and aromatic content typical of many SAF pathways generally improve atomization and evaporation processes, leading to more homogeneous fuel–air mixtures and reduced particulate matter emissions compared with conventional jet fuels. Overall, the review highlights the importance of integrating atomization and combustion studies into SAF development and certification, providing insights that can support the optimization of fuel formulations and contribute to the safe and efficient large-scale deployment of sustainable aviation fuels.
Consolidated bioprocessing (CBP) by cellulolytic fungi offers a direct route for cellulosic ethanol production, but the process still relies heavily on costly complex nitrogen sources. Replacing complex nitrogen with inorganic nitrogen is a potential strategy to reduce the cost of the medium, yet it often leads to impaired fermentation performance, and the metabolic basis for this limitation remains unclear. Replacement of yeast extract with inorganic nitrogen was evaluated in the engineered thermophilic fungus Myceliophthora thermophila YL913, a CBP strain capable of producing ethanol directly from cellulose. Ammonium sulfate supported higher ethanol production than nitrate, but still resulted in a lower titer than yeast extract. Under ammonium sulfate, transcriptomic and metabolomic analyses revealed altered amino acid metabolism together with reduced carbohydrate metabolic and cellulose catabolic functions, while supplementation with selected amino acids provided only limited improvement. Supplementation with 5 mg/L thiamine pyrophosphate (TPP) increased ethanol production under ammonium sulfate from 14.3 g/L to 23 g/L, reaching a level comparable to that obtained with yeast extract. Moreover, α-ketoglutarate was identified as a responsive metabolic node associated with the altered fermentation state. Stepwise strengthening of the endogenous TPP biosynthetic pathway progressively improved ammonium-based ethanol fermentation, and additional overexpression of pyruvate decarboxylase further enhanced ethanol production. In a 5-L fermenter, the final engineered strain produced 49.5 g/L ethanol under ammonium sulfate without exogenous TPP, close to the 51.2 g/L obtained with the TPP-supplemented parental strain. This study establishes ammonium sulfate as a feasible low-cost alternative to yeast extract for CBP-based cellulosic ethanol production by the engineered M. thermophila strain. These findings provide a practical strategy for developing low-cost fungal CBP processes for cellulosic ethanol production supported by inorganic nitrogen.
Free fatty acids are considered a potential source of biodiesel because they are excreted by engineered cyanobacterial cells, eliminating the processing costs associated with cell recovery and extraction. Although the cyanobacterial mutants engineered for the production of free fatty acids do excrete significant amounts of the product, the yield per dry cell weight is low due to their rapid proliferation and the consequent substantial cell biomass, which makes it impossible to support sustainable biofuel production. Development of strategies to increase the production of free fatty acids per cell is urgently needed. To increase the per-cell production of free fatty acids, we attempted to increase the allocation of photosynthetically fixed carbon into fatty acid production by restricting cell proliferation. Growth limitation of free fatty acid-producing cells was achieved by cultivating dAS2T, a free fatty acid-producing mutant of Synechococcus elongatus PCC 7942 defective in active nitrate uptake, in media containing nitrate as the sole source of nitrogen. Although the nitrogen-limited cultivation increased the per-cell yield of free fatty acids, it significantly decreased the extracellular free fatty acid level, reducing the volumetric productivity. Overexpressing an endogenous free fatty acid-efflux pump in the dAS2T strain elevated extracellular free fatty acid level to those seen under nitrate-sufficient conditions, while maintaining slow cell growth. By decoupling the production of fatty acids from cell growth in this manner, excretion of 0.90 g of free fatty acids per g of dry cell weight was achieved in 240 h, with a free fatty acid productivity of 1.8 mg L−1 h−1. This represents the highest yield of free fatty acids obtained to date without significantly decreasing the volumetric productivity. Growth limitation and enhancement of excretion of the product are essential to increase the per-cell yield of the free fatty acid production system using cyanobacteria. The high yield and productivity of extracellular free fatty acids achieved in this study suggest that the cyanobacteria-based production of free fatty acids provides a reliable approach for sustainable biofuel production through the “milking” strategy.
