Biotechnology for Biofuels最新文献

Background

Aeration plays a critical role in the bioconversion of pretreated lignocellulose by enhancing lytic-polysaccharide-monooxygenase(LPMO)-supported enzymatic saccharification. However, its broader impact, particularly on fermentation inhibitors, remains insufficiently understood. The hypothesis that aeration not only promotes LPMO activity, which has been shown clearly in previous studies, but also affects fermentation inhibitors was investigated in experiments with softwood pretreatment liquids. The effects of aeration were explored through chemical analysis of fermentation inhibitors and through subsequent fermentations with the xylose-utilizing Saccharomyces cerevisiae yeast CelluX™4 to test the fermentability. Controls in which N₂ rather than air was supplied to the pretreatment liquids were used to distinguish between evaporation effects and effects caused by oxidation due to O₂ in air. In separate experiments, two redox-dependent detoxification methods, treatments with sulfite and laccase, were further investigated.

Results

While aeration had no negative effects on the subsequent fermentation of a sugar control, it compromised the fermentability of the pretreatment liquids. Compared to the N₂ control, subsequent fermentation of aerated samples showed reduced consumption of fermentable sugar (glucose, mannose, xylose) at 0.61 compared to 0.76 g L⁻¹ h⁻¹, and lower ethanol productivity (0.23 vs. 0.30 g L⁻¹ h⁻¹). Apart from more commonly studied pretreatment by-products (such as aliphatic carboxylic acids, furan aldehydes, and phenolics), methanol (~ 1 g L⁻¹) was detected in both pretreatment liquids. The methanol concentration decreased during gas addition, which was attributed to evaporation. Compared to the initial pretreatment liquid, aerated reaction mixtures exhibited slightly elevated levels of formaldehyde, but lower levels of furfural and vanillin. Sulfite detoxification was successful under both aeration and N₂ conditions. Treatment with laccase was found to have variable effects on the fermentability depending on the conditions applied.

Conclusions

The results underscore the dual role of aeration in softwood bioconversion, positive for promoting LPMO activity but potentially negative with respect to subsequent fermentability, and highlight the need to carefully tailor aeration strategies for the design of efficient biochemical processing of lignocellulosic feedstocks. Treatment with reducing agents, such as sulfite, emerges as a possibility to alleviate negative side-effects on the fermentability when aeration is used to promote LPMO activity.

Background

This study establishes a computational framework for predictive style modeling in tobacco formulation design, addressing the critical disconnect between empirical approaches and blended system complexity. Herein, "style" refers to the characteristic sensory profiles (e.g., aroma, taste, and physiological sensations) intrinsically linked to cultivation regions, which arise from the unique combination of local environmental factors, such as climate and soil composition. A convolutional neural network (CNN) framework was developed to integrate conventional chemical indicators with thermogravimetric analysis-derived features from 434 geographically authenticated tobacco leaf samples. Through regionally constrained Monte Carlo sampling of composition ratios, 304,800 formulation data sets simulating real-world blending constraints were generated to enable robust model training.

Results

The leaf-centric CNN demonstrated remarkable region-style classification accuracy (99.54% via fivefold cross-validation), outperforming conventional machine learning models and revealing thermal–chemical complementarity in regional style characterization. However, direct application to blended formulations revealed a critical limitation: only 50.91% of blended formulations maintained stylistic consistency with their primary source leaves, underscoring the inadequacy of single-leaf model for blended systems. To overcome this, a unified CNN framework was trained on a consolidated multi-source data set encompassing both raw leaves and engineered blends, leveraging their shared feature space. This hybrid learning model achieved dual breakthroughs in regional style identification accuracy (90.09%) and leaf-to-blend style consistency (87.90%). Mechanistic analysis identified a nonlinear threshold effect, showing that primary source leaves maintained 99.91% stylistic dominance when exceeded 90% composition, decreasing to 67.90% at 30% composition. Significant formulation style deviation risks emerged when compositional gaps between principal and secondary source leaves narrowed below 10%.

Conclusions

Building on these insights, a probabilistic style modulation strategy was proposed and validated through case applications, transforming theoretical discoveries into actionable design strategies. This innovation establishes region ratio constraints based on threshold-defined boundaries, creating a data-driven framework that systematically achieves target formulation style through the threshold's predictive capacity. This framework advances tobacco engineering from empirical practices to predictive digital transformation, providing a template for agricultural product manufacturing systems facing similar formulation challenges.

Background

Incorporating the production of related ligninolytic enzymes into industrial filamentous fungus Aspergillus niger will enhance the bioconversion of lignocelluloses to various chemical products.

Results

In this study, transgenic expression of Phanerochaete chrysosporium manganese peroxidases (mnps) and Trametes sp. C30 laccase hybrid Lac131 (lac131) were examined and optimized in A. niger 11414 prtT∆ strain. Five mnps (mnp1, mnp2, mnp3, mnp4, and mnp5) and lac131 genes were expressed separately or in combination. The transgenic strain containing the entire mnp2 genomic coding sequence (gmnp2) exhibited the highest mnP activity among the five mnp over-expression strains in the modified minimal medium (mMM) with addition of 5 g/L bovine hemoglobin (bHg). We examined the effects of hemin and bHg on mnP production in the gmnp2 strain cultures and found that at least 1 g/L bHg was required, while hemin was not. Culture conditions for mnP production were further optimized for the gmnp2 strain and the highest mnP activities were detected in the cultures grown at 25 °C and 200 rpm with an initial pH of 4.5. Effects of soy protein, skim milk, and bovine serum albumin on mnP production were investigated; 5 g/L of soy proteins or skim milk had comparable effects to 2.5 g/L bHg, while cultures with bovine serum albumin had diminished mnP activity. Disruption of both prtT and vsm1 substantially augmented the mnP production and its activity reached 575 U/L. Trametes sp. C30 laccase hybrid lac131 was strongly expressed in either A. niger gmnp2 (1975 U/L) or 11414prtT∆ (3895 U/L) strain. Both mnP and laccase in the culture supernatants effectively decolorized selected phenolic compounds (dyes) and cleaved tagged model lignin dimers.

Conclusion

The mnP was successfully produced in A. niger by optimizing the culture conditions and host strain. Co-expression of all four mnp genes in the same expression host by multiplex CRISPR will lead to the mnP production reaching levels comparable to P. chrysosporium, while only requiring 36 h at 25 °C. The Lac131 activity in transgenic A. niger strain is 4- to 7-times higher than that in previous studies. Co-production of mnP and laccase in A. niger will enhance the lignin bioconversion efficiency.

Background

Oleaginous microorganisms contain oil and fat at amounts greater than 20% of their biomass weight, with fatty acid and chemical compositions often similar to those of vegetable oil and animal fat. Oleaginous yeasts, including Lipomyces starkeyi, are particularly promising because of their high oil accumulation capacity, broad sugar utilization, and ability to use non-edible biomass, making them suitable for large-scale, cost-effective oil production. However, reducing production costs remains a major challenge, as media costs account for the majority of total microbial oil production costs. Okara, a byproduct of tofu and soy milk production, is a potential low-cost nitrogen source. Although previous study have reported the use of solid okara for oil production and its lower oil yield than that of yeast extract medium, the potential benefits of adding inorganic nitrogen to improve yield have not been fully explored.

Results

We tested the effect of inorganic nitrogen addition on oil production in okara medium using culture experiments and found that the addition of ammonium sulfate significantly increased not only the cell concentration but also the oil yield by 1.61-fold (19.7 ± 0.44 [g/L]). In addition, the presence of both sulfate and ammonium ions was important for increasing the oil production. Metabolome analysis of the culture supernatant showed that sulfate ions contribute to glutathione synthesis, whereas ammonium ions provide nitrogen and affect the glutathione synthesis pathway through the ammonia assimilation pathway, which may result in increased oil productivity.

Conclusions

The use of okara medium supplemented with ammonium sulfate can reduce the cost of nitrogen source materials to a level of several percentages of that of conventional yeast extract medium, presenting the possibility of inexpensive oil production by L. starkeyi. In addition, the dual roles of ammonium sulfate in enhancing oil production were proposed. Furthermore, this is the first study to confirm the relationship between an enhanced glutathione synthesis pathway and increased oil production by L. starkeyi. These findings provide a foundation for the further development of sustainable and economically viable microbial oil production bioprocesses.

The shift toward sustainable biomanufacturing necessitates microbial platforms that efficiently convert low-cost, non-food feedstocks into high-value chemicals. Sucrose, a widely available and economical carbon source, remains underutilized in industrial Escherichia coli fermentation due to its low metabolic efficiency. This study investigates the production of L-lactic acid monomer in E. coli using sucrose, a cost-effective carbon source. Initially, we found that the recombinant strain 090S with the cscR gene knocked out exhibited an enhanced aerobic growth rate; however, during anaerobic fermentation for acid production, synthesis of the lactic acid monomer ceased after 3–4 h, indicating an impediment in sucrose metabolism under anaerobic conditions. Furthermore, we analyzed its transcriptional characteristics under aerobic-anaerobic phases through dynamic transcriptomic profiling and found significant differences. Specifically, for the csc operon, all three genes (cscB, cscK, and cscA) saw a significant decrease in expression when transferred into anaerobic conditions, retaining less than 10% of their aerobic expression levels. Here, we address this critical challenge by engineering optimized anaerobically active promoters to decouple sucrose utilization from native transcriptional constraints. Ultimately, the recombinant strain 091S, in which overexpresses the cscA and cscB genes by using the gapA promoter, produced 129.7 g/L of L-lactic acid in a 5-L bioreactor within 30 h of fermentation, with an average volumetric productivity of 4.32 g/(L·h), marking a 3.04-fold increase over the control. Additionally, an industrial fermentation process was simulated in a 30-L bioreactor under scaled-up conditions, resulting in a higher L-lactic acid yield of 145.7 g/L and a productivity of 4.96 g/(L·h), which was similar to that of glucose as a carbon source. This study elucidates the impact of oxygen content changes on gene transcription levels during the fermentation of E. coli using sucrose as a carbon source, offering a scalable and economically viable strategy for the efficient production of bio-products from sucrose or sucrose-rich feedstocks by E. coli.

Renowned for their distinctive aromas, terpenoid flavor compounds and their precursors are widely used in medicine, food, and the flavor and fragrance industries. Rapid advances in synthetic biology, including the modification of microbial chassis cells, the design of synthetic pathways for novel target products, and the integration of large-scale microbial fermentation, have enabled the development of microbial cell factories for the green and efficient production of terpenoid flavor compounds and their precursors, offering broader market potential. This review examines common biosynthetic mechanisms, recent progress in the field, and strategies for enhancing the biosynthetic efficiency of terpenoid flavor compounds and their precursors. This study aims to support the advancements of sustainable production technologies and promote industrial application within the flavor and fragrance sector.

The rising demand for natural products is accelerating research into sustainable methods for producing bio-based flavourings like ethyl butyrate. In this study, ethyl butyrate was successfully produced through the enzymatic esterification of butyric acid and ethanol, which were derived from the co-cultivation of Clostridium tyrobutyricum and Saccharomyces cerevisiae. Initial monoculture experiments with both strains were performed to investigate compromised fermentation conditions for co-cultivation. Based on these findings, anaerobic co-cultivation conditions were established at 37 °C and 150 rpm, with the pH controlled at 6. The effects of varying inoculation times in co-culture were examined, considering the solvent and acid tolerance of both strains. Due to the limited acid tolerance of S. cerevisiae, with significant inhibition at butyric acid concentrations above 10 g L¯¹, a time-delayed inoculation with C. tyrobutyricum was implemented. In batch experiments, the final concentrations of butyric acid and ethanol were 13.98 ± 3.06 g L¯¹ and 21.43 ± 1.66 g L¯¹, respectively. Further enhancement of product concentrations was explored through a fed-batch cultivation strategy yielding up to 45.62 ± 3.82 g L¯¹ of butyric acid and 18.61 ± 4.11 g L¯¹ of ethanol. Ethyl butyrate was formed from the fermentation products by lipase-catalysed enzymatic esterification in a two-phase system through the addition of an organic phase. The ester concentration in the organic phase reached a maximum of 23.93 ± 0.68 g L¯¹ (esterification yield 25%). This study presents a viable approach to the production of bio-based ethyl butyrate offering a sustainable alternative to traditional chemical synthesis methods.

Graphical Abstract

Waste activated sludge (WAS) represents a significant operational and environmental challenge for wastewater treatment plants (WWTPs) due to its low biodegradability, attributed to extracellular polymeric substances (EPS) that hinder enzymatic hydrolysis. Electrochemical (EC) pretreatment has shown promise in improving organic matter solubilization. However, conventional systems often face limitations related to high energy demand, mineralization of organic matter and electrode degradation. This study evaluates EC pretreatment with two dimensionally stable anodes, Ti/RuO₂ and Ti/RuO₂–ZrO₂–Sb₂O₅, as scalable alternatives for improving WAS biodegradability and energy recovery. The EC with both electrodes using WAS as the sole electrolyte, with an applied current of 10 mA/cm² for 30 min, achieved significantly enhanced solubilization with minimal mineralization. This effectively enhances the anaerobic biodegradability of WAS and increases methane recovery while maintaining low energy consumption and avoiding chemical additives. Methane yields increased to 168 and 342 N-L_CH4/kg_VS for WAS pretreated with Ti/RuO₂ and Ti/RuO₂–ZrO₂–Sb₂O₅, respectively, compared to 85 N-L_CH4/kg_VS for untreated sludge. Energy analysis revealed a net gain of 1.64 kW-h/kg_VS, outperforming other EC systems reported in the literature. In this sense, the implementation of this process could be integrated at an industrial scale in WWTPs as a cost-effective strategy for sludge valorization and resource recovery, in line with circular economy principles.

Gangliosides are essential glycosphingolipids critical in neurodevelopment and cell signaling. Traditionally sourced from animal tissues, their production raises ethical concerns and faces challenges in scalability and cost. Chemoenzymatic methods have emerged as alternatives but lack flexibility and broad industrial applicability of microbial systems. However, complete microbial biosynthesis remains challenging due to the complexity of reconstructing the biosynthetic pathway in non-native hosts. We report the first successful complete microbial synthesis of gangliosides by engineering the industrial filamentous fungus Ashbya gossypii. Using modular metabolic engineering, we heterologously expressed human and yeast enzymes to reconstruct a functional ganglioside biosynthetic pathway. Pathways for producing activated N-acetylneuraminic acid, lactosylceramide, and sialylated intermediates were integrated, yielding GM3 and GD3 at milligram-per-liter levels. These titers were further enhanced by introducing a heterologous Leloir pathway for galactose metabolism. This work represents a foundational advance in microbial glycoengineering, offering a scalable, animal-free microbial platform for ganglioside production with broad applications.

Background

Biological deconstruction of lignocellulose for sustainable chemical production offers an opportunity to harness evolutionarily specialized enzymes and organisms for industrial bioprocessing. While hydrolysis of cellulose and hemicellulose by CAZymes yields fermentable sugars, ligninolysis releases a heterogeneous mix of aromatic compounds that likely play a crucial role in shaping microbial communities and microbial metabolism. Here, we interrogated the metabolomic and transcriptomic response of a lignocellulolytic anaerobic fungus, Neocallimastix californiae, to a heterogeneous mixture of aromatic compounds derived from lignin.

Results

Through exposing the fungus to both a concentration it might experience in its native environment and an elevated concentration of alkaline lignin, we observe that N. californiae transforms vanillin and that supplying alkaline lignin at 0.125 g/L, alongside cellulose, enhances the growth and polysaccharide-degrading activity of N. californiae. Altogether, our results further suggest that vanillin consumption, increased polymer-degrading activity, increased metabolic activity, and transcriptomic remodeling of amino acid synthesis genes all coincide with increased melanin production by fungal cells. These observations challenge previous notions that aromatics from lignocellulose only inhibit the growth and polymer deconstruction capabilities of the biomass-degrading anaerobic fungi (Neocallimastigomycetes).

Conclusions

This study demonstrates that anaerobic fungi have a complex relationship with aromatic chemicals derived from lignin and hemicellulose and shift their metabolism in response to the addition of lignocellulose-derived aromatics to their growth medium. Further, as no known pathways for the biochemical transformation of aromatics were detected in these organisms despite observed transcriptome remodeling in the presence of aromatics, we suggest they might encode novel biochemical routes for scavenging amino acid building blocks from aromatic monomers derived from hemicellulose side chains and lignin.