Biotechnology for Biofuels最新文献

Background

The pattern of xylan backbone substitution by 4-O-methylglucuronic acid (mGlcA) and O-acetyl groups generated during biosynthesis in the Golgi apparatus plays a key role in molecular interactions between secondary cell wall polymers in hardwoods, thus regulating biorefinery properties of woody biomass. This study aims at comparing the effects of two different approaches to reduce xylan glucuronidation, targeting either xylan biosynthesis by RNAi suppression of GLUCURONIC SUBSTITUTION OF XYLAN1 genes (denoted Gux1RNAi) or by xylan post-synthetic modification in the secondary cell wall by apoplastic wood-specific expression of a fungal α-glucuronidase from family GH67 (denoted WP:GH67) in hybrid aspen (Populus tremula L. x tremuloides Michx.). The effects of reducing xylan glucuronidation by these two approaches on xylan acetylation and solubility, and recalcitrance of wood to enzymatic hydrolysis were compared.

Results

Both approaches reduced mGlcA/xylose (Xyl) ratio by approx. 30% without altering Xyl content in alkali extracts, while xylan acetyl content in subcritical water extracts was increased only in Gux1RNAi lines. The Gux1RNAi lines showed reduced glucuronidation in the major xylan domain containing evenly-spaced mGlcA, whereas the WP:GH67 lines were affected in both odd and even glucuronidation patterns. Interestingly, the arrays containing mGlcA every second xylosyl unit were not affected by any modification, whereas an increase in xylo-oligosaccharides containing O-2,3 diacetylated Xyl was found in Gux1RNAi lines only. Neither modification consistently affected glucose or Xyl yields in saccharification without or with acid pretreatment, whereas xylan extractability in subcritical water was increased in Gux1RNAi, but not in WP:GH67 lines.

Conclusion

Our study supports the hypothesis that acetylation competes with glucuronidation by GUX1 during biosynthesis, and increased acetylation caused by GUX1 suppression rather than reduced xylan glucuronidation per se improves xylan extractability. The 30% reduction in glucuronidation did not affect enzymatic saccharification, supporting the concept that cell wall structure and recalcitrance in transgenic lines are maintained by xylan domains which were not affected by either transgenic modification.

Background

Short-chain primary amines are a versatile group of molecules in biological systems, derived from the decarboxylation of amino acids or the transamination of carbonyl-containing molecules. Isobutylamine, the decarboxylation product of l-valine, has applications in chemical synthesis of pharmaceuticals, herbicides, fungicides and specialized chemicals, where it is especially interesting as a substitute for petrochemical compounds. Since the chemical synthesis of short-chain primary amines is hazardous and environmentally unsustainable, we established an efficient biosynthetic approach in this work.

Results

The natural amino acid producer Corynebacterium glutamicum is an ideal chassis for the production of amino acid-derived molecules. In this study, we assessed the toxicity of several relevant short-chain primary amines on C. glutamicum. A crude extract assay demonstrated the decarboxylation of l-valine to isobutylamine by the l-valine decarboxylase VlmD_Sv from Streptomyces viridifaciens. This enzyme was instrumental to extend production of pyruvate in three engineered strains to yield l-valine and subsequently isobutylamine. To ensure intracellular l-valine supply, the extracellular l-valine accumulation was decreased up to 20-fold by deletion of the genes for the branched-chain amino acid exporter BrnFE_Cg and/or overexpression of the l-valine importer gene brnQ_Cg. Surprisingly, expression of the transhydrogenase genes pntAB_Ec from Escherichia coli did not improve but drastically reduced isobutylamine production. Biochemical analysis revealed that pntAB_Ec expression depolarized the membrane potential.

Conclusions

This study demonstrated the highest production of isobutylamine and the highest production of any microbial short-chain primary amine in minimal medium. In shake flask cultivation, an isobutylamine titer of 12.1 g/L and a yield of 0.74 mol isobutylamine/mol glucose were obtained. Transfer to a 3 L fed-batch bioreactor fermentation was successful and a titer of 33.2 g/L was reached within 64 h with a yield of 0.47 mol/mol and a volumetric productivity of 0.52 g/l/h. Moreover, isobutylamine production on a second-generation feedstock was shown, highlighting the potential of sustainable bio-based production of isobutylamine.

Chitinous wastes, particularly shrimp shell residues, represent a major underutilized biomass resource with significant potential for bioconversion into high-value products. In this study, four thermophilic bacterial strains (Bacillus amyloliquefaciens BT 2022, Bacillus licheniformis Basma87, Priestia megaterium AMD 2024, and Streptomyces maritimus MSQ-2021) were screened for their ability to produce chitinase using shrimp shell waste (SSW) as the sole carbon and nitrogen source. Among these, P. megaterium AMD 2024 exhibited the highest chitinolytic activity. Optimization of fermentation parameters was initially performed using the one-variable-at-a-time (OVAT) approach, identifying 5% SSW, 60° C, 72 h incubation, pH 7.0, 200 rpm shaking, and 2% inoculum size as optimal conditions, yielding 93.24 U/mL chitinase activity. Subsequent statistical optimization using response surface methodology (RSM) with Plackett–Burman design (PBD) and central composite design (CCD) significantly enhanced enzyme production, achieving maximum activity of 273.3 U/mL, representing a 2.93-fold increase compared to OVAT. Partial purification via ammonium sulphate saturation (60–80%) resulted in 260.0 U/mL activity with 95.13% relative activity. Cytotoxicity assays using normal human skin cell line HFB4 confirmed the biocompatibility of the purified enzyme at concentrations up to 260.0 U/mL. These findings demonstrate that shrimp shell waste can be efficiently valorized into chitinase through microbial fermentation, providing an eco-friendly solution for waste management and offering safe, sustainable applications in agriculture, industry, and cosmetics.

Graphical Abstract

Based on the efficient digestive system of the larva of the sun beetle (Pachnoda marginata) in utilizing lignocellulose-rich biomass, a reactor system was designed to partially mimic processes that occur in the larval gut. We aimed developing a process with this system for anaerobic mono-digestion of wheat straw to produce volatile fatty acids (VFAs) and biogas. Three stirred tank reactors connected in series were customized with polyurethane foam and operated at 37 °C. The first two reactors were designed to mimic the midgut of the larva with enhanced VFA production and the third one to resemble the hindgut and optimized for methanogenesis. The system was fed in a semi-continuous regime with ground wheat straw in an alkaline medium. Enrichment cultures from midgut and hindgut of the larva were used as inocula and three conditions with increasing organic loading rate were tested. The highest biomass to methane conversion occurred at the lowest organic loading rate and highest retention time, when a methane yield of 148 mL_Norm g⁻¹_VS was obtained and VS degradation reached 44%. The microbial community was enriched toward cellulose and hemicellulose degrading taxa, including Dysgonomonadaceae, Lachnospiraceae, Marinilabiliaceae and Ruminococcaceae. The methanogenic community shifted from predominance of Methanosarcina to Methanobacterium and Methanoculleus. A comparison with other studies investigating straw utilizing bioreactors confirmed that a core microbiome composed of the phyla Firmicutes, Bacteroidetes and Proteobacteria effectuates the anaerobic digestion of lignocellulose-rich materials, regardless of the source of the inoculum and the process conditions. The designed process was stable and capable to convert wheat straw into methane and carboxylates. Further adaptations are suggested to improve the anaerobic digestion and decrease assembly and operating costs.

Background

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.

Results

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% and molecular sieve addition equal to substrate mass), quantitative levulinic acid conversion and levulinate yield (~ 98–99%) were achieved within 5 h. In comparison, the secondary alcohol 2-butanol, evaluated for mechanistic comparison, reached a lower conversion (~ 70%) under otherwise identical conditions, consistent with its greater steric hindrance within the enzyme active site. Molecular docking simulations supported experimental observations by elucidating substrate–enzyme interactions and differences in binding affinity. The immobilized enzyme exhibited excellent operational stability, maintaining over 97% LA conversion over five consecutive cycles, and successful operation at 100 mL in a bioreactor highlighted the potential for further scale-up. The solvent-free system reduces process complexity, facilitates downstream processing and enhances process sustainability.

Conclusion

Overall, this work demonstrates an efficient strategy for valorizing fusel oil side stream into high-value levulinate esters, contributing to circular bioeconomy approaches and the development of renewable fuel oxygenates and chemical platforms.

Graphical abstract

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.

Background

Interest in single-cell oils from oleaginous yeasts is growing with respect to their potential as sustainable alternatives to plant- and fossil-based oils. The yeast Cutaneotrichosporon oleaginosus is a promising candidate for microbial lipid production due to its broad substrate range and high lipid accumulation capacity. Among potential feedstocks, volatile fatty acids (VFAs) derived from organic waste streams have attracted increasing attention as low-cost, sustainable carbon sources. These VFA mixtures typically contain C2–C6 volatile fatty acids, including acetic, propionic, butyric, valeric, isovaleric, and caproic acids. However, a systematic understanding of how individual VFAs affect growth, lipid accumulation, and fatty acid composition in C. oleaginosus is still lacking.

Results

This study investigates the effects of individual VFAs in fed-batch fermentations with C. oleaginosus ATCC 20509 under controlled, elemental carbon-equivalent conditions, using acetic, propionic, butyric, valeric, and caproic acid as well as the branched-chain acids 2-methylpropionic, 2-methylbutyric, and 3-methylbutyric acid. It was demonstrated that carbon chain length strongly influences these parameters, with growth and lipid production generally decreasing as chain length increases. The highest lipid titers were observed in acetic acid- and butyric acid-based fermentations. Even-chain VFAs primarily resulted in the synthesis of even-chain fatty acids. In contrast, odd-chain VFAs promoted the formation of odd-chain fatty acids, with propionic acid emerging as a particularly promising substrate for the targeted production of odd-chain fatty acids. Fermentations with branched VFAs revealed that growth and lipid accumulation are strongly reduced, potentially due to steric effects associated with methyl substituents. However, the presence of the methyl group did not significantly alter the overall fatty acid profile compared to unbranched carbon chains, as confirmed by NMR analysis.

Conclusion

These findings demonstrate that the choice of substrate has a major impact on targeted lipid production. By enabling the modulation of lipid composition and supporting efficient biomass and lipid accumulation, volatile fatty acids offer a high-value alternative to conventional carbon sources. Overall, these results highlight the potential of VFAs to contribute to a circular bioeconomy by converting low-value waste streams into valuable microbial oils.

Graphical Abstract

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% GC content, comprising 5393 coding sequences and complete glycolytic and fermentative pathways. Functional annotation and KEGG pathway mapping identified genes associated with ethanol metabolism and protein quality-control systems involved in stress adaptation. Although several ethanol-related enzymes were detected, a complete canonical AdhE/PFOR ethanol fermentation pathway was not reconstructed. Quantitative gene expression analysis (qRT-PCR) under fermentative and ethanol-stress conditions showed significant upregulation of key fermentative genes (adhE, pdhA) and protein quality-control genes (groEL, groES), whereas the small heat-shock protein hsp20 was selectively downregulated. This transcriptional expression profile suggests differential involvement of chaperone systems during ethanol stress adaptation. Moderate ethanol exposure (5%) was associated with increased expression of groEL and selected metabolic genes, indicating a potential relationship between protein quality-control processes and ethanol-associated adaptation. The contrasting regulation of hsp20 and groES suggests differential transcriptional responses to ethanol exposure. However, these observations are based on targeted gene expression analyses and should be considered preliminary until validated through broader functional and systems-level investigations. Comparative analysis with supported possibilities of ethanologens underscores the metabolic versatility and robust stress-management capacity of strain GRJBSBT-1. These findings provide genomic and transcriptional insights into ethanol-associated metabolism and stress adaptation in B. cereus GRJBSBT-1 and may serve as a foundation for future investigations aimed at evaluating its biotechnological potential.

Graphical abstract

Background

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.

Results

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%, demonstrating a strong synergistic relationship between LPMOs and CDHs even in a native CDH-lacking host.

Conclusion

These findings define the core oxidative machinery underlying biomass deconstruction in T. reesei, revealing the major cellulose-oxidative role of TrLPMO9A and the importance of a cooperative redox network for efficient lignocellulose depolymerization. Moreover, successful reconstitution of CDH activity in a naturally CDH-deficient host establishes redox engineering as a promising strategy to enhance industrial biomass conversion.

Background

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.

Results

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.

Conclusions

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.