Biotechnology for Biofuels最新文献

Background

Efficient enzymatic hydrolysis of lignocellulosic biomass (LCB) is essential for maximizing the recovery of fermentable sugars for diverse biotechnological applications. However, pretreatment by-products including lignin interact with hydrolytic enzymes, blocking their access to substrates and leading to poor monomeric sugar recovery. This study evaluated the effects of all 20 exogenous amino acids (AAs) as additives to block lignin active sites and facilitate enzyme access to polysaccharide substrates for enhanced sugar recovery. The hydrolysates were subsequently tested for microbial lipid production by Rhodotorula toruloides CGMCC 2.1389 as a model application.

Results

The study found that most AAs enhanced enzymatic hydrolysis of 1% (w/v) H₂SO₄- and Na₂CO₃-pretreated corn stover (CS), with l-proline (Pro) increasing total reducing sugar (TRS) recovery by over 20%. Lipid production by R. toruloides on the hydrolysates was verified under single-stage and two-stage culture conditions. The lipid yield reached over 17 g/100 g TRS with some AAs, despite lower titers. Moreover, the dominance of C16 and C18 fatty acids in the lipids suggest no adverse effects of AAs on the yeast's metabolism.

Conclusion

Exogenous AA addition during enzymatic hydrolysis enhanced sugar recovery; however, its impact on R. toruloides lipid production varies with culture conditions, where a two-stage process with nutrient limitation could be more favorable for high lipid production. While this strategy proved to be more effective for enhanced sugar recovery, future studies are expected to uncover the underlying mechanisms that drive this improvement.

Background

Liquid fuels from lignocellulosic feedstocks are required for transition to a sustainable bioeconomy. However, the recalcitrance of carbon-containing feedstock cell walls to deconstruction poses a barrier to cost effective biological conversion of plant biomass to biofuels. One-step consolidated bioprocessing (CBP) in which anaerobic thermophilic bacteria convert lignocellulosic biomass into liquid fuels is a platform for overcoming the recalcitrance of plant biomass.

Results

The amounts of hemicellulosic and pectic polysaccharides, two complex cell wall glycans that contribute to plant biomass recalcitrance and that are partially solubilized during CBP of switchgrass aerial biomass by Clostridium thermocellum were evaluated in the liquor, solid residues and residue washate recovered during a 120-h CBP process. After 120 h, 24% of milled switchgrass was solubilized in the C. thermocellum CBP platform. Higher concentrations of arabinose, xylose, galactose, and glucose accumulated in the CBP-fermentation liquor and washate compared to fermentation controls without C. thermocellum, indicating that C. thermocellum solubilized hemicelluloses, but did not fully metabolize them. After five days of fermentation, the relative amount of rhamnose in the solid residues increased by 16% compared to controls, and CBP solid residues had more than 23% increased reactivity against RG-I reactive monoclonal antibodies, indicating that the pectic polymer rhamnogalacturonan I (RG-I) was not effectively solubilized from switchgrass biomass by C. thermocellum CBP. Similarly, the amount of mannose (Man) in the CBP solid residues increased by 7% and reactivity against galactomannan reactive antibodies increased by greater than 14%, indicating that the hemicellulosic polymer galactomannan was also resistant to degradation by C. thermocellum during CBP fermentation.

Conclusions

These findings show that C. thermocellum is unable to effectively degrade RG-I pectic and galactomannan hemicellulosic components in switchgrass biomass. Targeting these polymers for improved solubilization could enhance the efficiency of conversion of grass biomass to biofuels.

Background

Plant-based materials have the potential to replace some petroleum-based products, offering compostability and biodegradability as critical advantages. Xylan-rich biomass sources are gaining recognition due to their abundance and underutilization in current industrial applications. Research of potential xylan applications has been complicated by the complex and heterogeneous structure that varies for different xylan feedstocks. Acylation is a broadly used reaction in functionalization of polysaccharides at an industrial scale. However, the efficiency of this reaction varies with the xylan source. To optimize xylan valorization, a systematic understanding of structure–reactivity relationships is essential.

Results

This study explores, characterizes, and compares various xylan feedstocks in the acylation process. Xylan feedstocks were analyzed for their chemical composition, degree of polymerization, branching, solubility, and presence of impurities. These features were correlated with xylan glycotypes’ reactivity toward functionalization with succinic anhydride in an optimized DMSO/KOH condition, achieving carboxyl contents of up to 1.46. We used principal component analysis and hierarchical clustering to identify key structural features of xylan that promote its reactivity. Our findings reveal that xylans with higher xylose content and lower degrees of branching exhibit enhanced reactivity, achieving higher carboxyl content and yields. Structural analyses confirmed successful modification, and light scattering analyses showed dramatic changes in the solution properties. Succinylation improves the solubility and film-forming properties of native xylans.

Conclusions

This study shows key structure–reactivity relationships in xylan succinylation, establishing that low branching, high xylose content, and reduced lignin impurity enhance chemical functionalization. The results offer a framework for selecting optimal biomass feedstocks and support future efforts in genetic and synthetic biology to design plants with tunable xylan architectures. These findings advance the hemicellulose valorization for applications in coatings and packaging.

Background

Polyhydroxyalkanoates (PHAs), biodegradable polymers, can be synthesised and degraded by a number of bacteria. With a range of monomer composition and molecular weight, these polymers can be used for packaging to medical applications. However, the production cost, inadequate mechanical properties, and challenging melt processing properties are major impediments.

Understanding and harnessing the regulatory networks underpinning PHA production in a model organism Pseudomonas putida KT2440 is an invaluable tool to increase PHA production and alter polymer properties for specific applications.

Results

The small RNAs CrcY and CrcZ, key components of the carbon catabolite repression (CCR) system, are implicated in PHA metabolism in P. putida KT2440. Their in trans overexpression in P. putida KT2440 shows a 1.3- to 3.5-fold increase in PHA titre (g/L), using glucose or octanoate as feedstocks. This is accompanied by a decrease in the Mw of the synthesised polymer. Among the proteins showing differential expression in response to CrcY and CrcZ overexpression, glutaryl-CoA dehydrogenase GcdH, involved in the catabolism of lysine, hydroxylysine, and tryptophan, and gamma-glutamyl transpeptidase GGT, involved in glutathione metabolism, showed a consistent increase in abundance across different conditions. It also appears that CrcY and CrcZ can compensate for each other, as only when both sRNAs are removed is a 2.5-fold decrease in PHA observed. We also show that these sRNAs require other CCR elements, Hfq and Crc, for their role in PHA metabolism.

Conclusions

One strategy to overcome poor mechanical properties of PHAs is to blend them with a second polymer. Medium chain length (mcl)-PHA acts as a plasticiser when blended with poly-3-hydroxybutyrate (PHB), the most widespread used PHA resin. Here we show a clear effect of the overexpression of CCR elements CrcY and CrcZ in P. putida KT2440 on the amount of the accumulated mcl-PHA and its Mw, making this tool valuable to produce mcl-PHA-based additives.

These findings highlight the complementary regulatory roles of CrcY and CrcZ in modulating CCR to optimise PHA production. This study provides insights into leveraging CCR elements to enhance the efficiency of PHA biosynthesis, contributing to the development of sustainable bioplastic production.

Xylitol, a five-carbon sugar alcohol, is recognized as a desirable sugar alternative due to its low-calorie content and metabolism independent of insulin. Its commercial production generally involves the chemical hydrogenation of D-xylose, an approach that is energy-demanding and environmentally unfriendly. Although fermentation offers a biological alternative, it often suffers from low conversion efficiency and limited yields. However, xylitol is an intermediate metabolite in various microbial species, and its biosynthesis can be enhanced through metabolic engineering. Genetically modifying microbial cell factories—such as bacteria, fungi, and yeast—has shown significant improvements in xylitol production. Furthermore, the precursor xylose, which is utilized by microbes, can be derived from lignocellulosic biomass through hydrolysis, offering a more sustainable and cost-effective production route. This review discusses recent advances in the bioproduction of xylitol and highlights various metabolic engineering strategies employed to enhance xylitol yield in microbial cell factories.

Graphical Abstract

Background

The degradation of agricultural wastes is crucial for sustainable economic and environmental development, necessitating efficient cellulolytic enzymes to enable high-value bioconversion. The filamentous fungus Trichoderma reesei is a widely used cellulase producer for deconstructing agricultural wastes in biomass conversion. However, its enzyme system remains suboptimal and requires further refinement to achieve economical bioconversion of agricultural wastes.

Results

Herein, a hyper-cellulolytic T. reesei mutant strain CU7-4 derived from the industrial strain RUT-C30 was obtained by UV mutagenesis. When degrading the different pretreated corncob residues, CU7-4 exhibited a 20% improvement in saccharification efficiency compared to the parental strain RUT-C30. Furthermore, comparative proteomics was employed to decipher the variation between the secretomes of CU7-4 and RUT-C30. It was found that the discrepancy of the protein proportion between the secretomes may enable the changed saccharification efficiency towards the pretreated corncob residues. Then, three small secreted proteins (SSP1, EPL1, CUT1) and two β-glucosidases (Cel3H, Cel3F) were identified through the significant differences analysis in protein abundance between CU7-4 and RUT-C30, combined with responding to the essential transcriptional regulator Xyr1. Further investigation of these five proteins was conducted. Deletion of SSP1 and EPL1 was certified to facilitate degrading corncob residues and corn stover. Overexpression of Cel3F improved the activities of cellobiohydrolase and β-glucosidase, and the in vitro addition of Cel3F significantly promoted the saccharification efficiency of RUT-C30 toward corncob residues.

Conclusions

This study not only expands the protein functions for deciphering the mechanism of lignocellulose degradation, but also provides valuable protein targets for engineering the robust and powerful lignocellulolytic enzyme system, thereby facilitating the efficient degradation of agricultural wastes.

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.