Biotechnology for Biofuels最新文献

Background

Biohydrogen production from agricultural waste is a promising strategy to address climate change and energy challenges. This study aimed to optimize the process parameters for biohydrogen production from watermelon peels (WMP) by Clostridium butyricum NE133 using statistical optimization techniques. Initial screening of eight significant variables influencing hydrogen production including, initial pH, incubation temperature, WMP concentration, inoculum volume, yeast extract, tryptone, sodium acetate, and ammonium acetate concentration was conducted by a Plackett–Burman (PB) design.

Results

The results showed that four variables including, initial pH (P < 0.001), WMP concentration (P < 0.001), sodium acetate (P = 0.023), and ammonium acetate (P = 0.048) had statistically significant effects on hydrogen production. The model curvature (P = 0.040) indicated that it was significant. Box–Behnken (BB) design under response surface methodology (RSM) was employed to optimize the four selected variables to maximize hydrogen production. The optimal conditions for maximizing hydrogen production from WMP by C. butyricum were: initial pH of 8.98, WMP concentration of 44.75%, sodium acetate 4.49 gL⁻¹, and ammonium acetate 1.15 gL⁻¹ at with predicted H_max of 4703.23 mLL⁻¹. The determination coefficient R² of the model was 0.9902 with the lack of fit F-value was 1.86.

Conclusions

The confirmation experiment revealed only a 0.59% difference between the predicted and experimental hydrogen production, indicating that the optimum conditions were actual with the least error. Improvement of about 103.25% in hydrogen production from WMP by C. butyricum NE133 was achieved after the optimization process.

Graphical Abstract

Paddy straw (PS), a by-product of rice production, has a large volume, low economic value, and environmental impact due to burning, contributing to pollution and health hazards. This manuscript highlights the combined effect of acid treatments and enzymatic hydrolysis of paddy straw to produce fermentable hydrolysate, a potential biofuel. This study uses response surface methodology (RSM) with a Box–Behnken design to optimize process parameters (acid concentration, temperature, and duration of hydrolysis), thereby improving the efficiency of converting paddy straw into fermentable sugars. The efficacy of pretreatment was evaluated based on cellulose content and lignin reduction. The optimal conditions of 1% H₂SO₄, 80 °C, and 20 min resulted in effective cellulose enrichment (95.4%) and lignin reduction (38.2%), promoting efficient enzymatic hydrolysis. The enzymatic hydrolysis used cellulase from Trichoderma reesei, yielding high glucose concentrations of 225.2 mg glucose ml⁻¹ g⁻¹ paddy straw. Using Brunauer–Emmett–Teller (BET) analysis and morphology of pretreated and raw PS samples, the surface modification was validated for the optimized hydrolysis conditions. Surface area and pore volume for optimized condition decreased by 58.6% and 25% respectively. However, mean pore diameter increased by 87.9%. Herein, this study offers a more efficient, optimized, and sustainable pathway for converting paddy straw into biofuel using cellulase, with broader implications for agricultural waste management and renewable energy production.

Graphical Abstract

Background

Microbial lipid extraction is a critical process in the production of biofuels and other valuable chemicals from oleaginous microorganisms. The process involves the separation of lipids from microbial cells. Given the complexity of microbial cell walls and the demand for efficient and environmentally friendly extraction methods, further research is still needed in this area. This study aims to pursue the extraction of intracellular lipids from oleaginous yeasts using inexpensive solvents, without disrupting the cells and even maintaining a certain level of cell viability.

Results

The study used fresh fermentation broth of Rhodotorula toruloides as the lipid extraction target and employed a binary solvent of methyl tert-butyl ether (MTBE) and n-hexane for lipid extraction. The effects of extraction time and solvent ratio on cell viability, lipid extraction efficiency, and fatty acid composition were analyzed. Conditions that balanced lipid yield and cell survival were selected for lipid extraction.

Specifically, using a binary solvent (with 40% MTBE) to extract an equal volume of R. toruloides fermentation broth achieved a total lipid extraction rate of 60%, while maintaining a 5% cell survival rate (the surviving cells served as the seed for the second round of lipid production). After separating the solvent phase and supplementing the lipid-extracted cells with carbon sources and a small amount of nitrogen sources, the cells gradually regained biomass and produced lipids. Repeating this "gentle" extraction on surviving and regrown cells and adding carbon and nitrogen sources can enable a second round of growth and lipid production in these cells.

Conclusions

This is an interesting finding that may potentially encompass the extraction mechanisms of polar/nonpolar solvents and the phenomenon of yeast autophagy. This method does not require the destruction of the cell wall of oleaginous yeast. The separation after extraction is simple, and both the cells and solvents can be recycled. It provides a possible approach for simultaneous fermentation and lipid extraction.

ABRE BINDING FACTOR 4 (ABF4) is a pivotal regulatory gene in the abscisic acid (ABA) signaling pathway, and changes in its expression levels can modulate the plant's stress resistance. To further explore the specific regulatory mechanisms of alternative splicing (AS) in the ABA signaling pathway and to identify new breakthroughs for breeding high stress-resistant varieties of Brassica napus, we identified 17 homologous genes of ABF4 in the genome. Utilizing bioinformatics techniques, we analyzed their motifs, conserved domains, and cis-acting elements of their promoters. Through transcriptome data from the stress-tolerant dwarf strain ndf2 and its parental line 3529, we uncovered a significantly differentially expressed ABF4 gene, which we named BnABF4L. Subsequently, we analyzed the AS events of BnABF4L under normal growth conditions and different abiotic stresses, as well as the impact of different transcript variants' 5’ untranslated region (5'UTR) on gene translation. BnABF4L undergoes alternative 3' splice site (A3SS) selection to produce three transcripts (V1-V3) with divergent 5'UTRs. While V1 translation is suppressed by upstream ORFs (uORFs), V2/V3 exhibit enhanced translational efficiency. Under stress, ndf2 shifts splicing toward V3, circumventing uORF-mediated repression to upregulate stress-adapted isoforms. We validated the inhibitory effect of upstream open reading frames (uORFs) on protein-coding open reading frame (pORFs) and, based on the collective experimental results, proposed the flexible regulatory mechanism of AS events of BnABF4L in response to stress. Our findings provide new insights for future studies on stress resistance in rapeseed as well as for research on the regulation of alternative splicing mechanisms in the ABA signaling pathway.

Background

High temperature and ethanol are two critical stress factors that significantly challenge bioethanol production using Saccharomyces cerevisiae. In this study, the tolerance mechanisms of the multi-tolerant S. cerevisiae strain E-158 to heat stress and combined heat-ethanol stress were investigated using comparative transcriptomics.

Results

Under heat stress at 44 °C, glucose transport and reactive oxygen species (ROS) scavenging were significantly upregulated, while gluconeogenesis, acetate formation, and dNDP formation showed significant downregulation. Under combined heat (43 °C) and ethanol (3% v/v) stress, glucose transport, glycolysis, acetate formation, peroxisome activity, ROS scavenging, and ribosome synthesis were significantly upregulated, while glycerol formation, cellular respiration and dNDP formation exhibited significant downregulation. Fourteen transcription factors (TFs), considered to play a key role in both stress conditions, were individually overexpressed and deleted in S. cerevisiae strain KF-7 in this study. Among these TFs, Gis1p, Crz1p, Tos8p, Yap1p, Dal80p, Uga3p, Mig1p, and Opi1p were found to contribute to enhanced heat tolerance in S. cerevisiae. Compared with KF-7, strains overexpressing DAL80 and CRZ1 demonstrated markedly improved fermentation performance under stress conditions. Under heat stress at 44 °C, glucose consumption increased by 10% and 12%, respectively, for strains KF7DAL80 and KF7CRZ1, while ethanol production increased by 12% and 15%, respectively, compared to KF-7. Under combined stress conditions of 43 °C and 3% (v/v) ethanol, glucose consumption increased by 67% and 44%, ethanol production by 116% and 77%, and ethanol yield by 29% and 22%, respectively, for KF7DAL80 and KF7CRZ1 compared to KF-7. KF7CRZ1 performs comparably to E-158, while KF7DAL80 outperforms E-158.

Conclusions

This study provides valuable theoretical insights and identifies critical TF targets, contributing to the development of robust S. cerevisiae strains for improved bioethanol production.

Background

Due to its ability to grow fast on CO₂, CO and H₂ at high temperatures and with high energy efficiency, the thermophilic acetogen Thermoanaerobacter kivui could become an attractive host for industrial biotechnology. In a circular carbon economy, diversification and upgrading of C1 platform feedstocks into value-added products (e. g., ethanol, acetone and isopropanol) could become crucial. To that end, genetic and bioprocess engineering tools are required to facilitate the development of bioproduction scenarios. Currently, the genome editing tools available for T. kivui present some limitations in speed and efficiency, thus restricting the development of a powerful strain chassis for industrial applications.

Results

In this study, we developed the versatile genome editing tool Hi-TARGET, based on the endogenous CRISPR Type I-B system of T. kivui. Hi-TARGET demonstrated 100% efficiency for gene knock-out (from both purified plasmid and cloning mixture) and knock-in, and 49% efficiency for creating point mutations. Furthermore, we optimized the transformation and plating protocol and increased transformation efficiency by 245-fold to 1.96 × 10⁴ ± 8.7 × 10³ CFU μg⁻¹. Subsequently, Hi-TARGET was used to demonstrate gene knock-outs (pyrE, rexA, hrcA), a knock-in (ldh::pFAST), a single nucleotide mutation corresponding to PolC^C629Y, and knock-down of the fluorescent protein pFAST. Analysis of the ∆rexA deletion mutant created with Hi-TARGET revealed that the transcriptional repressor rexA is likely involved in the regulation of the expression of lactate dehydrogenase (ldh). Following genome engineering, an optimized curing procedure for edited strains was devised. In total, the time required from DNA to a clean, edited strain is 12 days, rendering Hi-TARGET a fast, robust and complete method for engineering T. kivui.

Conclusions

The CRISPR-based genome editing tool Hi-TARGET developed for T. kivui can be used for scarless deletion, insertion, point mutation and gene knock-down, thus fast-tracking the generation of industrially-relevant strains for the production of carbon-negative chemicals and fuels as well as facilitating studies of acetogen metabolism and physiology.

Microalgae have received a lot of interest as a sustainable solution for carbon dioxide fixation due to their great efficiency in capturing CO₂ and converting it into valuable biomass, making them a promising tool for mitigating climate change and expanding carbon capture technology. This study examines the efficacy of fixed shaped portable conical helix baffles (PCHB) in enhancing gas–liquid mixing to promote microalgal growth in column photobioreactors (PBRs). Flat (90° angle from cone surface), round, and inclined (60° angle from cone surface) baffles were compared for performance. Modeling the gas flow indicated that round PCHB produced more spiral vortices and achieved better mixing performance than flat and inclined designs. Increasing the baffle size from 3 to 7 cm resulted in a 21% higher mass transfer coefficient. The simulation was verified by experiments. Notably, the implementation of a PCHB with a round helix-shaped structure (5 cm) led to a 33% (2.102 ± 0.08 g/L) and 17% (2.419 ± 0.07 g/L) dry mass increase of Limnospira fusiformis when compared to flat and incline-shaped baffles, respectively. Our study revealed that using a round-shaped PCHB resulted to higher spiral movement, which in turn increases CO₂ utilization and cell proliferation. Our approach demonstrates high potential to further optimize industrial PBRs, thereby facilitating CO₂ sequestration during microalgal cultivation to combat global warming.

Background

The pulp and paper industry is under increasing pressure to adopt sustainable solutions that address its substantial energy consumption and environmental impact. One of the most energy-intensive operations is the thermal drying, which presents significant opportunities for efficiency improvements. This study evaluates a cell-free mild enzyme pretreatment, utilizing a cocktail of cellulases and xylanases, combined with cationic starch, to enhance dewatering efficiency and improve paper strength utilizing bleached hardwood pulp fibers. Life cycle and economic analysis were also conducted to quantify the environmental impact and economic benefits, with a particular focus on direct greenhouse gas emissions. Enhanced water removal during pressing can significantly reduce energy consumption during thermal drying, facilitating the decarbonization of the paper industry.

Results

The cell-free enzyme pretreatment, applied with mild refining and cationic starch, achieved significant improvements in dewatering efficiency and paper strength. The treatment led to an 11% point increase in solids and a 25% improvement in tensile strength. Morphological analyses revealed no changes in fiber length and width; however, reductions in kink and curl indexes indicated enhanced fiber flexibility and bonding potential. Furthermore, the enzyme–starch combination decreased water retention value by 27%, including substantial reductions in bound and hard-to-remove water content. Environmental assessments estimated a 12% reduction in global warming potential (GWP), with the technology yielding net savings of $11.29 per air-dried ton of paper through reduced natural gas consumption.

Conclusions

This study demonstrates the technical feasibility and economic viability of incorporating enzyme and cationic starch treatments into papermaking. The treatment improves paper quality while reducing energy consumption, costs, and carbon emissions. These findings support the broader adoption of enzyme-based innovations for sustainable manufacturing, aligning with decarbonization goals and industry demands for greater efficiency. The results highlight a promising avenue for achieving significant environmental and economic benefits in the pulp and paper sector.

Graphical Abstract

Lignocellulosic biomass offers a vast and renewable resource for biofuel production and carbon management solutions. The effective conversion of lignocellulosic biomass into economically competitive biofuels and bioproducts demands a comprehensive understanding of its complex structure and composition, often requiring a range of analytical tools to achieve meaningful insights. However, for the analysis of rigid solids, many traditional methods necessitate dissolution or chemical/physical modification of the sample, which limit our ability to capture an intact view of its structural components. This highlights the need for non-destructive approaches, such as solid-state nuclear magnetic resonance (ssNMR), which preserves the sample’s natural state while providing deep, molecular-level insights. While advanced multi-dimensional ssNMR on ¹³C-enriched materials has recently proven exceptionally valuable for elucidating the complex macrostructure of biomass, isotopic enrichment is expensive, laborious and is clearly infeasible at large scales. In this review, we explore the role of solid-state NMR methods at natural isotopic abundance as essential tools for the non-destructive, in-depth characterization of lignocellulosic biomass and bioenergy materials in their native and unaltered state. After a brief introduction to the basic principles of solid-state NMR, we first describe the acquisition and interpretation of routine 1D ¹³C ssNMR spectra of lignocellulose and other related biopolymers and products. We then delve into more advanced ssNMR approaches, including key spectral editing techniques, probing polymer dynamics, and various 2D methods applicable at natural abundance. Understanding of domain miscibility as observed from proton-based spin diffusion effects is a theme throughout. Our aim is to highlight key examples where ssNMR provides valuable insights into the composition, structure, dynamics, and morphology of rigid biomaterials relevant to the bioenergy economy, revealing both the native structures and fundamental transformations that occur across conversion and decomposition pathways. We hope that this review encourages a broader adoption of ssNMR methods in bioenergy research, where it can serve as a pivotal analytical tool for achieving sustainable biomass utilization and advancing a carbon-efficient bioeconomy.

Background

Syntrophic propionate- and acetate-oxidising bacteria (SPOB and SAOB) play a crucial role in biogas production, particularly under high ammonia conditions that are common in anaerobic degradation of protein-rich waste streams. These bacteria rely on close interactions with hydrogenotrophic methanogens to facilitate interspecies electron transfer and maintain thermodynamic feasibility. However, the impact of mixing-induced disruption of these essential syntrophic interactions in biogas systems remains largely unexplored. This study investigates how magnetic stirring and orbital shaking influence degradation dynamics, microbial community composition, and gene expression in syntrophic enrichment communities under high-ammonia conditions.

Results

Stirring significantly delayed the initiation of propionate degradation in one culture and completely inhibited it in the other two parallel cultures, whereas acetate degradation was less affected. Computational fluid dynamics modelling revealed that stirring generated higher shear rates (~ 20 s⁻¹) and uniform cell distribution, while shaking led to lower shear rates and cell accumulation at the bottom of the culture bottle. Visual observations confirmed that stirring inhibited floc formation, while shaking promoted larger flocs compared to the static control condition, which formed smaller flocs and a sheet-like biofilm. Microbial community analysis identified substrate type and degradation progress as primary drivers of community structure, with motion displaying minimal influence. However, metatranscriptomic analysis revealed that motion-induced gene downregulation was associated with motility, surface sensing, and biofilm formation in SAOB and another bacterial species expressing genes for the glycine synthase reductase pathway. Stirring also suppressed oxalate–formate antiporter expression in SPOB, suggesting its dependence on spatial proximity for this energy-conserving mechanism. The strongest gene expression changes of stirring were observed in methanogens, indicating a coupling of the first and last steps of hydrogenotrophic methanogenesis, likely an adaptive strategy for efficient energy conservation. Other downregulated genes included ferrous iron transporters and electron transfer-associated enzymes.

Conclusions

This study highlights that stirring critically disrupts the initial syntrophic connection between SPOB and methanogens, whereas SAOB communities exhibit greater tolerance to shear stress and disruptive conditions that inhibits aggregate formation. These findings emphasize the importance of carefully managing mixing regimes, especially when attempting to reactivate ammonia-tolerant syntrophic propionate degraders in biogas systems experiencing rapid propionate accumulation under high-ammonia conditions.

Graphical abstract