Biotechnology for biofuels and bioproducts最新文献

Background: Anaerobic microbes produce multienzyme complexes known as cellulosomes to enhance the degradation of cellulosic substrates. These complexes localize diverse enzymes onto a protein scaffold, where proteins are anchored by dockerin domains. Although the cellulosomes of anaerobic fungi incorporate a broad array of cellulolytic enzymes, they remain largely unexplored. Notably, some fungal cellulosomes reportedly comprise expansin-like proteins with potential to disrupt cellulose networks. While two bacterial cellulosomal expansin-like proteins have been characterized, no fungal cellulosomal expansin-like proteins have been functionally characterized to date.

Results: Sequence analyses of expansin-like proteins from the anaerobic fungus Neocallimastix californiae revealed similar N-terminal domains among proteins with or without appended dockerins. Those without dockerins, however, consistently lacked the first conserved aromatic residue that forms the substrate binding surface of the C-terminal family 63 carbohydrate binding module. One cellulosomal expansin-like protein from N. californiae (NcaEXLX1) was recombinantly expressed with and without (NcaEXLX1tr) the dockerin domains. The adsorption characteristics of NcaEXLX1 and NcaEXLX1tr, and impact on cellulase (Cel7B) activity, were then investigated using quartz crystal microbalance with dissipation (QCM-D). NcaEXLX1 exhibited higher binding to cellulose nanofibrils (CNF) compared to NcaEXLX1tr. Despite the lower binding of NcaEXLX1tr to CNF, both NcaEXLX1 and NcaEXLX1tr enhanced the action of Cel7B to similar extents.

Conclusions: This study reports the production and characterization of a fungal cellulosomal expansin-like protein. The corresponding NcaEXLX1 protein and truncated variant were shown to enhance the activity of an endoglucanase, similar to observations made with non-cellulosomal expansin-like proteins. Notably, the improvement in cellulase activity upon the addition of NcaEXLX1 or NcaEXLX1tr was not correlated to extent of substrate binding.

Background: The growing demand for sustainable alternatives to fossil-based chemicals has increased interest in platform chemicals derived from renewable biomass sources, such as malic acid. This C4 dicarboxylic acid is valued for its diverse application potential in food, pharmaceuticals, and bioplastics. Sustainable platform chemicals remain commercially uncompetitive primarily due to high production costs driven by high substrate costs. Microbial production using more cost-effective feedstocks like sugar beet molasses shows promise. However, it faces challenges from high osmolality, growth inhibitors, and predetermined substrate composition during fermentation, as well as elevated pigmentation that complicates downstream processing. Moreover, the separation techniques typically used for highly polar carboxylic acids face considerable yield limitations due to the high solubility of malic acid and its salts.

Results: This study developed an all-encompassing production process for malic acid from untreated sugar beet molasses. Fermentative malic acid production with Ustilago trichophora was investigated in batch, fed-batch, and pulsed batch in shake flask scale, followed by a scale-up into 150 L pilot scale. A total of 15.7 kg malic acid was produced in a repeated pulsed batch with membrane-based cell retention with a titer of 108 g/L, a yield of 0.50 g/g, and a space-time yield of 0.66 g/L/h (max. 1.1 g/L/h). In addition, the byproduct succinic acid was detected in concentrations of up to 22.9 g/L. In the subsequent downstream processing, activated carbons were used for two-stage product capture, solvent change, and decolorization, followed by crystallization of the products malic acid and succinic acid. Based on experimental results, an Aspen Plus model was developed to estimate the overall process yields of 0.43 g malic acid (98% purity) and 0.10 g succinic acid per gram sucrose equivalent. A techno-economic analysis suggests production costs within the range of current market prices.

Conclusion: Agricultural residue streams are often proposed as cost-effective alternatives for fermentative platform chemical production, although the challenges addressed hamper the direct transfer of process strategies from established organic acid production. By presenting a holistic approach explicitly tailored to malic acid production from untreated molasses, this work demonstrates the techno-economic feasibility of the developed process at a meaningful scale.

Bioaugmentation, the intentional introduction of specific microorganisms into anaerobic digestion (AD) systems, has shown promise in enhancing methane production and in mitigating stressful conditions, particularly in systems operating below optimal performance. This review presents a systematic literature review (SLR) of research on bioaugmentation in AD. This review identified and analysed studies meeting predefined eligibility criteria through a structured methodology involving research protocol, search, appraisal, synthesis, analysis, and reporting. A notable innovation of this review is its comprehensive critical comparison of different controls used in bioaugmentation studies, which has been inadequately addressed in previous literature. To facilitate the functional understanding, strains for bioaugmentation were grouped into the four phases of anaerobic digestion (hydrolysis, acidogenesis, acetogenesis and methanogenesis). A highly diverse set of microbes has been described for bioaugmentation, especially from the families Clostridiaceae, Pseudomonadaceae and Syntrophomonadaceae. Most works are related to hydrolysis. The few works that address acidogenesis are mostly related to dark fermentation. Several studies used methanogenic archaea as well as syntrophic acetate oxidising bacteria, despite the difficulties in culturing them. On the other hand, studies applying strains for acetogenesis were largely underrepresented. Especially works on syntrophic propionate and butyrate oxidation (SPO and SBO) were missing.

The adoption of electric vehicles (EVs) in rural areas is constrained by limited charging infrastructure, while small-scale dairy farms remain significant sources of greenhouse gas (GHG) emissions from manure management. This study addresses both challenges by demonstrating a dispatchable renewable electricity generation system that integrates anaerobic digestion (AD) with an external-combustion, Stirling-type combined heat and power (CHP) unit to support rural EV charging. A trailer-based system incorporating a 2.25 m³ AD reactor and a 5.6 kW CHP unit was constructed and operated using 28 kg/day of dairy manure. Results from a 50-day demonstration informed scaling and modeling for a representative 30-cow dairy farm. The AD process achieved a methane productivity of 232 L/kg volatile solids, with electrical and thermal conversion efficiencies of 15.05% and 37.11%, respectively. When scaled to a 30-cow farm, the system produced 46.46 kWh/day of net electricity and 10.43 kWh/day of recoverable heat, sufficient to meet realistic rural EV charging demands. Techno-economic analysis estimated a total capital investment of $99,000, annual operating costs of $2,000, and combined energy revenues of $7,797/year, corresponding to a 28-year payback period. Sensitivity analysis indicated that system economics are most strongly influenced by revenue generation. Life cycle assessment showed substantial environmental benefits relative to conventional manure management, including annual reductions of 324.44 tons CO₂-eq in global warming potential. Compared with stand-alone photovoltaic EV charging systems that require large battery storage to ensure winter reliability in northern climates, the biogas-based system leverages biogas storage as a low-cost energy buffer, enabling on-demand electricity generation with lower capital intensity. Overall, this work demonstrates a compact and scalable pathway for integrating manure-derived biogas with EV charging to advance rural electrification and circular bioeconomy goals.

Background: Methylococcus species utilize methane as the sole carbon and energy source, converting it into biomass and other metabolic end products. Owing to this metabolic capacity, they hold particular promise in industrial C1 biotechnology, especially for the production of protein-rich feed. However, the industrial cultivation of Methylococcus-based consortia on methane is inherently nonsterile, exposing the process to potential biological risks that may compromise the stability, duration and productivity of cultivation. One of the most critical threats is bacteriophage infection, whose triggers for rapid phage-mediated lysis and resulting economic losses remain incompletely understood. Elucidating these processes is paramount for devising strategies to mitigate or prevent detrimental outcomes.

Results: In this investigation, nine publicly accessible genomes of Methylococcus species were examined, culminating in the identification of eleven prophage sequences distributed variably among the genomes. Sequence annotations revealed that nine prophages are potentially functional and intact, whereas the rest carry incomplete gene sets indicative of nonviability. Phylogenetic analyses corroborated the substantial diversity of prophages, which formed distinct clusters related to γ-proteobacteria phages. Furthermore, comparative genomic analyses demonstrated a high degree of structural conservation despite the presence of rearrangements. The annotation of the CRISPR‒Cas systems provided insights into additional dimensions of phage‒bacteria interactions. Examination of prophage integration sites did not reveal any disruption of metabolic gene structures, thus suggesting minimal risk of deleterious phenotypic outcomes.

Conclusions: These findings considerably advance the current understanding of the genetic diversity and biological properties of prophages infecting Methylococcus species, underscoring the importance of holistic approaches for the detection and analysis of these elements. Our findings underscore the need for routine prophage monitoring in industrial methanotrophic consortia, with the pipeline established here serving as a foundational framework for future refinement and industrial adaptation.

Background: Artificial microbial consortium has been widely employed to improve the production of fengycin, a natural lipopeptide. Kinetic models are essential for understanding and predicting the dynamic behavior of metabolic systems, especially microbial consortia. Since the time evolution of metabolite concentrations and biomass is continuous and dynamic, systems of ordinary differential equations (ODEs) provide a natural and effective framework for capturing such interactions. In this work, a kinetic model based on ODEs was established to describe a multi-strain artificial consortium for fengycin synthesis, utilizing Bacillus subtilis, Yarrowia lipolytica, and Corynebacterium glutamicum as target strains.

Results: The model captures microbial growth, intermediate metabolite formation, final product synthesis, and substrate consumption. It was successfully applied to analyze and interpret the cultivation data of target strains on various substrates. The model explicitly incorporates the accumulation of amino acids synthesized by C. glutamicum, the accumulation of fatty acids synthesized by Y. lipolytica, and the process in which B. subtilis utilized amino acids and fatty acids as partial precursors for fengycin production. The mathematical model ended up as a nonlinear ordinary differential system, which was solved with an adaptive step-size Runge-Kutta method, coupled with a genetic algorithm to roughly estimate the optimal model parameters associated with cellular growth, substrate consumption, and product level in fermentation broths.

Conclusions: The numerical results of the kinetic model agreed well with experimental data, and all seven sets of experimental conditions were fitted with overall relative errors ranging from 7.4 to 15.1%. This kinetic modeling provided a meaningful tool for the rational design and construction of further artificial consortia.

Background: Efficient degradation of cellulose is a key bottleneck in the industrialization of biofuels. While fungi achieve substrate conversion through precise regulation of cellulase systems, the systematic mechanisms underlying efficient degradation (encompassing gene transcription, extracellular protein cooperation, and product metabolism) remain unclear in specific fungi, especially thermophilic fungi critical for industrial production.

Results: (1) C. thermophilum did not induce cellulases under cellobiose, while microcrystalline cellulose (MCC) strongly activated degradation. CtClr-2 acts as a core transcription factor, directly driving the co-expression of key genes including LPMOs, CDH, and CBH; its deletion reduces MCC degradation efficiency by 30%. (2) Enzyme secretion may follow a three-stage cascade pattern (CBH1-A → LPMOs/CDH-1 → CBH1/2-B), where the selective secretion and temporal synergy of oxidases and hydrolase increase the reducing sugar yield by 60.6%. (3) The sugar acid metabolic network may enable efficient utilization of degradation products and potentially help maintain extracellular pH.

Conclusions: This study reveals the efficient "transcriptional regulation-enzyme secretion adaptation" synergistic mechanism in C. thermophilum. CtClr-2 coordinates key genes, and staged enzyme secretion optimizes synergy, while sugar acid metabolism ensures homeostasis. These insights advance thermophilic cellulolysis understanding and provide targets for engineering industrial strains through synthetic biology (for example, enhancing enzyme yield and optimizing degradation efficiency), aiding cost reduction in biofuel production.

Lignocellulosic biomass is an abundant renewable carbon source for biofuel production, but its conversion to fermentable sugars is hindered by poor cellulase activity on highly crystalline and insoluble cellulose. While pretreatment makes biomass more amenable to enzymatic degradation, several issues linger related to productive enzyme binding and efficient catalytic turnover. To address this bottleneck, we employed protein supercharging to rationally design a glycosyl hydrolase (GH) family-6 exocellulase (Cel6B) and its native family-2a carbohydrate binding module (CBM2a) from the thermophilic cellulolytic microbe Thermobifida fusca. A chimeric library of 32 supercharged constructs rationally designed across both GH/CBM domains was synthesized and expressed in E. coli. Screening of the entire library of supercharged enzymes on several cellulosic substrates identified one key construct, D5 CBM2a-WT Cel6B, containing a positively supercharged CBM2a that showed 2-threefold higher activity on all substrates tested at pH 5.5. Purified enzyme assays confirmed that exocellulases behave quite differently from their endocellulase counterparts when supercharged using similar protocols. Still, the purified D5 CBM2a-WT Cel6B mutant showed a 2.3-fold improvement in specific activity compared to native enzyme on crystalline cellulose. Analysis of melt curves depicts that, while all other constructs tested have one distinct melt peak near the expected CBM melting point, domain melting is decoupled for the D5 CBM2a mutant. This effect reveals an intrinsic melting temperature of the Cel6B CD nearly 18 °C higher than the coupled melting temperature of the full-length enzyme. This unexpected stabilization effect of supercharged CBM2a domain is likely the driving force for activity improvements seen for this exocellulase that is otherwise prone to stalling and denaturation on the cellulose surface during processive catalytic turnover cycles. When combining this supercharged exocellulase construct with its endocellulase counterpart, our results showed that supercharged enzymes, exhibiting the highest activity alone, produced the best synergistic partners. This study highlights another successful implementation of protein supercharging for cellulases and provides another key piece toward building an effective synergistic cellulase cocktail for lignocellulosic biomass deconstruction.

Arabinoxylans are complex polysaccharides found in the secondary cell walls of plants, and their substitution patterns of arabinose (Ara) and glucuronic acid (GlcA) significantly affect their properties for structural and bio-functional applications. In this study, we engineered xylan side chains by overexpressing the arabinosyltransferase AtXAT2 in both wild-type Arabidopsis and a glucuronic acid-deficient mutant (atdgux). The genetically modified xylan substrates were subsequently utilized to prepare nanocrystals and Pickering emulsions. Notably, nanocrystals derived from lower-substituted xylan displayed more ordered arrangements and higher crystallinity, while conventional xylan substrates exhibited superior emulsifying properties. Furthermore, the modification of xylan side chains significantly influenced the alignment and crystallinity of cellulose microfibrils, enhancing biomass saccharification and reducing cellulose nanocrystal dimensions. This study thus illustrates an effective strategy for achieving diverse and valuable bioproduction through precise genetic engineering of xylan in plants and provides valuable insights into the dynamic mediation of xylan and the construction of cell wall networks.

Glucoamylases capable of hydrolyzing raw starch at moderate temperatures exhibit significant potential for the direct and efficient hydrolysis of raw starch. Using bioinformatics-assisted mining strategies, four candidate glucoamylases were initially identified from the GenBank database. Among these candidates, SeGA demonstrated high specific activities of 412.94 U/mg toward soluble starch and 143.51 U/mg toward raw corn starch. The half-life of SeGA was 192 h at 40 °C. Using SeGA as the initial enzyme, FuncLib design was performed to enhance its activity. The mutant SeGA-21 exhibited higher specific activity of 466.22 U/mg toward soluble starch and 173.98 U/mg toward raw corn starch, representing increases of 1.13-fold and 1.21-fold compared to SeGA, respectively. The half-life of SeGA-21 at 40 °C increased to 240 h, representing a 25% increase compared with SeGA. SeGA-21 can efficiently hydrolyze 30% raw corn starch, achieving a hydrolysis rate of approximately 31% after a 9-h reaction. When working synergistically with amylolytic enzymes, a hydrolysis rate of 71.5% was achieved for 30% raw corn starch at 40 °C within 15 h of reaction. Due to its ability to hydrolyze raw starch, moderate reaction temperature requirements, and excellent thermostability, SeGA-21 can be regarded as an efficient enzyme for the cold hydrolysis of raw starch and starch modification.