Efficient and sustainable pretreatment of lignocellulosic biomass is critical for biofuel and biochemical production, yet its optimization is often hindered by slow, labor-intensive experimental methods. Here, we report the first demonstration of a custom-built, miniaturized, high-throughput screening platform integrated with one-pot enzymatic saccharification, enabling parallel evaluation of solvent type, feedstock, and temperature with minimal material use and high reproducibility. As a proof-of-concept, the HTX platform was used to screen five amine-functionalized solvents, including isopropanolamine, butylamine, N-methylbutylamine, ethanolamine, and ethanolamine acetate across three bioenergy crops (sorghum, poplar, and switchgrass) and pretreatment temperatures ranging from 80 to 140 °C. Vacuum drying successfully removed more than 99% of the solvents from the pretreated biomass, eliminating the need for water washing prior to saccharification. Isopropanolamine and N-methylbutylamine yielded the highest glucose (70-80%) and xylose (58-67%) release, with trends reflecting feedstock recalcitrance. The produced hydrolysates supported robust growth of an engineered strain of the yeast Rhodosporidium toruloides, confirming biocompatibility. This high-throughput platform provides a scalable, feedstock-agnostic framework for rapid pretreatment screening, accelerating solvent-feedstock pairing and process optimization. Its ability to integrate pretreatment, solvent removal, saccharification, and microbial conversion in a miniaturized format offers significant advantages for cost-competitive biorefinery development.

A metallic-electron donation system (MDS) was developed that supplies intracellular reducing equivalents via zero-valent iron enclosed in a retrievable "tea-bag", coupled with active pH readjustment, to relieve redox and cost constraints in glycerol-to-1,3-propanediol fermentation. This approach provided consistent and stable intracellular reducing equivalents, significantly enhancing the PDO yield from 0.38 +/- 0.01 to 0.77 +/- 0.01 mol/mol (PDO produced per glycerol consumed). The robustness and scalability of the developed MDS approach were then validated by successfully scaling up PDO production to a 5 L bioreactor, which demonstrated excellent operational stability and reproducibility. Preliminary techno-economic analysis indicated a minimum selling price (MSP) of similar to 0.65 USD kg(-1) under optimized batch conditions (pH 7.5 with ZVI and pH readjustment) and similar to 0.61 USD kg(-1) for a continuous surrogate parameterized from the 5 L run (with a target internal rate of return of 10 %), highlighting the economic potential of the proposed MDS approach. Compared to conventional electro-fermentation methods, the MDS simplifies operational complexity, significantly reduces capital and operational expenditures, and can be retrofitted into conventional stirred-tank bioreactors with minimal hardware changes, adding a new operational axis-direct redox control-without electrodes or external power. Oxidized ZVI can be reduced and recycled, improving the cost-effectiveness and sustainability of the proposed MDS with minimal process complexity. Collectively, the results indicate that the proposed MDS is a robustly scalable bioprocessing platform for efficient and economically viable microbial fermentation. By targeting glycerol-a common biodiesel by-product-and grounding feasibility through techno-economic analysis, the MDS outlines a practical circular-bioeconomy route to PDO that is readily retrofittable into existing assets.

Benzylamines are key intermediates in pharmaceuticals, agrochemicals, and polymers, but their conventional production relies on benzyl chloride - a petroleum-derived compound with high toxicity and energy demands. Lignin, accounting for up to 30% of plant biomass, is the largest renewable source of aromatic carbon on Earth. However, its highly complex and recalcitrant structure poses a major barrier to efficient conversion into high-value chemicals. Here, we developed a catalytic approach to convert commercial kraft lignin into phenolic benzylamines through selective depolymerization and subsequent functionalization. We systematically evaluated the effects of three alcohol solvents, formic acid (FA), and a ruthenium-on-carbon (Ru/C) catalyst on monophenol yield and selectivity. Up to 6.5 wt% monophenol yield was achieved using methanol (MeOH), FA, and Ru/C at 300 degrees C for 2 h. Quantum thermodynamic simulations based on the COSMO-RS model confirmed the superior solvation and reactivity of the MeOH + FA system, rationalizing observed product yield. The purified monophenolic products, primarily guaiacol and alkyl guaiacols, were then converted into functionalized benzylamines with >90% yield via a multicomponent Mannich reaction under mild conditions. Techno economic analysis (TEA) and life cycle assessment (LCA) underscore the importance of improving lignin depolymerization yields and expanding biorefinery scale. Solvent-only configurations outperform other options in both cost and emissions, with the methanol-only case performing the best ($105 /kg and 26 kg CO2e/kg) at a large-scale facility. This study establishes a scalable, bio-based pathway for producing benzylamines from commercial kraft lignin, advancing lignin valorization and offering a sustainable alternative to produce petrochemical-based benzylamines.

Textile waste, dominated by polyester-cotton blends, largely evades recycling and ends up in landfills or incinerators. Here, we demonstrate an aqueous ionic liquid–mediated route for selective depolymerization of textile waste, including polyester and polycotton (polyester/cotton blend). Using aqueous cholinium lysinate ([Ch][Lys]), we depolymerized poly(ethylene terephthalate) (PET) textiles with over 95 % terephthalic acid (TPA) yields, comparable to virgin PET resin. Notably, colored fabrics showed no inhibitory effect from dyes. While protic ionic liquids such as ethanolammonium acetate showed limited efficiency, their precursor amine enabled near-complete PET conversion—ethanolamine promoted aminolysis with byproduct formation whereas butylamine achieved higher TPA recovery through combined hydrolysis and aminolysis. Importantly, polycotton blends achieved complete PET depolymerization with TPA yield above 85 % and preserved cotton. These results establish aqueous ionic liquids as efficient, selective agents for deconstructing polyester in blended textiles, offering flexible product and ionic liquid management pathways toward scalable and facile textile recycling.

Exploring the feasibility of applying alkanolamines as biomass pretreatment solvents for the deconstruction of biofuels is useful, owing to their ease of accessibility and their potential to serve as a low energy-intense, cost-effective downstream conversion. For the current study, we have selected a wide range of biomass feedstocks and evaluated biomass deconstruction efficiency using dual-functional solvents, ethanolamine and ethanolammonium acetate, and comparing two different solvent recovery approaches - water washing and solvent evaporation. Pretreatment conditions for both processes included 15% solid loading of 2 mm size ground and homogeneously mixed biomass with pure ethanolamine (EA) and ethanolammonium acetate (EAA) at 140 degrees C for 3 h. For the first solvent removal process, the pretreated biomass was washed until the pH reached 7.0, and for the second solvent removal process, solvent evaporation was performed in a vacuum oven set at 80 degrees C and 140 degrees C for EA and EAA respectively, followed by pH adjustment to 5.0. The next step involved saccharification using Cellic (R) enzymes to liberate glucose and xylose from the pretreated solids. Enzymatic hydrolysis of coconut chips, hay, rice hulls and a pelletized 4-crop blended mix (corn stover, switchgrass, pine, and eucalyptus) revealed significantly higher sugar release through the solvent evaporation route as compared to the washing process as washing led to high solid losses. Through this study, we demonstrated the effective use of alkanolamines as biomass pretreatment solvents relevant to a commercial biorefinery setting, as well as that vacuum-based solvent removal is a better strategy for improved release of fermentable sugars that also enables facile solvent removal.

Lignin plays a vital role in the economics of biorefineries, serving as a source of process energy and a feedstock for sustainable fuels and chemical production. While understanding lignin's chemical composition is crucial, emerging evidence suggests that a more comprehensive understanding of its macromolecular structure is critical to explaining its complex behavior in the biorefinery. This study investigated the collapse of the lignin network in corn stover feedstock after harvest and storage as a result of the microbial digestion of hemicellulose. Fluorescence microscopy was used to detect the collapse of lignin by the changes in lignin's fluorescence lifetime, anisotropy, and the number of effective emitters. Our in situ microscopic results revealed lignin's coil-globule transition phenomena, which was only previously predicted by molecular dynamics modeling of extracted lignin in solvent. This collapse of lignin macromolecular structure was supported by results from NMR, IR, Raman, and powder X-ray diffraction. Our study revealed that the two major approaches for lignin valorization in the lignin-first biorefinery model, namely, monomer extraction and milled wood lignin extraction, were negatively impacted by the lignin collapse. As changes during storage are a source of feedstock variability, our study highlights the importance of understanding the effect of feedstock handling on biorefinery operations and economics.

Transforming agricultural waste into biofuels and bioproducts is crucial to advancing a low-carbon bioeconomy. However, the inherent variability in the composition and quality introduces uncertainties in the conversion efficiency and poses challenges in process development. Through integrating a high-throughput conversion system, material characterization techniques, and advanced data analysis tools, this study investigates the variability of corn stover and its subsequent impacts on carbohydrate conversion. The findings reveal that indoor storage substantially reduces the moisture and ash content and soil contamination, while other properties remain largely unchanged. Self-degradation due to microbial activity during storage decreases the carbohydrate content of corn stover but enhances glucose and xylose yields. A negative correlation is observed between sugar yields and lignin content across samples with varying ash and moisture content. The inhibitory effect of lignin diminishes in self-degraded samples likely due to the disrupted cell wall structure. Although self-degradation slightly increases cellulose crystallinity, no strong correlation was observed between the crystallinity and sugar yield. Hot water pretreatment under mild conditions effectively mitigates inherent variability, consistently improving the sugar yield from corn stover by up to 50%. By elucidating the feedstock variability and its impact on convertibility, these findings offer valuable insights into appropriate feedstock handling and management, highlighting potential strategies to address variability challenges.

Exploring the potential of advanced distillable solvents as efficient biomass pretreatment agents is critical for biorefineries, enhancing fermentable sugar yields while enabling solvent recovery and recycling without suffering significant losses. Here, we employ distillable amine-based solvents for pretreating a wide range of lignocellulosic feedstocks, aiming to facilitate the industrial release of fermentable sugars from diverse feedstocks through enzymatic hydrolysis. Twenty-two diverse feedstocks, sourced from different geographical regions and representing various biomass categories, were surveyed for chemical (mainly carbohydrates and lignin) and lignin (S, G, and H units) profiles. Several solvents, including ethanolamine, ethanolammonium acetate, butylamine, butylammonium acetate, and triethylamine, were tested for the pretreatment of eight selected biomasses. Among these solvents, butylamine emerged as the most effective due to its favorable sugar release, excellent solvent removal rate, and low boiling point, facilitating solvent recovery and recycling. Extending butylamine pretreatment to all 22 feedstocks demonstrated desirable sugar yields and highly efficient solvent removal in the majority of the biomass sources tested. Agricultural residues and their mixtures showed particularly favorable sugar release. Despite minimal changes in cellulose crystallinity, XRD characterization of sorghum, poplar, and pine before and after butylamine pretreatment showed a decrease in intensity and a slight shift of certain peaks, indicating alterations in cellulose structure. Fourier-transform infrared spectroscopy and thermogravimetric analysis analyses suggested disruption of biomass linkages in hemicellulose and lignin, enhancing enzymatic digestibility. Scale-up experiments of the mixed agricultural feedstocks in a 1 L Parr reactor achieved over 90% glucose liberation and more than 99% butylamine removal, highlighting the scalability of the method. The resulting hydrolysates supported the growth of diverse bacterial and fungal strains, indicating downstream compatibility with commercial fermentation processes. This study presents butylamine as an effective, recoverable pretreatment solvent for a wide range of lignocellulosic feedstocks, offering a promising solution to key biorefinery challenges. The demonstrated scalability and compatibility with various biomass types and blends underscore its potential for industrial application, advancing sustainable biofuel and biochemical production.

Biological hydrogen production from lignocellulosic biomass sustainably couples organic waste reduction with renewable energy generation. Efficient conversion is challenged by the structural complexity of lignocellulose and resulting recalcitrance to enzymatic degradation. Clostridium thermocellum natively breaks down biomass with highly effective hemi-/cellulases systems (i.e., cellulosomes) and generates hydrogen in anaerobic cultivation, creating a compelling platform for lignocellulosic biohydrogen production. Achieving commercially viable production rates requires balancing high biomass loading and throughput against uniform mixing conditions required for enzyme dispersion, pH and temperature control, and efficient hydrogen and metabolite removal in continuous operation. To address these barriers to process intensification, we implemented novel reactor and process designs for high-solids lignocellulosic biomass fermentations using the C. thermocellum KJC19-9 strain, genetically engineered for co-utilization of cellulose and hemicellulose sugars (i.e., xylose). Via computational fluid dynamics (CFD) modeling and experimental validation, we achieved a >50% improvement in biohydrogen production with an improved anchor-type impeller morphology, coupled to a threefold reduction in agitation rate. To further reduce rheological constraints and accumulation of toxic metabolites, we then transitioned the process to sequencing fed-batch operation. The resulting process generated 24.87 L H-2 L-1 from 160 g L-1 of deacetylated and mechanically refined (DMR)-pretreated corn stover biomass over 16 days while solubilizing >95% of influent cellulose and hemicellulose, setting a new performance benchmark for continuous production of biohydrogen from lignocellulose.

Transitioning to a bioeconomy that makes use of low-emission and waste feedstocks requires greater flexibility to accommodate seasonal variations and mitigate long-term storage challenges, such as material loss and fire risk. To achieve this goal, biomass deconstruction technologies must efficiently handle diverse feedstocks. Here, we assess the cost of using butylaminea distillable solventto deconstruct 22 different biomass feedstocks: 7 herbaceous, 9 woody, 4 food processing residues, and 2 blends. Lignocellulosic sugar production costs, based on current empirical data, range from $1.3 to 6.1/kg, suggesting that substantial improvements are required to compete with conventional sugars. The high solvent loading (850 g/kg of whole slurry) is a process bottleneck. Lowering the solvent loading to 59 g/kg of whole slurry, demonstrated in an L-scale reactor using poplar biomass, reduces the minimum sugar selling price by 33%. Solvent loading and recovery, solid loading, sugar yield, enzyme use, and delivered biomass cost all play key roles in reaching sugar production costs of $0.45-0.79/kg. Strategic feedstock blending to maximize carbohydrate content, process optimization to improve conversion efficiency, and the selection of low-cost feedstocks are important to advancing feedstock-flexible biorefineries.

Converting polyethylene (PE) into valuable materials, particularly ones that are better for the environment than the incumbent plastics, not only helps mitigate environmental issues caused by plastic waste but also alleviates the long-standing problem of microbial fermentation competing with food supplies. However, the inherent robustness of PE due to its strong carbon-carbon bonds and high molecular weight necessitates harsh decomposition conditions, resulting in diverse decomposition outcomes that present significant challenges for downstream applications, especially for bioconversion. In this study, we demonstrate a hybrid biological-chemical conversion process for PE, converting its decomposition products, namely short-chain diacids, into a monomer, β-keto-δ-lactone (BKDL), for highly recyclable polydiketoenimine plastics using engineered Corynebacterium glutamicum. Since BKDL synthesis requires a substantial supply of malonyl-CoA, we employed an alternative biosynthesis pathway that leverages C. glutamicum's natural proficiency in amino acid production. We optimized this pathway in vivo by minimizing carbon loss to CO2 and byproducts, improving the transporter system, and maximizing co-factor regeneration. Furthermore, we co-optimized the PE deconstruction process to produce predominantly C4 to C6 diacids and integrated three catabolic pathways into the engineered strain to enhance diacid utilization, maximizing the carbon conversion from PE. Finally, an engineered polyketide synthase was introduced into C. glutamicum to enable BKDL synthesis. This work demonstrates the potential of a chemo-biological hybrid strategy for recycling plastic waste, highlighting its promise in addressing environmental challenges and promoting sustainable materials.

Butylamine is an effective in-and-out pretreatment solvent for hybrid poplar. It penetrates the cell walls and breaks ester cross-linkages, facilitating lignin release and improving enzymatic digestion.

Microbial conversion of lignocellulosic biomass represents an alternative route for production of biofuels and bioproducts. While researchers have mostly focused on engineering strains such as Rhodotorula toruloides for better bisabolene production as a sustainable aviation fuel, less is known about the impact of the feedstock heterogeneity on bisabolene production. Critical material attributes like feedstock composition, nutritional content, and inhibitory compounds can all influence bioconversion. Further, the given feedstocks can have a marked influence on selection of suitable pretreatment and hydrolysis technologies, optimizing the fermentation conditions, and possibly even modifying the microorganism's metabolic pathways, to better utilize the available feedstock. This work aimed to examine and understand how variations in corn stover batches, anatomical fractions, and storage conditions impact the efficiency of bisabolene production by R. toruloides. All of these represent different facets of feedstock heterogeneity. Deacetylation, mechanical refining, and enzymatic hydrolysis of these variable feedstocks served as the basis of this research. The resulting hydrolysates were converted to bisabolene via fermentation, a sustainable aviation fuel precursor, using an engineered R. toruloides strain. This study showed that different sources of feedstock heterogeneity can influence microbial growth and product titer in counterintuitive ways, as revealed through global analysis of protein expression. The maximum bisabolene produced by R. toruloides was on the stalk fraction of corn stover hydrolysate (8.89 +/- 0.47 g/L). Further, proteomics analysis comparing the protein expression between the anatomic fractions showed that proteins relating to carbohydrate metabolism, energy production, and conversion as well as inorganic ion transport metabolism were either significantly upregulated or downregulated. Specifically, downregulation of proteins related to the iron-sulfur cluster in stalk fraction suggests a coordinated response by R. toruloides to maintain overall metabolic balance, and this was corroborated by the concentration of iron in the feedstocks.One-Sentence Summary This study elucidates the effects of different sources of corn stover on bisabolene production by engineered Rhodotorula toruloides, highlighting the importance of understanding feedstock variability to enhance bioprocess efficiency and economic outcomes. Graphical Abstract

Phototrophic bacteria and algae are primarily responsible for photosynthesis in aerobic environments, directly harnessing light energy to convert CO2 into chemical energy. Artificial photosynthesis mimics this process through strategies like the solar-to-fuel cycle, chemically reducing CO2 into various chemicals. Electrochemical systems, leveraging photovoltaic cells, have been developed to facilitate the transmission of electrons and H2, generated from water electrolysis, to microbial catalysts for fuel production. Cupriavidus metallidurans CH34, a versatile facultative chemolithotrophic microbe, utilizes Fe-Ni hydrogenase for H2 dependency. This study delved into the aerobic-autotrophic growth of C. metallidurans under electro-fermentation conditions using a single electrolyzer setup. To demonstrate mimicking photosynthesis based on an electrolyzer, we developed a scalable all-in-one single electrolyzer and applied RppA protein expressed C. metallidurans CH34 strains to confirm malonyl-CoA level measurement. As a result, Single cylinder electrolyzer fermentation appeared to demonstrate the well autotrophic growth, and it produced the malonyl-CoA, which derived chemical as flaviolin was accumulated at 695.9 ± 66.1 mg/L

Derived from renewable feedstocks, such as biomass, polylactic acid (PLA) is considered a more environmentally-friendly plastic than conventional petroleum-based polyethylene terephthalate (PET). However, PLA must still be recycled and its growing popularity and mixture with PET plastics at the disposal stage poses a cross-contamination threat in existing recycling facilities and results in low-value and low-quality recycled products. Hybrid upcycling has been proposed as a promising sustainable solution for mixed plastic waste; but its techno-economic and lifecycle environmental performance remain understudied. Here we propose a hybrid upcycling approach using a biocompatible ionic liquid (IL) to first chemically depolymerize plastics, then convert the depolymerized stream via biological upgrading with no extra separation. We show that over 95% of mixed PET/PLA was depolymerized into their respective monomers, which then served as the sole carbon source for the growth of Pseudomonas putida, enabling the conversion of the depolymerized plastics into biodegradable polyhydroxyalkanoates (PHA). In comparison to conventional commercial PHA, the estimated optimal production cost and carbon footprint are reduced by 62% and 29%, respectively.

Clostridium thermocellum is a desirable biocatalyst for biohydrogen production, with a native ability to simultaneously saccharify cellulose and to metabolize released cellodextrins for hydrogen production. During fermentation with C. thermocellum, partial pressures of two gases - CO2 and H2 - are critical drivers of overall reaction kinetics. Biohydrogen production is enhanced by maintaining a low hydrogen partial pressure, while high concentrations of dissolved CO2 promote microbial biomass synthesis. Our study evaluates the inherent trade-offs between hydrogen stripping and inorganic carbon supply for optimized biohydrogen synthesis. We find that nitrogen sparging at low flow rates increases hydrogen production when compared with an equivalent nitrogen overlay, but that high rates of nitrogen sparging inhibit cell growth and hydrogen production. Decreasing dissolved hydrogen partial pressure via nitrogen sparging also lowers the production of reduced metabolites, including lactate and ethanol. To address potential stripping of inorganic carbon from the production medium, we supplemented CO2 to the sparging gas and co-optimized for gas flow rate and for the CO2 fraction of the sparging gas. Total hydrogen production increased from 50 mmol center dot L- 1 in the base condition, when the bioreactor was sparged with 0.1 LPM of pure nitrogen, to 181.3 mmol center dot L-1 when sparged with 1.3 LPM of 33 % CO2, demonstrating that biohydrogen production is highly sensitive to both parameters. Fine sensitivity of biohydrogen production to sparging conditions highlights the critical importance of bioreactor design and operation to achieve maximum H2 removal without compromising inorganic carbon supply to bacterial central metabolism.

This article outlines the technical and economic potentials of lignin in unlocking sustainable biorefineries. The benefits of using this highly functionalized biopolymer for the growth of sustainable economy have been highlighted. But practically, the possibility of commercially substituting petroleum oil with lignin is still not very high as the estimated biofuel production cost is 2–3 times higher than the former one. However, with the advancement in technology and more efficient measures by biorefineries such as storing and processing the biomass near the field so as to reduce the transportation cost, it is possible to gain higher profits. Companies like Domtar, Stora Enso, Borregaard’s LignoTech, VITO, and Chemelot InSciTe have been promoting commercial value of lignin. The growth of lignin market after the start-up production at various sites has been discussed in this review. Combining the complete “start-to-finish” analysis with economic evaluation gives a pragmatic overview of the possibilities whether lignin will join petroleum oil as an efficient and cost-effective renewable source.

Sugar derived from lignocellulosic biomass is an important intermediate product, often used for the production of biofuels and value-added chemicals. It is therefore essential to understand and reduce the production costs of sugar derived from lignocellulosic biomass. This study evaluates the economic feasibility of a biorefinery producing sugar from lignocellulosic biomass using a combination of autohydrolysis and mechanical refining pretreatment. This biorefinery is self-sufficient in energy, producing excess electricity for sale. The minimum sugar selling price (MSSP) that achieves a zero net present value is $446/metric ton. The possibility of using the two main by-products, dissolved hemicellulose and lignin, for the production of high-value products, xylitol and polyol, is also evaluated. When dissolved hemicellulose is used for xylitol production, the MSSP decreases to $347/metric ton. When lignin is liquefied to produce polyol, the MSSP is further reduced to $342/metric ton. External energy (natural gas) must be purchased if xylitol and polyol are produced. An analysis of capital and operating costs reveals that, although the production of xylitol and polyol incurs additional capital investment, their correspondingly high values outweigh the additional capital costs. Sensitivity analysis results indicate that efficient recovery and recycling of expensive chemicals like acetone and glycerol are key to the success of the biorefinery. Yields of xylitol and polyol also have strong impacts on the process economics. (c) 2020 Society of Industrial Chemistry and John Wiley & Sons Ltd

Biological lignin valorization has emerged as a major solution for sustainable and cost-effective biorefineries. However, current biorefineries yield lignin with inadequate fractionation for bioconversion, yet substantial changes of these biorefinery designs to focus on lignin could jeopardize carbohydrate efficiency and increase capital costs. We resolve the dilemma by designing 'plug-in processes of lignin' with the integration of leading pretreatment technologies. Substantial improvement of lignin bioconversion and synergistic enhancement of carbohydrate processing are achieved by solubilizing lignin via lowering molecular weight and increasing hydrophilic groups, addressing the dilemma of lignin- or carbohydrate-first scenarios. The plug-in processes of lignin could enable minimum polyhydroxyalkanoate selling price at as low as $6.18/kg. The results highlight the potential to achieve commercial production of polyhydroxyalkanoates as a co-product of cellulosic ethanol. Here, we show that the plug-in processes of lignin could transform biorefinery design toward sustainability by promoting carbon efficiency and optimizing the total capital cost.