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.

The conversion of biomass feedstocks into fuels and chemicals using fast pyrolysis is a promising approach to renewable energy. Feed screws that convey biomass to pyrolysis reactors, however, often encounter plugging. Indirect heating of the feed screw occurs due to contact with the pyrolysis chamber, resulting in a heating gradient ranging from ambient temperature (22 °C) to reactor temperature (500 °C). Given that major cell wall macromolecules, such as lignin, cellulose, and hemicellulose, begin to produce bio-oils and volatile resin acid compounds within this temperature gradient, we hypothesized that indirect heating during feed screw conveyance is sufficient to cause premature degradation of biomass. We characterized this degradation by observing increases in surface roughness, changes in overall particle morphology, and the production and deposition of bio-oils on biomass particle surfaces. Correlative analysis between optical in situ hot-stage microscopy, confocal Raman spectroscopy, and SEM analysis revealed that heating at temperatures as low as 375 °C caused significant increases in surface roughness, with large fissures forming between and within cell walls. Additionally, droplets of bio-oil were observed on particles, especially in the bark and cambium samples. This work suggests that these phenomena contribute to particle agglomeration, leading to feed screw plugging, and that engineering a solution to cool the feed screw could prevent particle agglomeration and reduce plugging incidents, thereby increasing biomass processing efficiency.

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.

Microplastics have emerged as major environmental hazards that require efficient, cost-effective, and sustainable remediation technologies. This study introduces an integrative platform for the remediation and upcycling of microplastics by algae, while synergizing with plastic upcycling, wastewater treatment, and algal production. The strategy employs a mechanism that enhances hydrophobic interactions between the cell surface and microplastics, enabling rapid aggregation and removal. The platform achieves a superior microplastic removal efficiency of 91.4% within 1 hour, with a capacity of 0.1-gram microplastic per gram of biomass. Furthermore, the study demonstrates an upcycling strategy that converts microplastics-enriched cyanobacteria into plastic composites with unique performance. This work also integrates microplastic removal with cyanobacterial bioproduction and wastewater treatment, offering an approach that synergizes remediation with these value-added processes. Ultimately, this platform provides a viable and sustainable pathway to address microplastic pollution by creating value through plastic upcycling, wastewater nutrient removal, and CO2-based bioproduction.

Stimulated Raman scattering (SRS) microscopy using picosecond near-IR pulses has provided a great penetration depth with reduced fluorescence interference when imaging biological samples for bioenergy applications. These tools have provided insight into 1) tracking the degradation of chemical composites in biomass feedstocks to investigate the recalcitrant factors during the deconstruction processes, 2) monitoring the production of chemicals in photosynthetic plants and wood-digesting microorganisms, and 3) probing plant-bacteria interactions. However, the above processes are usually slow and require continuous imaging for an extended period. This is challenging for classic SRS because the laser power needed to achieve enough sensitivity causes photodamage in the samples during such long experiments. Quantum-squeezed light with reduced noise in the intensity quadrature can improve the sensitivity of classic SRS microscopy beyond the shot noise limit. The successful squeezing of one of the picosecond pulses in the above SRS will improve sensitivity and reduce photodamage, greatly expanding the range of studies available to SRS microscopy.

Algal biofuel is regarded as one of the ultimate solutions for renewable energy, but its commercialization is hindered by growth limitations caused by mutual shading and high harvest costs. We overcome these challenges by advancing machine learning to inform the design of a semi-continuous algal cultivation (SAC) to sustain optimal cell growth and minimize mutual shading. An aggregation-based sedimentation (ABS) strategy is then designed to achieve low-cost biomass harvesting and economical SAC. The ABS is achieved by engineering a fast-growing strain, Synechococcus elongatus UTEX 2973, to produce limonene, which increases cyanobacterial cell surface hydrophobicity and enables efficient cell aggregation and sedimentation. SAC unleashes cyanobacterial growth potential with 0.1 g/L/hour biomass productivity and 0.2 mg/L/hour limonene productivity over a sustained period in photobioreactors. Scaling-up the SAC with an outdoor pond system achieves a biomass yield of 43.3 g/m 2 /day, bringing the minimum biomass selling price down to approximately $281 per ton.

A simple and highly selective analytical procedure is presented for the determination of cellulosic glucan content in samples that contain both cellulose and starch. This method eliminates the unacceptably large compounding errors of current two-measurement methods. If both starch and cellulose are present before analytical hydrolysis, both will be hydrolyzed to glucose causing bias and inaccuracy in the method. To prevent this interference, the removal of starch prior to cellulosic quantification is crucial. The method presented here is a concise in-series procedure with minimal measurements, eliminating large compounding errors. Sample preparation consists of a starch extraction employing enzymatic hydrolysis followed by a simple filtration and wash. The samples are then subjected to a two-stage acid hydrolysis. The concentration of glucose is determined by ion exchange high-performance liquid chromatography with a Pb 2+ column and a refractive index detector. The cellulosic glucan content is calculated based on the initial dry weight of the starting material. Data for the native biomass materials studied show excellent reproducibility, with coefficients of variance of 3.0% or less associated with the method. This selectivity for cellulosic glucan by the procedure was validated with several analytical techniques such as liquid chromatography coupled with mass spectrometry (LC–MS), Raman spectroscopy, and nuclear magnetic resonance.

The variability of chemical, physical, and mechanical properties of lignocellulosic biomass feedstocks has a major impact on the efficiency of biomass processing and conversion to fuels and chemicals. Storage conditions represent a key source of variability that may contribute to biomass quality variations from the time of harvest until delivery to the biorefinery. In some cases, substantial microbial degradation can take place during storage. In this work, we investigate how degradation during storage affects the surface texture, surface energy, and porosity of different corn stover anatomical fractions (e.g., leaf, stalk, and cob). Understanding any potential changes in surface properties is important because interparticle interactions during bioprocessing cause aggregation and blockages that lead to at least process inefficiency and at most complete equipment failure. The surface roughness and texture parameters of corn stover with variable degrees of microbial degradation were calculated directly from stereomicroscopy and scanning electron microscopy micrographs. Surface energy and porosity were measured by inverse gas chromatography. The results show differing trends in the impact of increasing biological heating and degradation depending on the specific corn stover tissue type that was analyzed. These results also indicate that biomass surface properties are scale-dependent and that the scale, which is most industrially relevant, may depend on the specific unit operation within the biorefinery being considered.

Feedstock variability is a significant barrier to the scale-up and commercialization of lignocellulosic biofuel technologies. Variability in feedstock characteristics and behavior creates numerous challenges to the biorefining industry by affecting continuous operation and bioffiels yields. Currently, feedstock variability is understood and explained largely on the basis of chemical composition. Physical and mechanical properties and behavior of lignocellulosic feedstock in various unit operations, studied through advanced analytical methods, can further explain variability. Such studies will enable us in developing processes and designing equipment to improve operation and conversion performance. In this perspective, we review several advanced analytical methods that measure density, moisture content, thermal properties, flowability, grindability, rheology properties, and micromorphological characteristics. We also discuss the correlations and interactions among these properties that reflect the complexity of lignocellulosic biomass as a feedstock and the associated quality metrics and logistics of supplying consistent quality feedstock to a biorefinery. We also examine methods that have not traditionally been used to characterize lignocellulosic feedstocks but have the potential to bridge the gap in our explanation of feedstock variability.

Visualization of lignin in plant cell walls, with both spatial and chemical resolution, is emerging as an important tool to understand lignin's role in the plant cell wall's nanoscale architecture and to understand and design processes intended to modify the lignin. As such, this chapter reviews recent advances in advanced imaging methods with respect to lignin in plant cell walls. This review focuses on the importance of lignin detection and localization for studies in both plant biology and biotechnology. Challenges going forward to identify and delineate lignin from other plant cell wall components and to quantitatively analyze lignin in whole cell walls from native plant tissue and treated biomass are also discussed.

Traditional bioproduct engineering focuses on pathway optimization, yet is often complicated by product inhibition, downstream consumption, and the toxicity of certain products. Here, we present the co-compartmentation of biosynthesis and storage via a synthetic droplet as an effective new strategy to improve the bioproduct yield, with squalene as a model compound. A hydrophobic protein was designed and introduced into the tobacco chloroplast to generate a synthetic droplet for terpene storage. Simultaneously, squalene biosynthesis enzymes were introduced to chloroplasts together with the droplet-forming protein to co-compartmentalize the biosynthesis and storage of squalene. The strategy has enabled a record yield of squalene at 2.6 mg/g fresh weight without compromising plant growth. Confocal fluorescent microscopy imaging, stimulated Raman scattering microscopy, and droplet composition analysis confirmed the formation of synthetic storage droplet in chloroplast. The co-compartmentation of synthetic storage droplet with a targeted metabolic pathway engineering represents a new strategy for enhancing bioproduct yield.

Understanding plant cell wall cross-linking chemistry and polymeric architecture is key to the efficient utilization of biomass in all prospects from rational genetic modification to downstream chemical and biological conversion to produce fuels and value chemicals. In fact, the bulk properties of cell wall recalcitrance are collectively determined by its chemical features over a wide range of length scales from tissue, cellular to polymeric architectures. Microscopic visualization of cell walls from the nanometer to the micrometer scale offers an in situ approach to study their chemical functionality considering its spatial and chemical complexity, particularly the capabilities of characterizing biomass non-destructively and in real-time during conversion processes. Microscopic characterization has revealed heterogeneity in the distribution of chemical features, which would otherwise be hidden in bulk analysis. Key microscopic features include cell wall type, wall layering, and wall composition—especially cellulose and lignin distributions. Microscopic tools, such as atomic force microscopy, stimulated Raman scattering microscopy, and fluorescence microscopy, have been applied to investigations of cell wall structure and chemistry from the native wall to wall treated by thermal chemical pretreatment and enzymatic hydrolysis. While advancing our current understanding of plant cell wall recalcitrance and deconstruction, microscopic tools with improved spatial resolution will steadily enhance our fundamental understanding of cell wall function.

Here, we examine grain boundaries (GBs) with respect to non-GB regions (grain surfaces (GSs) and grain interiors (GIs)) in high-quality micrometer-sized perovskite CH3NH3PbI3 (or MAPbI3) thin films using high-resolution confocal fluorescence-lifetime imaging microscopy in conjunction with kinetic modeling of charge-transport and recombination processes. We show that, contrary to previous studies, GBs in our perovskite MAPbI3 thin films do not lead to increased recombination but that recombination in these films happens primarily in the non-GB regions (i.e., GSs or GIs). We also find that GBs in these films are not transparent to photogenerated carriers, which is likely associated with a potential barrier at GBs. Even though GBs generally display lower luminescence intensities than GSs/GIs, the lifetimes at GBs are no worse than those at GSs/GIs, further suggesting that GBs do not dominate non-radiative recombination in MAPbI3 thin films.

It is known that plant growth promoting bacteria (PGPB) elicit positive effects on plant growth and biomass yield. However, the actual mechanism behind the plant-PGPB interaction is poorly understood, and the literature is scarce regarding the thermochemical pretreatability and enzymatic degradability of biomass derived from PGPB-inoculated plants. Most recent transcriptional analyses of PGPB strain Burkholderia phytofirmans PsJN inoculating potato in literature and Arabidopsis in our present study have revealed the expression of genes for ferritin and the biosynthesis and transport of siderophores (i.e., the molecules with high affinity for iron), respectively. The expression of such genes in the shoots of PsJN-inoculated plants prompted us to propose that PsJN-inoculation can improve the host plant's iron uptake and accumulation, which facilitates the downstream plant biomass pretreatment and conversion to simple sugars. In this study, we employed B. phytofirmans PsJN to inoculate the Arabidopsis thaliana plants, and conducted the first investigation for its effects on the biomass yield, the anatomical organization of stems, the iron accumulation, and the pretreatment and enzymatic hydrolysis of harvested biomass. The results showed that the strain PsJN stimulated plant growth in the earlier period of plant development and enlarged the cell size of stem piths, and it also indeed enhanced the essential metals uptake and accumulation in host plants. Moreover, we found that the PsJN-inoculated plant biomass released more glucose and xylose after hot water pretreatment and subsequent co-saccharification, which provided a novel insight into development of lignocellulosic biofuels from renewable biomass resources.

BACKGROUND:Plant hemicellulose (largely xylan) is an excellent feedstock for renewable energy production and second only to cellulose in abundance. Beyond a source of fermentable sugars, xylan constitutes a critical polymer in the plant cell wall, where its precise role in wall assembly, maturation, and deconstruction remains primarily hypothetical. Effective detection of xylan, particularly by in situ imaging of xylan in the presence of other biopolymers, would provide critical information for tackling the challenges of understanding the assembly and enhancing the liberation of xylan from plant materials.RESULTS:Raman-based imaging techniques, especially the highly sensitive stimulated Raman scattering (SRS) microscopy, have proven to be valuable tools for label-free imaging. However, due to the complex nature of plant materials, especially those same chemical groups shared between xylan and cellulose, the utility of specific Raman vibrational modes that are unique to xylan have been debated. Here, we report a novel approach based on combining spectroscopic analysis and chemical/enzymatic xylan removal from corn stover cell walls, to make progress in meeting this analytical challenge. We have identified several Raman peaks associated with xylan content in cell walls for label-free in situ imaging xylan in plant cell wall.CONCLUSION:We demonstrated that xylan can be resolved from cellulose and lignin in situ using enzymatic digestion and label-free SRS microscopy in both 2D and 3D. We believe that this novel approach can be used to map xylan in plant cell walls and that this ability will enhance our understanding of the role played by xylan in cell wall biosynthesis and deconstruction.

In higher plant cells, lignin provides necessary physical support for plant growth and resistance to attack by microorganisms. For the same reason, lignin is considered to be a major impediment to the process of deconstructing biomass to simple sugars by hydrolytic enzymes. The in situ variation of lignin in plant cell walls is important for better understanding of the roles lignin play in biomass recalcitrance.

In lignocellulosic biomass, lignin is the second most abundant biopolymer. In plant cell walls, lignin is associated with polysaccharides to form lignin-carbohydrate complexes (LCC). LCC have been considered to be a major factor that negatively affects the process of deconstructing biomass to simple sugars by cellulosic enzymes. Here, we report a micro-spectroscopic approach that combines fluorescence lifetime imaging microscopy and Stimulated Raman Scattering microscopy to probe in situ lignin concentration and conformation at each cell wall layer. This technique does not require extensive sample preparation or any external labels. Using poplar as a feedstock, for example, we observe variation of LCC in untreated tracheid poplar cell walls. The redistribution of LCC at tracheid poplar cell wall layers is also investigated when the chemical linkages between lignin and hemicellulose are cleaved during pretreatment. Our study would provide new insights into further improvement of the biomass pretreatment process.