Production of deuterated biomass by cultivation of Lemna minor (duckweed) in D2O.

MAIN CONCLUSION: Common duckweed Lemna minor was cultivated in 50% D2O to produce biomass with 50-60% deuterium incorporation containing cellulose with degree of polymerization close (85%) to that of H2O-grown controls. The small aquatic plant duckweed, particularly the genus Lemna, widely used for toxicity testing, has been proposed as a potential source of biomass for conversion into biofuels as well as a platform for production of pharmaceuticals and specialty chemicals. Ability to produce deuterium-substituted duckweed can potentially extend the range of useful products as well as assist process improvement. Cultivation of these plants under deuterating conditions was previously been reported to require addition of kinetin to induce growth and was hampered by anomalies in cellular morphology and protein metabolism. Here, we report the production of biomass with 50-60% deuterium incorporation by long-term photoheterotrophic growth of common duckweed Lemna minor in 50% D2O with 0.5% glucose. L. minor grown in 50% D2O without addition of kinetin exhibited a lag phase twice that of H2O-grown controls, before start of log phase growth at 40% of control rates. Compared to continuous white fluorescent light, growth rates increased fivefold for H2O and twofold for 50% D2O when plants were illuminated at higher intensity with a metal halide lamp and a diurnal cycle of 12-h light/12-h dark. Deuterium incorporation was determined by a combination of 1H and 2H nuclear magnetic resonance (NMR) to be 40-60%. The cellulose from the deuterated plants had an average-number degree of polymerization (DPn) and polydispersity index (PDI) close to that of H2O-grown controls, while Klason lignin content was reduced. The only major gross morphological change noted was root inhibition.

A concerted systems biology analysis of phenol metabolism in Rhodococcus opacus PD630.

Rhodococcus opacus PD630 metabolizes aromatic substrates and naturally produces branched-chain lipids, which are advantageous traits for lignin valorization. To provide insights into its lignocellulose hydrolysate utilization, we performed 13C-pathway tracing, 13C-pulse-tracing, transcriptional profiling, biomass composition analysis, and metabolite profiling in conjunction with 13C-metabolic flux analysis (13C-MFA) of phenol metabolism. We found that 1) phenol is metabolized mainly through the ortho-cleavage pathway; 2) phenol utilization requires a highly active TCA cycle; 3) NADPH is generated mainly via NADPH-dependent isocitrate dehydrogenase; 4) active cataplerotic fluxes increase plasticity in the TCA cycle; and 5) gluconeogenesis occurs partially through the reversed Entner-Doudoroff pathway (EDP). We also found that phenol-fed R. opacus PD630 generally has lower sugar phosphate concentrations (e.g., fructose 1,6-bisphosphatase) compared to metabolite pools in 13C-glucose-fed Escherichia coli (set as internal standards), while its TCA metabolites (e.g., malate, succinate, and α-ketoglutarate) accumulate intracellularly with measurable succinate secretion. In addition, we found that phenol utilization was inhibited by benzoate, while catabolite repressions by other tested carbon substrates (e.g., glucose and acetate) were absent in R. opacus PD630. Three adaptively-evolved strains display very different growth rates when fed with phenol as a sole carbon source, but they maintain a conserved flux network. These findings improve our understanding of R. opacus' metabolism for future lignin valorization.

Multi-omic elucidation of aromatic catabolism in adaptively evolved Rhodococcus opacus.

Lignin utilization has been identified as a key factor in biorefinery profitability. However, lignin depolymerization generates heterogeneous aromatic mixtures that inhibit microbial growth and the conversion of lignocellulose to biochemicals. Rhodococcus opacus is a promising aromatic-catabolizing, oleaginous bacterium, but mechanisms for its aromatic tolerance and utilization remain undercharacterized. To better understand these mechanisms, we adaptively evolved R. opacus for improved utilization of 32 combinations of diverse aromatic compounds. Evolved R. opacus mutants showed up to 1900% growth improvement in the utilization of phenol, guaiacol, 4-hydroxybenzoate, vanillate, and benzoate compared to the wild-type strain. Whole genome sequencing revealed several redox-related genes with mutations shared across multiple adapted mutants. PVHG6, the mutant with the most improved growth on a mixture of multiple aromatic compounds, showed 56% lower superoxide dismutase activity than the wild-type strain, suggesting that redox reactions are important for aromatic tolerance and utilization. Comparative transcriptomics revealed by-product detoxification pathways and five aromatic funneling pathways that were upregulated in response to specific aromatic compounds. Gene knockout experiments confirmed the two degradation routes of the ß-ketoadipate pathway for five aromatic compounds. These results provide an improved understanding of aromatic bioconversion and facilitate development of R. opacus as a biorefinery host.

Comparative transcriptomics elucidates adaptive phenol tolerance and utilization in lipid-accumulating Rhodococcus opacus PD630.

Lignin-derived (e.g. phenolic) compounds can compromise the bioconversion of lignocellulosic biomass to fuels and chemicals due to their toxicity and recalcitrance. The lipid-accumulating bacterium Rhodococcus opacus PD630 has recently emerged as a promising microbial host for lignocellulose conversion to value-added products due to its natural ability to tolerate and utilize phenolics. To gain a better understanding of its phenolic tolerance and utilization mechanisms, we adaptively evolved R. opacus over 40 passages using phenol as its sole carbon source (up to 373% growth improvement over wild-type), and extensively characterized two strains from passages 33 and 40. The two adapted strains showed higher phenol consumption rates (∼20 mg/l/h) and ∼2-fold higher lipid production from phenol than the wild-type strain. Whole-genome sequencing and comparative transcriptomics identified highly-upregulated degradation pathways and putative transporters for phenol in both adapted strains, highlighting the important linkage between mechanisms of regulated phenol uptake, utilization, and evolved tolerance. Our study shows that the R. opacus mutants are likely to use their transporters to import phenol rather than export them, suggesting a new aromatic tolerance mechanism. The identified tolerance genes and pathways are promising candidates for future metabolic engineering in R. opacus for improved lignin conversion to lipid-based products.

Production of deuterated switchgrass by hydroponic cultivation.

MAIN CONCLUSION: The bioenergy crop switchgrass was grown hydroponically from tiller cuttings in 50 % D 2 O to obtain biomass with 34 % deuterium substitution and physicochemical properties similar to those of H 2 O-grown switchgrass controls. Deuterium enrichment of biological materials can potentially enable expanded experimental use of small angle neutron scattering (SANS) to investigate molecular structural transitions of complex systems such as plant cell walls. Two key advances have been made that facilitate cultivation of switchgrass, an important forage and biofuel crop, for controlled isotopic enrichment: (1) perfusion system with individual chambers and (2) hydroponic growth from tiller cuttings. Plants were grown and maintained for several months with periodic harvest. Photosynthetic activity was monitored by measurement of CO2 in outflow from the growth chambers. Plant morphology and composition appeared normal compared to matched controls grown with H2O. Using this improved method, gram quantities of switchgrass leaves and stems were produced by continuous hydroponic cultivation using growth medium consisting of basal mineral salts in 50 % D2O. Deuterium incorporation was confirmed by detection of the O-D and C-D stretching peaks with FTIR and quantified by (1)H- and (2)H-NMR. This capability to produce deuterated lignocellulosic biomass under controlled conditions will enhance investigation of cell wall structure and its deconstruction by neutron scattering and NMR techniques.

4-O-methylation of glucuronic acid in Arabidopsis glucuronoxylan is catalyzed by a domain of unknown function family 579 protein.

The hemicellulose 4-O-methyl glucuronoxylan is one of the principle components present in the secondary cell walls of eudicotyledonous plants. However, the biochemical mechanisms leading to the formation of this polysaccharide and the effects of modulating its structure on the physical properties of the cell wall are poorly understood. We have identified and functionally characterized an Arabidopsis glucuronoxylan methyltransferase (GXMT) that catalyzes 4-O-methylation of the glucuronic acid substituents of this polysaccharide. AtGXMT1, which was previously classified as a domain of unknown function (DUF) 579 protein, specifically transfers the methyl group from S-adenosyl-L-methionine to O-4 of α-D-glucopyranosyluronic acid residues that are linked to O-2 of the xylan backbone. Biochemical characterization of the recombinant enzyme indicates that GXMT1 is localized in the Golgi apparatus and requires Co(2+) for optimal activity in vitro. Plants lacking GXMT1 synthesize glucuronoxylan in which the degree of 4-O-methylation is reduced by 75%. This result is correlated to a change in lignin monomer composition and an increase in glucuronoxylan release during hydrothermal treatment of secondary cell walls. We propose that the DUF579 proteins constitute a previously undescribed family of cation-dependent, polysaccharide-specific O-methyl-transferases. This knowledge provides new opportunities to selectively manipulate polysaccharide O-methylation and extends the portfolio of structural targets that can be modified either alone or in combination to modulate biopolymer interactions in the plant cell wall.

Genetic manipulation of lignin reduces recalcitrance and improves ethanol production from switchgrass.

Switchgrass is a leading dedicated bioenergy feedstock in the United States because it is a native, high-yielding, perennial prairie grass with a broad cultivation range and low agronomic input requirements. Biomass conversion research has developed processes for production of ethanol and other biofuels, but they remain costly primarily because of the intrinsic recalcitrance of biomass. We show here that genetic modification of switchgrass can produce phenotypically normal plants that have reduced thermal-chemical (≤180 °C), enzymatic, and microbial recalcitrance. Down-regulation of the switchgrass caffeic acid O-methyltransferase gene decreases lignin content modestly, reduces the syringyl:guaiacyl lignin monomer ratio, improves forage quality, and, most importantly, increases the ethanol yield by up to 38% using conventional biomass fermentation processes. The down-regulated lines require less severe pretreatment and 300-400% lower cellulase dosages for equivalent product yields using simultaneous saccharification and fermentation with yeast. Furthermore, fermentation of diluted acid-pretreated transgenic switchgrass using Clostridium thermocellum with no added enzymes showed better product yields than obtained with unmodified switchgrass. Therefore, this apparent reduction in the recalcitrance of transgenic switchgrass has the potential to lower processing costs for biomass fermentation-derived fuels and chemicals significantly. Alternatively, such modified transgenic switchgrass lines should yield significantly more fermentation chemicals per hectare under identical process conditions.

Tunable Viscoelasticity of Alginate Hydrogels via Serial Autoclaving.

Alginate hydrogels are widely used as biomaterials for cell culture and tissue engineering due to their biocompatibility and tunable mechanical properties. Reducing alginate molecular weight is an effective strategy for modulating hydrogel viscoelasticity and stress relaxation behavior, which can significantly impact cell spreading and fate. However, current methods like gamma irradiation to produce low molecular weight alginates suffer from high cost and limited accessibility. Here, a facile and cost-effective approach to reduce alginate molecular weight in a highly controlled manner using serial autoclaving is presented. Increasing the number of autoclave cycles results in proportional reductions in intrinsic viscosity, hydrodynamic radius, and molecular weight of the polymer while maintaining its chemical composition. Hydrogels fabricated from mixtures of the autoclaved alginates exhibit tunable mechanical properties, with inclusion of lower molecular weight alginate leading to softer gels with faster stress relaxation behaviors. The method is demonstrated by establishing how viscoelastic relaxation affects the spreading of encapsulated fibroblasts and glioblastoma cells. Results establish repetitive autoclaving as an easily accessible technique to generate alginates with a range of molecular weights and to control the viscoelastic properties of alginate hydrogels, and demonstrate utility across applications in mechanobiology, tissue engineering, and regenerative medicine.

Reuse of Lignin to Synthesize High Surface Area Carbon Nanoparticles for Supercapacitors Using a Continuous and Single-Step Aerosol Method.

There is a growing demand for the synthesis of high surface area carbons, also known as carbon nanoparticles (CNPs). Existing synthesis methods for high surface area carbons have limited environmental benignity and economic viability due to the requirement of multistep and batch processes and harsh activating and/or templating chemicals. Herein, we demonstrate the synthesis of high surface area CNPs from lignin, a waste byproduct, through a single-step, continuous gas phase aerosol technique without the use of activating or templating chemicals. This continuous approach requires significantly less time for synthesis: on the order of seconds in comparison to hours for conventional methods. Properties of carbon materials synthesized from lignin are controlled by temperature and residence time, and the role of these parameters inside the aerosol reactor on carbon nanoparticle size, morphology, molecular structure, and surface area is systematically investigated. Furthermore, the as-obtained carbon nanoparticles are tested for specific capacitance, and the best-performing material (surface area 925 m2/g) exhibited a specific capacitance of 247 F/g at 0.5 A/g with excellent capacity retainment of over 98% after 10,000 cycles. This is a clear demonstration of their superior performance compared with supercapacitors synthesized earlier from lignin. Overall, the simple (single-step, continuous, and rapid) operation and the avoidance of the use of activating/templating chemicals make the aerosol technique a promising candidate for the scalable and sustainable synthesis of CNPs from lignin.

Genetically Engineered Protein-Based Bioadhesives with Programmable Material Properties.

Silk-amyloid-mussel foot protein (SAM) hydrogels made from recombinant fusion proteins containing ß-amyloid peptide, spider silk domain, and mussel foot protein (Mfp) are attractive bioadhesives as they display a unique combination of tunability, biocompatibility, bioabsorbability, strong cohesion, and underwater adhesion to a wide range of biological surfaces. To design tunable SAM hydrogels for tailored surgical repair applications, an understanding of the relationships between protein sequence and hydrogel properties is imperative. Here, we fabricated SAM hydrogels using fusion proteins of varying lengths of silk-amyloid repeats and Mfps to characterize their structure and properties. We found that increasing silk-amyloid repeats enhanced the hydrogel's ß-sheet content (r = 0.74), leading to higher cohesive strength and toughness. Additionally, increasing the Mfp length beyond the half-length of the full Mfp sequence (1/2 Mfp) decreased the ß-sheet content (r = -0.47), but increased hydrogel surface adhesion. Among different variants, the hydrogel made of 16xKLV-2Mfp displayed a high ultimate strength of 3.0 ± 0.3 MPa, an ultimate strain of 664 ± 119%, and an attractive underwater adhesivity of 416 ± 20 kPa to porcine skin. Collectively, the sequence-structure-property relationships learned from this study will be useful to guide the design of future protein adhesives with tunable characteristics for tailored surgical applications.

A High-Quality Genome-Scale Model for Rhodococcus opacus Metabolism.

Rhodococcus opacus is a bacterium that has a high tolerance to aromatic compounds and can produce significant amounts of triacylglycerol (TAG). Here, we present iGR1773, the first genome-scale model (GSM) of R. opacus PD630 metabolism based on its genomic sequence and associated data. The model includes 1773 genes, 3025 reactions, and 1956 metabolites, was developed in a reproducible manner using CarveMe, and was evaluated through Metabolic Model tests (MEMOTE). We combine the model with two Constraint-Based Reconstruction and Analysis (COBRA) methods that use transcriptomics data to predict growth rates and fluxes: E-Flux2 and SPOT (Simplified Pearson Correlation with Transcriptomic data). Growth rates are best predicted by E-Flux2. Flux profiles are more accurately predicted by E-Flux2 than flux balance analysis (FBA) and parsimonious FBA (pFBA), when compared to 44 central carbon fluxes measured by 13C-Metabolic Flux Analysis (13C-MFA). Under glucose-fed conditions, E-Flux2 presents an R2 value of 0.54, while predictions based on pFBA had an inferior R2 of 0.28. We attribute this improved performance to the extra activity information provided by the transcriptomics data. For phenol-fed metabolism, in which the substrate first enters the TCA cycle, E-Flux2's flux predictions display a high R2 of 0.96 while pFBA showed an R2 of 0.93. We also show that glucose metabolism and phenol metabolism function with similar relative ATP maintenance costs. These findings demonstrate that iGR1773 can help the metabolic engineering community predict aromatic substrate utilization patterns and perform computational strain design.

13C cell wall enrichment and ionic liquid NMR analysis: progress towards a high-throughput detailed chemical analysis of the whole plant cell wall.

The ability to accurately and rapidly measure plant cell wall composition, relative monolignol content and lignin-hemicellulose inter-unit linkage distributions has become essential to efforts centered on reducing the recalcitrance of biomass by genetic engineering. Growing (13)C enriched transgenic plants is a viable route to achieve the high-throughput, detailed chemical analysis of whole plant cell wall before and after pretreatment and microbial or enzymatic utilization by (13)C nuclear magnetic resonance (NMR) in a perdeuterated ionic liquid solvent system not requiring component isolation. 1D (13)C whole cell wall ionic liquid NMR of natural abundant and (13)C enriched corn stover stem samples suggest that a high level of uniform labeling (>97%) can significantly reduce the total NMR experiment times up to ~220 times. Similarly, significant reduction in total NMR experiment time (~39 times) of the (13)C enriched corn stover stem samples for 2D (13)C-(1)H heteronuclear single quantum coherence NMR was found.

Deuterium incorporation in biomass cell wall components by NMR analysis.

A commercially available deuterated kale sample was analyzed for deuterium incorporation by ionic liquid solution (2)H and (1)H nuclear magnetic resonance (NMR). This protocol was found to effectively measure the percent deuterium incorporation at 33%, comparable to the 31% value determined by combustion. The solution NMR technique also suggested by a qualitative analysis that deuterium is preferentially incorporated into the carbohydrate components of the kale sample.

Biopolymer nanocomposite films reinforced with nanocellulose whiskers.

A xylan nanocomposite film with improved strength and barrier properties was prepared by a solution casting using nanocellulose whiskers as a reinforcing agent. The 13C cross-polarization magic angle spinning (CP/MAS) nuclear magnetic resonance (NMR) analysis of the spectral data obtained for the NCW/xylan nanocomposite films indicated the signal intensity originating from xylan-cellulose interactions. Qualitatively, the spectral data obtained for the NCW/xylan nanocomposite films indicated that the amount of xylan adsorbed to cellulose increases with the addition of NCW. In an attempt to quantify this effect, non-linear least-squared spectral line fitting was used to deconvolute the adsorbed xylan peak at -82 ppm. The peak intensity ratio of adsorbed xylan peak and xylan C1 peak, which represents the total amount of xylan increases suggesting that upon the addition of NCW, the amount of adsorbed xylan increases. In an effort to further infer the structure-property relationships associated with the observed strength and barrier properties, 1H NMR T2 relaxation experiments were also conducted to investigate the change in the nature of carbohydrate-water interactions as a result of NCW incorporation. Water adsorbed into the 50% nanocomposite film had significantly shorter relaxation times with respect to the control xylan/sorbitol and all other NCW/xylan nanocomposite films. Additionally, X-ray diffraction of the nanocomposite films showed increased levels of crystalline material in the nanocomposites due to NCW addition.

Correlated mechanochemical maps of Arabidopsis thaliana primary cell walls using atomic force microscope infrared spectroscopy.

Spatial heterogeneity in composition and organisation of the primary cell wall affects the mechanics of cellular morphogenesis. However, directly correlating cell wall composition, organisation and mechanics has been challenging. To overcome this barrier, we applied atomic force microscopy coupled with infrared (AFM-IR) spectroscopy to generate spatially correlated maps of chemical and mechanical properties for paraformaldehyde-fixed, intact Arabidopsis thaliana epidermal cell walls. AFM-IR spectra were deconvoluted by non-negative matrix factorisation (NMF) into a linear combination of IR spectral factors representing sets of chemical groups comprising different cell wall components. This approach enables quantification of chemical composition from IR spectral signatures and visualisation of chemical heterogeneity at nanometer resolution. Cross-correlation analysis of the spatial distribution of NMFs and mechanical properties suggests that the carbohydrate composition of cell wall junctions correlates with increased local stiffness. Together, our work establishes new methodology to use AFM-IR for the mechanochemical analysis of intact plant primary cell walls.

SANS study of cellulose extracted from switchgrass.

Lignocellulosic biomass, which is an abundant renewable natural resource, has the potential to play a major role in the generation of renewable biofuels through its conversion to bioethanol. Unfortunately, it is a complex biological composite material that shows significant recalcitrance, making it a cost-ineffective feedstock for bioethanol production. Small-angle neutron scattering (SANS) was employed to probe the multi-scale structure of cellulosic materials. Cellulose was extracted from milled native switchgrass and from switchgrass that had undergone a dilute acid pretreatment method in order to disrupt the lignocellulose structure. The high-Q structural feature (Q > 0.07 Å(-1)) can be assigned to cellulose fibrils based on a comparison of cellulose purified by solvent extraction of native and dilute acid pretreated switchgrass and a commercial preparation of microcrystalline cellulose. Dilute acid pretreatment results in an increase in the smallest structural size, a decrease in the interconnectivity of the fibrils and no change in the smooth domain boundaries at length scales larger than 1000 Å.

Poly(methyl vinyl ether-co-maleic acid)-polyethylene glycol nanocomposites cross-linked in situ with cellulose nanowhiskers.

Nanocomposites were developed by cross-linking cellulose nanowhiskers with poly(methyl vinyl ether-co-maleic acid) and polyethylene glycol. Nuclear magnetic resonance (NMR) studies showed cross-linking occurs between the matrix and cellulose nanowhiskers via an esterification reaction. Proton NMR T(2) relaxation experiments provided information on the mobility of the polymer chains within the matrix, which can be related to the structure of the cross-linked nanocomposite. The nanocomposite was found to consist of mobile chain portions between cross-linked junction points and immobilized chain segments near or at those junction points, whose relative fraction increased upon further incorporation of cellulose nanowhiskers. Atomic force microscopy images showed a homogeneous dispersion of nanowhiskers in the matrix even at high nanowhisker content, which can be attributed to cross-linking of the nanowhiskers in the matrix. Relative humidity conditions were found to affect the mechanical properties of the composites negatively while the nanowhiskers content had a positive effect. It is expected that the cross-links between the matrix and the cellulose nanowhiskers trap the nanowhiskers in the cross-linked network, preventing nanowhisker aggregation subsequently producing cellulose nanocomposites with unique mechanical behaviors. The results show that in situ cross-linking of cellulose nanowhiskers with a matrix polymer is a promising route to obtain nanocomposites with well dispersed nanowhiskers, tailored nanostructure, and mechanical performance.

Breakdown of cell wall nanostructure in dilute acid pretreated biomass.

The generation of bioethanol from lignocellulosic biomass holds great promise for renewable and clean energy production. A better understanding of the complex mechanisms of lignocellulose breakdown during various pretreatment methods is needed to realize this potential in a cost and energy efficient way. Here we use small-angle neutron scattering (SANS) to characterize morphological changes in switchgrass lignocellulose across molecular to submicrometer length scales resulting from the industrially relevant dilute acid pretreatment method. Our results demonstrate that dilute acid pretreatment increases the cross-sectional radius of the crystalline cellulose fibril. This change is accompanied by removal of hemicellulose and the formation of R(g) ∼ 135 A lignin aggregates. The structural signature of smooth cell wall surfaces is observed at length scales larger than 1000 A, and it remains remarkably invariable during pretreatment. This study elucidates the interplay of the different biomolecular components in the breakdown process of switchgrass by dilute acid pretreatment. The results are important for the development of efficient strategies of biomass to biofuel conversion.

Rapid quantitative analytical tool for characterizing the preparation of biodiesel.

A novel qualitative method has been developed for the determination of the various types of hydroxyl groups present in biodiesel production streams. The use of 2-chloro-4,4,5,5-tetramethyl-1,3,2-dioxaphospholane as a phosphitylation reagent for quantitative (31)P NMR analysis of the hydroxyl groups in biodiesel process samples has been fully developed. This experimental protocol allows for rapid analysis of biodiesel mixtures of alcohols, fatty acids, glycerol, and mono- and disubstituted glycerides. Characteristic chemical shift ranges were developed with model compounds and used to fully characterize the conversion of triglyceride samples to biodiesel for two commercial production processes.

Kinetics of Secondary Reactions Affecting the Organosolv Lignin Structure.

Many valorization approaches for lignin rely on its organic solvent (organosolv) extraction. However, the severity of the extraction conditions required to obtain high lignin extraction generally results in low-quality lignin for downstream processing. To better understand the secondary reaction pathways and kinetics related to molecular alterations that result from organosolv extraction under extreme conditions, extractions were conducted at temperatures of 150, 180, and 210 °C. Lignin was collected at residence times between 0.25 and 18 h and analyzed by NMR techniques to quantify the concentrations of key chemical moieties that appear or disappear upon reactions of lignin molecules during and after their fractionation from biomass. The kinetics of chemical moiety evolution was modeled as processes in-series. In these models, pseudo first-order kinetics were used to describe the change in concentration of chemical moieties on extracted lignin as a function of residence time.