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The project will determine the detailed composition of process lignocellulosic biomass slurries and solids using NREL’s unique capabilities in the area.
Production of drop-in fuels from lignocellulose using Rhodococcus opacus PD630 (hereafter R. opacus) is a challenging goal. During the grant period we have pushed the field forward significantly in several areas of research. Towards the end goal of accelerating the adoption of R. opacus in biofuel production, during the grant period we have expanded the phenotypic characterization of R. opacus grown in single aromatic (model lignocellulosic) compounds or their mixtures, modeling the growth conditions in lignocellulosic biomass. Harnessing the power of adaptive evolution, we produced evolved R. opacus isolates with superior lignin valorization capabilities and identified differentially expressed genes and pathways after adaptation. We used next generation multi-omic techniques such as genomic, transcriptomic, and metabolomic analyses, to identify the catabolic pathways used by R. opacus to degrade aromatic compounds and funnel these degradation products into central metabolism, as well as the aromatic transport genes required for increased tolerance and utilization. Taking this information one step further, we identified endogenous transcription factors and regulatory mechanisms important for degradation of five model aromatic compounds. To accurately estimate R. opacus growth and consumption on model lignin compounds we pioneered the use of novel extraction procedures prior to GC-MS analysis. Alongside 13 C-metabolic flux analysis, we have elucidated the metabolic routes preferred by Rhodococcus opacus during aromatic compound degradation. Finally, we used in tandem lipidomics and high-resolution mass spectrometry to identify the modulation of mycolic acids and phospholipid membrane composition modification as a strategy for aromatic tolerance in R. opacus. Being a non-model organism, R. opacus lacks the breadth of tools and technical foundation which drive biofuel research in more well-understood microbes such as Escherichia coli. To reduce this burden for use, we designed and produced new tools for genomic manipulation and engineering in R. opacus. These engineering breakthroughs support efficient genomic editing, enabling gene overexpression, repression, and genetic alteration. Using these tools, we have generated synthetically engineered strains with increased lipogenesis and growth, both positive traits required for increased lignin valorization. Optimizing engineered strains for biofuel production from lignocellulose requires extremely sophisticated synthetic rewiring of metabolism. To facilitate systems-level reorganization of metabolism in R. opacus, we created a genome-scale model that accurately predicts metabolic flux and growth rates on the aromatic compound phenol. Lignin requires extensive pre-treatment before biological degradation by R. opacus. Towards an eventual goal of degrading real-world lignin, we developed new depolymerization processes to generate lignin breakdown products (LBP). We optimized LBP storage and composition analysis techniques, enabling accurate prediction of specific LBP compound integration into cell wall components. Overall, through the work funded by this grant we generated 20 manuscripts (17 published, 3 in review/preparation), methods for increased accuracy in metabolomics of aromatic compounds, multiple genetic tools for altering the R. opacus genome, genome scale models for predicting flux through metabolic pathways, as well as multi-omic data for community use. The work funded by this grant has increased the knowledge of aromatic degradation in bacteria and advanced our efforts to optimize R. opacus for lignin valorization.
Fast pyrolysis is traditionally defined as the rapid decomposition of organic material in the absence of oxygen to produce primarily a liquid product known as bio-oil. However, the introduction of small amounts of oxygen to the process holds prospects of internally generating the energy needed for pyrolysis. The present study investigates the partial oxidation of lignin-derived compounds during pyrolysis, which generates both carbon oxides and aromatic carbonyl compounds. Analysis of lignin derived phenolic compounds was performed to determine if the composition had changed under oxidative conditions. NMR analyses indicates aromatic carbonyls increased under oxidative conditions, with a corresponding decrease in phenolic hydroxyl groups. Finally, model phenolic compounds were pyrolyzed to help understand the role of partial oxidation during autothermal pyrolysis of lignocellulosic biomass.
Accurate representation of the chemical diversity of litter in ecosystem-scale models is critical for improving predictions of decomposition rates and stabilization of plant material into soil organic matter. In this contribution, we conducted a systematic review to evaluate how conventional characterization of plant litter quality using proximate analysis compares with molecular-scale characterization using 13 C NMR spectroscopy. Using a molecular mixing model, we converted chemical shift regions from NMR into fractions of carbon (C) in five organic compound classes that are major constituents of plant material: carbohydrates, proteins, lignins, lipids, and carbonylic compounds. We found positive correlations between the acid soluble fraction and carbohydrates, and between the acid insoluble fraction and lignins. However, the acid-soluble fraction underestimated carbohydrates, and the acid insoluble fraction overestimated lignins by 243%. We identified two sources of uncertainties: i) disparities between litter chemical composition based on hydrolysability and actual chemical composition obtained from NMR and ii) conversion factors to translate proximate fractions into organic constituents. Both uncertainties are critical, potentially leading to misinterpretations of decay rates in litter decomposition models. Consequently, we recommend including explicit substrate chemistry data in the next generation of litter decomposition models.
Reductive catalytic fractionation (RCF) is an effective lignin-first biorefining method to extract lignin as a stabilized oil from lignocellulosic biomass. To realize RCF at scale, process modeling has shown that minimizing the use of exogenous organic solvents is critical. To this end, here we investigate the ability of lignin-derived monomers to act as either solvents or cosolvents for RCF. We begin by examining the influence of lignin-derived aromatic compounds (4-propylguaiacol, 4-propylphenol, and propylbenzene) on RCF monomer yields and subsequently extend our analysis to mixtures of 4-propylguaiacol and either methanol or water. We demonstrate that 4-propylguaiacol is an effective solvent for lignin extraction and depolymerization during RCF, especially when used in combination with water as a cosolvent. Cosolvent mixtures of 4-propylguaiacol and water enable up to 81% lignin extraction, monomer yields up to 25 wt %, and postreaction phase separation. However, unlike methanol, water as a cosolvent fails to inhibit aromatic ring hydrogenation when conducted over Ru/C as a catalyst, potentially leading to excess hydrogen consumption in a process utilizing this approach. Nonetheless, these results suggest a promising strategy for eliminating external organic solvents from RCF by utilizing mixtures of lignin-derived compounds and water as alternative extraction solvents.
To explore the effect of lignin composition on cellulase adsorption, here dehydrogenation polymers (DHPs) were prepared from p-glucocoumaryl alcohol/coniferin/syringin, giving rise to H-DHP, G-DHP, and S-DHP, respectively. The structures of DHPs were thoroughly characterized and compared by GPC and NMR techniques, and the Langmuir isotherm protocol was applied to determine the cellulase adsorption behaviors of these different types of DHPs. The adsorption study indicated that the binding strength between the DHPs and cellulase varied in the following order: G-DHP > H-DHP > S-DHP. The inhibition of different types of DHPs on enzymatic hydrolysis of cellulose was in the same order as the cellulase adsorption, indicating that non-productive adsorption was the main way to influence cellulase. The correlation analysis results showed a positive association between the phenolic hydroxyl group content in DHPs and their maximum adsorption capacity toward enzymes. A negative correlation between the PDI and binding strength was also observed. It was also found that the adsorbed cellulase could be desorbed and retained normal enzyme activity, and so it was presumed that DHPs and cellulase were mainly linked by physisorption such as hydrogen bonding. This study clearly showed that the composition of lignin had a great impact on cellulase, and that G-type lignin exhibited the most detrimental effect. The results could provide useful information on the mechanism of cellulase adsorption onto lignin using DHPs as lignin model compounds.
Organosolv treatment is an efficient and environmentally friendly process to degrade lignin into small compounds. The capability of characterizing the individual compounds in the complex mixtures formed upon organosolv treatment is essential for the optimization of the further lignin conversion processes and for the rational genetic engineering of plants used to produce lignin in order to improve lignin properties. In this study, an organosolv poplar lignin sample was initially analyzed by high-resolution mass spectrometry coupled with negative-ion mode electrospray ionization ((-)ESI HRMS). Lignin monomers and dimers were found to constitute the majority of the compounds in the organosolv lignin sample. Larger lignin oligomers, such as trimers and tetramers, and some not lignin-related compounds, were also detected. A high-performance liquid chromatograph/linear quadrupole ion trap/orbitrap mass spectrometer capable of multi-stage high-resolution tandem mass spectrometry experiments (HRMS n ), equipped with an (-)ESI source (HPLC/(-)ESI HRMS n ), was employed to separate the unknown compounds in the organosolv mixture and to obtain structural information for the deprotonated compounds via collision-activated dissociation (CAD) HRMS n experiments. To improve the understanding of the CAD behavior of deprotonated lignin-related compounds, 16 deprotonated model compounds with different functionalities and linkage types were examined. This approach enabled the assignment of likely structures for several lignin monomers, dimers, trimers, and tetramers, and some not lignin-related compounds, most likely fatty acids. Based on the proposed structures, compounds in the organosolv lignin sample contain β-O-4, 5–5, β-5, and possibly also 4-O-5 linkages. Most compounds contain G- and S-monomeric units although a small amount of H-units were also detected.