Shangxian Xie

Synergistic enzymatic and microbial lignin conversion

The utilization of lignin for fungible fuels and chemicals represents one of the most imminent challenges in modern biorefineries. However, bioconversion of lignin is highly challenging due to its recalcitrant nature as a phenolic heteropolymer. This study addressed the challenges by revealing the chemical and biological mechanisms for synergistic lignin degradation by a bacterial and enzymatic system, which significantly improved lignin consumption, cell growth and lipid yield. The Rhodococcus opacus cell growth increased exponentially in response to the level of laccase treatment, indicating the synergy between laccase and bacterial cells in lignin degradation. Other treatments like iron and hydrogen peroxide showed limited impact on cell growth. Chemical analysis of lignin under various treatments further confirmed the synergy between laccase and cells at the chemical level. 31P nuclear magnetic resonance (NMR) suggested that laccase, R. opacus cell and Fenton reaction reagents promoted the degradation of different types of lignin functional groups, elucidating the chemical basis for the synergistic effects. 31P NMR further revealed that laccase treatment had the most significant impact for degrading the abundant chemical groups. The results were further confirmed by the molecular weight analysis and lignin quantification by the Prussian blue assay. The cell–laccase fermentation led to a 17-fold increase of lipid production. Overall, the study indicated that laccase and R. opacus can synergize to degrade lignin efficiently, likely through rapid utilization of monomers generated by laccase to promote the reaction toward depolymerization. The study provided a potential path for more efficient lignin conversion and development of consolidated lignin conversion.

Comparative genomic analysis of the endosymbionts of herbivorous insects reveals eco-environmental adaptations: biotechnology applications.

Metagenome analysis of the gut symbionts of three different insects was conducted as a means of comparing taxonomic and metabolic diversity of gut microbiomes to diet and life history of the insect hosts. A second goal was the discovery of novel biocatalysts for biorefinery applications. Grasshopper and cutworm gut symbionts were sequenced and compared with the previously identified metagenome of termite gut microbiota. These insect hosts represent three different insect orders and specialize on different food types. The comparative analysis revealed dramatic differences among the three insect species in the abundance and taxonomic composition of the symbiont populations present in the gut. The composition and abundance of symbionts was correlated with their previously identified capacity to degrade and utilize the different types of food consumed by their hosts. The metabolic reconstruction revealed that the gut metabolome of cutworms and grasshoppers was more enriched for genes involved in carbohydrate metabolism and transport than wood-feeding termite, whereas the termite gut metabolome was enriched for glycosyl hydrolase (GH) enzymes relevant to lignocellulosic biomass degradation. Moreover, termite gut metabolome was more enriched with nitrogen fixation genes than those of grasshopper and cutworm gut, presumably due to the termites adaptation to the high fiber and less nutritious food types. In order to evaluate and exploit the insect symbionts for biotechnology applications, we cloned and further characterized four biomass-degrading enzymes including one endoglucanase and one xylanase from both the grasshopper and cutworm gut symbionts. The results indicated that the grasshopper symbiont enzymes were generally more efficient in biomass degradation than the homologous enzymes from cutworm symbionts. Together, these results demonstrated a correlation between the composition and putative metabolic functionality of the gut microbiome and host diet, and suggested that this relationship could be exploited for the discovery of symbionts and biocatalysts useful for biorefinery applications.

Simultaneous conversion of all cell wall components by an oleaginous fungus without chemi-physical pretreatment

Lignin utilization during biomass conversion has been a major challenge for lignocellulosic biofuel. In particular, the conversion of lignin along with carbohydrate for fungible fuels and chemicals will both improve the overall carbon efficiency and reduce the need for chemical pretreatments. However, few biomass-converting microorganisms have the capacity to degrade all cell wall components including lignin, cellulose, and hemicellulose. We hereby evaluated a unique oleaginous fungus strain, Cunninghamella echinulata FR3, for its capacity to degrade lignin during biomass conversion to lipid, and the potential to carry out consolidated fermentation without chemical pretreatment, especially when combined with sorghum (Sorghum bicolor) bmr mutants with reduced lignin content. The study clearly showed that lignin was consumed together with carbohydrate during biomass conversion for all sorghum samples, which indicates that this organism has the potential for biomass conversion without chemical pretreatment. Even though dilute acid pretreatment of biomass resulted in more weight loss during fungal fermentation than untreated biomass, the lipid yields were comparable for untreated bmr6/bmr12 double mutant and dilute acid-pretreated wild-type biomass samples. The mechanisms for lignin degradation in oleaginous fungi were further elucidated through transcriptomics and chemical analysis. The studies showed that in C. echinulata FR3, the Fenton reaction may play an important role in lignin degradation. This discovery is among the first to show that a mechanism for lignin degradation similar to those found in white and brown rot basidiomycetous fungi exists in an oleaginous fungus. This study suggests that oleaginous fungi such as C. echinulata FR3 can be employed for complete biomass utilization in a consolidated platform without chemical pretreatment or can be used to convert lignin waste into lipids.

Quality carbon fibers from fractionated lignin

The application of lignin carbon fibers was hindered by their low quality and mechanical performance. We addressed this challenge by developing a new approach to fractionate and modify lignin to produce quality carbon fibers using an enzyme-mediator system, which derives lignin fractions with different molecular weights, functional groups, and interunitary linkages. The fractionated lignin in general improves the miscibility and spinnability of lignin. In particular, the insoluble lignin fraction renders carbon fibers with a significantly improved turbostratic carbon structure as revealed by XRD and Raman spectroscopy. The improvement in the carbon structure leads to the significantly improved elastic modulus. The results suggest that higher molecular weights, less –OH groups, and more linear structures may contribute to the improved crystallization and mechanical performance of lignin carbon fibers. The technical breakthrough produces lignin-based carbon fibers with a similar elastic modulus to commercial carbon fibers for the first time, and paves the path for replacing PAN with lignin for producing quality carbon fibers.

Molecular weight and uniformity define the mechanical performance of lignin-based carbon fiber

Lignin-based carbon fiber represents a renewable and low-cost alternative to petroleum-based carbon fiber. However, the poor mechanical performance of current lignin carbon fiber hinders its application. We hypothesized that the lower optimal mechanical performance is caused by the inherent heterogeneity of lignin. It is still unknown how the molecular weights (MWs) and lignin uniformity will impact the performance of lignin carbon fiber. We thereby addressed this hypothesis by fractionating lignin into fractions with different MWs and polydispersity indices (PDIs). An enzyme–mediator-based method and a dialysis method were developed to derive lignin fractions with increased MW and decreased PDI. Lignin fractions were electro-spun into fibers after blending with polyacrylonitrile (PAN) at 1 : 1 (w/w) ratio. The fractionation in general improved the spinnability of lignin to allow us to obtain finer lignin-based carbon fibers. The elastic modulus of lignin carbon fibers, as measured by nanoindentation, was increased as the lignin MW increased and as PDI decreased. The scatter plot and linear regression revealed very good correlation between the elastic modulus and PDI, as well as certain correlation between the elastic modulus and MW. XRD and Raman spectroscopy revealed that the crystallite size and the content of the pre-graphitic turbostratic carbon increased with higher lignin MW and lower PDI, revealing the mechanism of the improvement in carbon fiber mechanical performance. This study elucidated the impacts of lignin MW and uniformity on the mechanical properties of carbon fiber, and could thus guide the development of lignin processing technologies for quality lignin-based carbon fiber.

Genomic and molecular mechanisms for efficient biodegradation of aromatic dye

Understanding the molecular mechanisms for aromatic compound degradation is crucial for the development of effective bioremediation strategies. We report the discovery of a novel phenomenon for improved degradation of Direct Red 5B azo dye by Irpex lacteus CD2 with lignin as a co-substrate. Transcriptomics analysis was performed to elucidate the molecular mechanisms of aromatic degradation in white rot fungus by comparing dye, lignin, and dye/lignin combined treatments. A full spectrum of lignin degradation peroxidases, oxidases, radical producing enzymes, and other relevant components were up-regulated under DR5B and lignin treatments. Lignin induced genes complemented the DR5B induced genes to provide essential enzymes and redox conditions for aromatic compound degradation. The transcriptomics analysis was further verified by manganese peroxidase (MnP) protein over-expression, as revealed by proteomics, dye decolorization assay by purified MnP and increased hydroxyl radical levels, as indicated by an iron reducing activity assay. Overall, the molecular and genomic mechanisms indicated that effective aromatic polymer degradation requires synergistic enzymes and radical-mediated oxidative reactions to form an effective network of chemical processes. This study will help to guide the development of effective bioremediation and biomass degradation strategies.

Enhancement of Environmental Hazard Degradation in the Presence of Lignin: a Proteomics Study

Proteomics studies of fungal systems have progressed dramatically based on the availability of more fungal genome sequences in recent years. Different proteomics strategies have been applied toward characterization of fungal proteome and revealed important gene functions and proteome dynamics. Presented here is the application of shot-gun proteomic technology to study the bio-remediation of environmental hazards by white-rot fungus. Lignin, a naturally abundant component of the plant biomass, is discovered to promote the degradation of Azo dye by white-rot fungus Irpex lacteus CD2 in the lignin/dye/fungus system. Shotgun proteomics technique was used to understand degradation mechanism at the protein level for the lignin/dye/fungus system. Our proteomics study can identify about two thousand proteins (one third of the predicted white-rot fungal proteome) in a single experiment, as one of the most powerful proteomics platforms to study the fungal system to date. The study shows a significant enrichment of oxidoreduction functional category under the dye/lignin combined treatment. An in vitro validation is performed and supports our hypothesis that the synergy of Fenton reaction and manganese peroxidase might play an important role in DR5B dye degradation. The results could guide the development of effective bioremediation strategies and efficient lignocellulosic biomass conversion.

CHAPTER 13:Reverse Design of Natural Biomass Utilization Systems for Biomass Conversion

Bioethanol produced from lignocellulosic feedstocks, such as grass, wood, and agricultural residues, offer an attractive option for a sustainable energy source. Rapid development and deployment of cost-competitive 2nd generation biofuels has made considerable progress in recent years, however, key conversion steps remain technically challenging. Natural biomass utilization systems (NBUS) show potential for reverse-designing cost-effective biorefineries that consolidate biomass conversion steps – pretreatment, hydrolysis, and ethanol fermentation - into a single bioprocessing event. Insect digestive systems and those of ruminant animals represent the pinnacle of NBUS systems for elucidating lignocellulosic deconstruction mechanisms and novel biocatalyst discovery. Emergence of new ‘omics technologies and systems biology approaches have facilitated understanding of complex and dynamic symbiotic interactions between host and gut microbes. Furthermore, synthetic biology has demonstrated that biomass conversion metabolic processes can be integrated into organisms more suited for tolerating high temperatures and inhibitory compounds prevalent in current biomass conversion technologies.