Biomass Conversion Efficiency Research Articles

The family Thermoactinomycetaceae: an emerging microbial resource with high application value

In recent years, interest has increased in the use of microorganisms to obtain additional valuable resources for green and sustainable development. Preliminary functional analyses have indicated that members of the family Thermoactinomycetaceae have good application potential for the production of novel specific enzymes, high-value bioactive compounds, novel secondary metabolites and the promotion of efficient biomass conversion. Therefore, they can be considered a new class of microbial resources with potentially high value. However, the lack of culture and culture-independent techniques coupled with the uncertain taxonomic status of the family Thermoactinomycetaceae, has made exploring these potential applications challenging. This paper reviews the distribution characteristics and functional properties of the family Thermoactinomycetaceae, providing a detailed interpretation of the potential application value of this group and proposing a set of systematic resource development strategies based on a combination of culture-dependent and culture-independent strategies to exploit its potential for resource applications.

Electrocatalytic Biomass Oxidation via Acid-Induced In Situ Surface Reconstruction of Multivalent State Coexistence in Metal Foams.

Electrocatalytic biomass conversion offers a sustainable route for producing organic chemicals, with electrode design being critical to determining reaction rate and selectivity. Herein, a prediction-synthesis-validation approach is developed to obtain electrodes for precise biomass conversion, where the coexistence of multiple metal valence states leads to excellent electrocatalytic performance due to the activated redox cycle. This promising integrated foam electrode is developed via acid-induced surface reconstruction to in situ generate highly active metal (oxy)hydroxide or oxide (MOxHy or MOx) specieson inert foam electrodes, facilitating the electrooxidation of 5-hydroxymethylfurfural (5-HMF) to 2,5-furandicarboxylic acid (FDCA). Taking nickel foam electrode as an example, the resulting NiOxHy/Ni catalyst, featuring the coexistence of multivalent states of Ni, exhibits remarkable activity and stability with a FDCA yields over 95% and a Faradaic efficiency of 99%. In situ Raman spectroscopy and theoretical analysis reveal an Ni(OH)2/NiOOH-mediated indirect pathway, with the chemical oxidation of 5-HMF as the rate-limiting step. Furthermore, this in situ surface reconstruction approach can be extended to various metal foams (Fe, Cu, FeNi, and NiMo), offering a mild, scalable, and cost-effective method for preparing potent foam catalysts. This approach promotes a circular economy by enabling more efficient biomass conversion processes, providing a versatile and impactful tool in the field of sustainable catalysis.

Biocrude production via hydrothermal liquefaction of cycas circinalis seed shell: A machine learning approach

ABSTRACT Hydrothermal liquefaction (HTL) is a promising thermochemical method for converting biomass into bio-crude fuel. This study explores the HTL of Cycas circinalis seed shell (CSS), focusing on the impacts of reaction time, feed slurry concentration, and temperature on bio-crude yield. Experiments were conducted at temperatures ranging from 250 to 375°C, reaction times from 10 to 40 minutes, and feed slurry concentrations between 10% and 30%. A decision tree regression (DTR) model predicted the optimal bio-crude yield of 35% at 375°C, 30% feed slurry concentration, and 40 minutes, with high accuracy (R² = 0.9853, RMSE = 0.992). Results highlight temperature and time as key factors influencing bio-crude production.The bio-crude was characterized using Fourier-transform infrared spectroscopy (FTIR), thermogravimetric analysis (TGA), nuclear magnetic resonance (NMR), and gas chromatography-mass spectrometry (GC-MS). Degradation kinetics of bio-crude and CSS biomass were analyzed using the Coats-Redfern method at heating rates of 5, 10, and 20°C/min. Parameters such as activation energy (E), rate constant, pre-exponential factor (A), enthalpy, entropy, and Gibbs free energy were determined. This research advances hydrothermal technology and promotes the development of sustainable and efficient biomass conversion processes.

Functionalized covalent organic frameworks for catalytic conversion of biomass-derived xylan to furfural.

A direct synthesis strategy was employed to prepare functionalized COFs enriched with acidic sites, using various precursor monomers. The functionalized COFs were applied in the catalytic conversion of biomass-derived xylan to furfural in the liquid phase. The study further assessed the recyclability and reusability of these COFs, explored the relationship between their structural features and catalytic performance, and investigated the reaction mechanism underlying the COF-catalyzed conversion of xylan to furfural. The results revealed the successful solvothermal synthesis of four COFs (COF-LZU1, TP-PA, TB-DAB, and TP-DAB). Among them, TP-DAB exhibited the highest acidity, reaching 2.092mmol/g, due to its rich content of -OH and -COOH. Additionally, TP-DAB demonstrated a well-developed porous structure and excellent thermal stability, essential characteristics for a highly efficient solid acid catalyst. When employed as a catalyst in a mixed solvent system of H2O/THF at 160°C for 180min, TP-DAB achieved complete xylan conversion with furfural yield of 63.49%. Moreover, TP-DAB maintained good stability and reusability, being effective for at least five cycles. This study presents a novel and highly effective green catalyst for the sustainable conversion of biomass-derived sugars into furfural, providing new avenues for the efficient conversion of biomass into fine chemicals.

Simultaneous Down-Regulation of Dominant Cinnamoyl CoA Reductase and Cinnamyl Alcohol Dehydrogenase Dramatically Altered Lignin Content in Mulberry.

Mulberry (Morus alba L.) is a significant economic tree species in China. The lignin component serves as a critical limiting factor that impacts both the forage quality and the conversion efficiency of mulberry biomass into biofuel. Cinnamoyl CoA reductase (CCR; EC 1.21.1.44) and cinnamyl alcohol dehydrogenase (CAD; EC 1.1.1.95) are the key enzymes that catalyze the final two reductive steps in the biosynthesis of monolignols. In this study, we conducted a comprehensive functional analysis to validate the predominant CCR genes involved in monolignol biosynthesis. In this study, we initially validated the predominant CCR genes implicated in monolignol biosynthesis through an extensive functional analysis. Phylogenetic analysis, tissue-specific expression profiling and enzymatic assays indicated that MaCCR1 is the authentic CCR involved in lignin biosynthesis. Furthermore, the expression level of MaCCR1 exhibited a significant positive correlation with lignin content, and the down-regulation of MaCCR1 via virus-induced gene silencing resulted in altered lignin content in mulberry. The down-regulation of MaCCR1 and MaCAD3/4, both individually and concurrently, exhibited markedly different effects on lignin content and mulberry growth. Specifically, the simultaneous down-regulation of MaCCR1 and MaCAD3/4 significantly altered lignin content in mulberry, resulting in dwarfism of the plants. Conversely, the down-regulation of MaCAD3/4 alone not only decreased lignin content but also led to an increase in biomass. These findings offer compelling evidence elucidating the roles of MaCCRs in mulberry and identify specific target genes, thereby providing a crucial foundation for the genetic modification of lignin biosynthesis.

Is Kraft Pulping the Future of Biorefineries? A Perspective on the Sustainability of Lignocellulosic Product Development.

By reflecting on the history and environmental impact of conventional biorefining, such as kraft pulping, we aim to explore important questions about how natural polymers can be more sustainably sourced to develop bio-products and reduce reliance on plastics. Since the Industrial Revolution, chemical pulping processes have enabled the mass production of cellulosic products from woody biomass. Kraft pulping, which dominates within modern pulp and paper mills, has significantly contributed to environmental pollution and carbon emissions due to sulfurous byproducts and its high water and energy consumption. While chemical pulping technologies have advanced over time, with improvements aimed at enhancing sustainability and economic feasibility, conventional biorefineries still face challenges related to biomass conversion efficiency and environmental impact. For example, efforts to fully utilize wood resources, such as isolating lignin from black liquor, have made limited progress. This perspective provides a thoughtful examination of the growth of chemical pulping, particularly the kraft process, in the production of consumer goods and its environmental consequences. It also presents key insights into the bottlenecks in developing truly sustainable biomass conversion technologies and explores potential alternatives to traditional chemical pulping.

Process integration and intensification for efficient catalytic conversion of biomass

Process integration and intensification for efficient catalytic conversion of biomass

Synergistic enhancement of nickel-cobalt nano-catalysts for 5-hydroxymethylfurfural conversion via electronic structure engineering and solar intensification

The electrooxidation of 5-hydroxymethylfurfural (HMF) into vital chemical intermediates has received widespread attention. In order to achieve efficient conversion of HMF, the main focus has been on the design and development of delicate catalysts. In addition, the introduction of an external field in the reaction process can also improve the electrocatalytic efficiency. In this paper, nickel-cobalt composite oxide catalysts with controlled metal centers (Ni/Co) were synthesized by a simple “coating-calcination” method. The rational rearrangement of d-electron by Co-doping facilitated the improvement of HMF adsorption, which was further confirmed by Density Functional Theory calculations. The catalysts synthesized at the optimal Co-doping ratio could still achieve 100 % of HMF conversion, 95.65 % of 2,5-furandicarboxylic acid yield, and 92.97 % of Faraday efficiency after 25 electrochemical cycles. Crucially, we introduced solar illumination to further enhance the performance of electrooxidation HMF. The catalyst performance was further improved via local photothermal effect and additional photocurrent. Therefore, the yield of FDCA was increased by more than 27 % under 2.0 kW m−2 illuminations. An electrooxidation system constructed based on external field enhancement and catalyst design provides new insights into the efficient conversion of biomass.

Nanobiohybrid Extracellular Vesicle Nanoreactor with Improving Metabolical Activity for Biocatalytic Therapy.

Extracellular vesicles (EVs) hosting enzymatic activities that function as independent metabolic units are attractive natural biocatalytic platforms. However, directly using these metabolically active nanoreactors for effective biocatalytic applications remains challenging, mainly due to their constrained catalytic capabilities. Here, we construct an EV-templated nanobiohybrid system by engineering an EV surface with a photoresponsive zeolitic imidazolate framework (ZIF). The deposition of ZIF nanostructures on EVs not only contributes to improved biocatalytic stability but also enables interfacial coupling between photoexcited electrons from the ZIF and the enzymatic reaction of metabolically active EVs. Nearly 300% of biomass conversion efficiency increment could be achieved by EVs derived from macrophages. This enhanced biocatalysis, high catalytic stability, and low cytotoxicity endowed the EV@ZIF nanosystem with robust biosynthesis and antimicrobial activity. When evaluated in a mouse periodontitis model, we show that the autologous biocatalytic EV@ZIF demonstrated efficient therapeutic capability by killing bacteria and inhibiting inflammation. This nanoengineering strategy will benefit the future optimization of metabolically active EV nanoreactors as biocatalysts for a broad range of therapeutics.

Study on the Effect of n-Hexane Insolubles on Pyrolysis Product Yields Based on Statistical Regression Analysis

Pyrolysis technology has attracted much attention due to its efficient biomass conversion capability, but the significant impact of n-hexane insoluble matter (INS) concentration on pyrolysis product yield has not been fully studied. Therefore, it is of great significance to explore the impact of INS on the yields of tar, water and coke residue. This study developed a linear regression model to analyze the effect of INS concentration on these pyrolysis products. The results show that INS concentration has a significant positive impact on tar yield, but has a smaller impact on water and coke residue yields. In addition, this study constructed a generalized linear model (GLM) to examine the interaction between INS concentration and mixing ratio, and found that the interaction effect was not significant. These findings provide valuable insights into optimizing the co-pyrolysis process of biomass and coal, helping to improve energy conversion efficiency and reduce environmental impact.

Spontaneous Corrosion Induced Scalable Fabrication of Bayberry‐Like Ni@Ni3S2 Core–Shell Catalysts for 5‐Hydroxymethylfurfural Oxidation

AbstractElectrocatalytic oxidation of biomass‐derived 5‐hydroxymethylfurfural (HMF) presents a promising pathway for synthesizing the value‐added chemical, 2,5‐furandicarboxylic acid (FDCA). However, it is still lack of scalable strategies to fabricate electrocatalysts with high activity and selectivity. Herein, a bayberry‐like Ni@Ni3S2 core–shell catalyst is prepared via a spontaneous corrosion strategy for HMF electro‐oxidation. The Ni3S2 shells with abundant electron‐deficient Ni sites facilitate the generation of high‐valence Ni3+OOH active species. Additionally, the in situ oxidation of S2− to SO42− enhances the electron and proton transport capacity. Both high‐performance liquid chromatography (HPLC) and density functional theory (DFT) analyses reveal that Ni@Ni3S2 preferentially selects the 2,5‐dicarboxyfurylfuran (DFF) pathway due to the lower reaction energy barrier from HMF to DFF, and the rate‐determining step of DFF oxidation to generate 5‐formylfuran‐2‐carboxylic acid (FFCA) is significantly accelerated. Consequently, the yield of FDCA and Faraday efficiency are found to be 99.20% and 99.35%, respectively, and they still reach 88.79% and 90.33% after ten cycles, showing excellent stability. Moreover, a mass production of 13.97 g Ni@Ni3S2 is successfully synthesized using the spontaneous corrosion strategy. This work offers a valuable reference for designing scalable and high‐performance catalysts by manipulating reaction routes to achieve efficient biomass conversion.

Engineering Lewis-Acid Defects on ZnO Quantum Dots by Trace Transition-Metal Single Atoms for High Glycerol-to-Glycerol Carbonate Conversion.

Efficient conversion of biomass wastes into valuable chemicals has been regarded as a sustainable approach for green and circular economy. Herein, a highly efficient catalytic conversion of glycerol (Gly) into glycerol carbonate (GlyC) by carbonylation with the commercially available urea is presented using low-cost transition metal single atoms supported on zinc oxide quantum dots (M1-ZnO QDs) as a catalyst without using any solvent. A facile one-step wet chemical synthesis allows various types of metal single atoms to simultaneously dope and introduce Lewis-acid defects in the ZnO QD structure. It is found that doping with a trace amount of isolated metal atoms greatly boosts the catalytic activity with Gly conversion of 90.7%, GlyC selectivity of 100.0%, and GlyC yield of 90.6%. Congruential results from both Density Functional Theory (DFT)and in situ Diffuse Reflectance Infrared Fourier Transform Spectroscopy (in situ DRIFTS) studies reveal that the superior catalytic performance can be attributed to the enriched Lewis acid sites that endow optimal adsorption, formation of the intermediate for coupling between urea and Gly, and desorption of GlyC. Moreover, the tiny size of ZnO QDs efficiently promotes the accessibility of these active sites to the reactants.

Hybrid zeolites in 1,4-dioxane-mediated co-solvent efficiently convert glucose to the biofuel 5-ethoxymethylfurfural

Direct and efficient conversion of abundant renewable biomass carbohydrates to 5-ethoxymethylfurfural (EMF), a potential biofuel candidate, is highly desirable but remains challenging. We report here the catalytic transformation of glucose using hybrid Hβ and USY zeolites in 1,4-dioxane-contained ethanol solution, leading to EMF production with an excellent yield of 60 % within 1 h. Hybrid zeolites rational modulate the chemical space, acidity, and acid sites synergistically to drive the imperative cascade reactions during the conversion. Equally important, the introduction of low-polarity aprotic 1,4-dioxane co-solvent can effectively stabilize the obtained EMF because the preferential solubilization of EMF by 1,4-dioxane resulted in a reduction of their nucleophilic charge, which in turn lowers its susceptibility to nucleophilic attack and minimizes the undesirable reactions. This work not only develops a promising protocol for EMF production from glucose but also sheds light on the mechanistic role of hybrid zeolites and 1,4-dioxane co-solvent in the cascade transformation.

A miniaturized feedstocks-to-fuels pipeline for screening the efficiency of deconstruction and microbial conversion of lignocellulosic biomass.

Sustainably grown biomass is a promising alternative to produce fuels and chemicals and reduce the dependency on fossil energy sources. However, the efficient conversion of lignocellulosic biomass into biofuels and bioproducts often requires extensive testing of components and reaction conditions used in the pretreatment, saccharification, and bioconversion steps. This restriction can result in a significant and unwieldy number of combinations of biomass types, solvents, microbial strains, and operational parameters that need to be characterized, turning these efforts into a daunting and time-consuming task. Here we developed a high-throughput feedstocks-to-fuels screening platform to address these challenges. The result is a miniaturized semi-automated platform that leverages the capabilities of a solid handling robot, a liquid handling robot, analytical instruments, and a centralized data repository, adapted to operate as an ionic-liquid-based biomass conversion pipeline. The pipeline was tested by using sorghum as feedstock, the biocompatible ionic liquid cholinium phosphate as pretreatment solvent, a "one-pot" process configuration that does not require ionic liquid removal after pretreatment, and an engineered strain of the yeast Rhodosporidium toruloides that produces the jet-fuel precursor bisabolene as a conversion microbe. By the simultaneous processing of 48 samples, we show that this configuration and reaction conditions result in sugar yields (~70%) and bisabolene titers (~1500 mg/L) that are comparable to the efficiencies observed at larger scales but require only a fraction of the time. We expect that this Feedstocks-to-Fuels pipeline will become an effective tool to screen thousands of bioenergy crop and feedstock samples and assist process optimization efforts and the development of predictive deconstruction approaches.

Insights into lignin bioconversion: lignin-derived compounds treatment of a novel marine fungus K-2.

The potential for the efficient conversion of lignocellulosic biomass has been extensively explored to produce a wide range of bioproducts. Many approaches have been sought for the deep conversion of lignin to generate products that are toxin-free and beneficial for processing into high-value-added components. This study reported a fungus isolated from the deep sea with strong synthesis of multiple lignocellulases, conversion of lignin and guaiacol (0.1%) by 71.6% and 86.1% within 9 days at 30 °C respectively, and outstanding environmental adaptability (20-50 °C and pH 3-8). Metabolic pathway profiling showed that this fungus utilized lignin to rapidly activate multiple ring-opening reactions including the 2,3- and 3,4-cleavage pathways, with the 2,3-cleavage pathway predominating after 5 days. Conversion of metabolic intermediates confirmed the superb potential of this strain for lignin treatment. Meanwhile, its shikimic acid pathway was metabolically active under lignin. This further expands the potential to produce valuable bioproducts during lignin treatment, especially under ambient conditions, which can significantly enhance high-value precursor compound production. © 2024 Society of Chemical Industry.

Investigating structural disparities and catalytic depolymerization of larch lignin for high-value biomass conversion

Understanding the structural diversity of lignin is crucial for its efficient conversion into high-value aromatic compounds. However, the impact of lignin's structural characteristics from artificial forests on catalytic depolymerization is not well understood. This study investigates the structural differences in lignin from larch trees of varying ages (10, 20, and 40 years) and anatomical parts (heartwood, sapwood, canopy, bark) to understand how these variations influence catalytic depolymerization. Polyoxometalates (POMs) were selected as catalysts due to their high efficiency in facilitating the conversion of lignin into valuable chemical products. By utilizing POMs for catalytic oxidation, we explore the relationship between lignin structure and depolymerized products, providing insights into the structural features of larch lignin. Our findings reveal that the β-O-4 content of heartwood lignin is as high as 78.7 %, while that of the canopy is lower at 67.1 %. Additionally, the unique lignin structure significantly impacted the yield and selectivity of aromatic monomers, with the highest monomer yield from heartwood lignin reaching 15.64 %, compared to the lowest yield of 7.46 % from canopy lignin. These results lay a theoretical foundation for developing efficient biomass conversion technologies and offer novel strategies for sustainable biomass utilization and environmentally friendly production.

Thermodynamic evaluation of decarbonized power production based on solar energy integration

This study aims to create an environmentally friendly system by utilizing solar and biomass energies as renewable sources, employing high temperature and pressure ranges for analysis. A new energy system is introduced, using biomass to produce hydrogen and other valuable commodities. The study evaluates power and hydrogen production technologies from a thermodynamic perspective, including conventional biomass gasification, calcium and iron thermochemical looping cycles, and gasification with supercritical water, incorporating carbon capture techniques. It assesses and compares the performance of locally available biomass material, such as cotton stalk, during gasification. Results show that employing the supercritical water gasification cycle yields the highest hydrogen production (8330 kg/h) and carbon capture rate (92.91 %), with potential for significant reduction in CO2 emissions. Supercritical water gasification also demonstrates electricity, heating, and CO2 production rates of 51.48 MW, 23.3 MW, and 16.6 MW, respectively. Integrating supercritical water gasification with solar energy enhances syngas productivity and system efficiency. Additionally, the highest performance is demonstrated in the SCWG case, which exhibits the lowest irreversibility at 320.32 MW. In conclusion, the system with supercritical water gasification emerges as the most efficient biomass conversion method, with higher energy and exergy efficiencies of 67.59 % and 49.74 %, respectively, under specific operating conditions. The developed model facilitates integration of solar energy into biomass gasification, enabling prediction and comparison of performance in terms of energy and exergy efficiencies.

Study on Cracking/Oxidation/Integrated Reforming Reaction for Efficient Conversion of Biomass to High‐Quality Syngas

AbstractThe advanced gasification technology of coal is mainly based on oxidation reaction and high temperature but is not suitable for biomass conversion. High tar and CO2 content are the two main issues that affect the efficiency of biomass gasification. In order to deeply convert hydrocarbons/tar and CO2 simultaneously, and enhance syngas yield, the cracking/partial oxidation/reforming reactions and their integrated reaction routes are investigated from an interrelated view. The effects of each reaction on the distribution of C/H elements in hydrocarbons/tar and syngas are illustrated. By cracking and oxidation reaction, the syngas yield can only reach 0.93 Nm3 kg−1, about 58 % of the theoretical maximum value; a large proportion of residual C/H atoms existing in stable hydrocarbons/tar/CO2/H2O are not converted. Based on the concept of lattice O oxidation combined with dry reforming, it realizes syngas yield (CO+H2) 1.56 Nm3 kg−1 with 91.6 % concentration, demonstrating that tar/hydrocarbons and CO2/H2O are converted to syngas efficiently. The effects of [O]/C ratio on gas yield represent a synergistic coordination between lattice Os oxidation and catalytic reforming reaction. Oxidation‐reforming is the optimum route for biomass conversion to high‐quality syngas.

Production of syngas from oil palm shell biomass using microwave gasification

The research work on microwave gasification (MWG) is limited in the literature compared to pyrolysis, and little attention has been paid to its efficiency. The present study aims to investigate the effect of MW power (20–40 %) and absorber loading (0–15 wt%) on the temperature, gas composition, syngas yield, quality, and efficiencies during gasification of oil palm shell biomass in a 1.25 kW and 2.45 GHz lab-scale microwave (MW) system. Overall, the yield and quality of syngas increased with MW power, but it decreased after reaching optimum absorber loading. The highest product gas (63.3 wt%) with a heating value of 11.66 MJ/m3 was obtained at an optimum process condition (absorber loading of 10 wt% and MW power of 40 %). Under these conditions, the heating rate of 17 °C/s and maximum syngas (CO + H2) (76.5 vol%) were recorded. The specific energy consumption increased with MW power but dropped with increasing absorber loading, demonstrating MWG to be more energy efficient at higher biomass loading. MW process efficiency (19.7 %) and biomass conversion efficiency (55.6 %) were achieved at optimum process conditions while considering syngas only. The MWG system can become more energy and process-efficient if all byproducts are utilized and scaled up.

Supercharged cellulases show superior thermal stability and enhanced activity towards pretreated biomass and cellulose

Non-productive binding of cellulolytic enzymes to various plant cell wall components, such as lignin and cellulose, necessitates high enzyme loadings to achieve efficient conversion of pretreated lignocellulosic biomass to fermentable sugars. Protein supercharging was previously employed as one of the strategies to reduce non-productive binding to biomass. However, various questions remain unanswered regarding the hydrolysis kinetics of supercharged enzymes towards pretreated biomass substrates and the role played by enzyme interactions with individual cell wall polymers such as cellulose and xylan. In this study, CBM2a (from Thermobifida fusca) fused with endocellulase Cel5A (from T. fusca) was used as the model wild-type enzyme and CBM2a was supercharged using Rosetta, to obtain eight variants with net charges spanning −14 to +6. These enzymes were recombinantly expressed in E. coli, purified from cell lysates, and their hydrolytic activities were tested against pretreated biomass substrates (AFEX and EA treated corn stover). Although the wild-type enzyme showed greater activity compared to both negatively and positively supercharged enzymes towards pretreated biomass, thermal denaturation assays identified two negatively supercharged constructs that perform better than the wild-type enzyme (∼3 to 4-fold difference in activity) upon thermal deactivation at higher temperatures. To better understand the causal factor of reduced supercharged enzyme activity towards AFEX corn stover, we performed hydrolysis assays on cellulose-I/xylan/pNPC, lignin inhibition assays, and thermal stability assays. Altogether, these assays showed that the negatively supercharged mutants were highly impacted by reduced activity towards xylan whereas the positively supercharged mutants showed dramatically reduced activity towards cellulose and xylan. It was identified that a combination of impaired cellulose binding and lower thermal stability was the cause of reduced hydrolytic activity of positively supercharged enzyme sub-group. Overall, this study demonstrated a systematic approach to investigate the behavior of supercharged enzymes and identified supercharged enzyme constructs that show superior activity at elevated temperatures. Future work will address the impact of parameters such as pH, salt concentration, and assay temperature on the hydrolytic activity and thermal stability of supercharged enzymes.