Lignocellulosic biomass is a feedstock for fuels and chemicals that does not compete with food resources and has less contaminants than refuse-derived biomass feedstocks. To convert lignocellulosics to biofuels or value-added products, multiple processing steps are typically necessary. One method of producing biofuels from lignocellulosic biomass utilizes a deacetylation and mechanical refining pretreatment and an enzymatic hydrolysis reaction to produce fermentable sugars from cellulose and hemicellulose. The rheological properties of biomass, such as yield stress and plastic viscosity, change during enzymatic hydrolysis and alter the energy requirements for pumping and mixing, an important consideration for the design of processing equipment. The dynamic changes in rheological properties that occur in a corn stover feedstock undergoing enzymatic hydrolysis are characterized in this work, and the influence on pressure losses in piping systems is estimated. Two rheometer geometries were fabricated with stereolithography 3D printing to reduce wall slip and sample ejection. The slurries have complex rheological behaviors that include shear-thinning behavior. Shear stress ramps were performed on samples at 20 and 50 °C using the custom geometries, and the Herschel–Bulkley model was fit to the data. The dynamic nature of the rheological properties is correlated with changes in the average fiber length at various extents of reaction, and the influence of solids concentration on the observed rheology and piping pressure losses is discussed.

A deployable, continuous enzymatic hydrolysis (CEH) process can address cost and commercialization risks associated with second-generation (Gen2) biorefinery sugar/lignin/ethanol production while contributing to energy supply and security. Developments in commercial enzymatic hydrolysis formulations targeting Gen2 pretreated biomass such as deacetylated mechanically refined (DMR) biomass necessitate a reassessment of the existing hybrid simultaneous saccharification and fermentation (SSF) approach. Notably, the practice of "finishing hydrolysis" in SSF has become problematic with the introduction of oxidative enzymes, such as lytic polysaccharide monooxygenases (LPMOs), into commercial cellulase formulations as these require specific redox conditions and cofactor. Moreover, continuous SSF has not been demonstrated at commercial scale, limiting deployment and the associated economic benefits to farmers, producers, and support industries. Continuous enzymatic hydrolysis (CEH) was demonstrated at bench scale to enable optimal saccharification performance of deacetylated mechanically refined (DMR) pretreated biomass. Diafiltration was demonstrated to retain pretreated biomass solids and enzymes for continuous reaction while removing solubilized product sugars in situ. A significant breakthrough afforded by the CEH process is its ability to achieve equivalent endpoint conversions with approximately 50

Protein engineering and screening of processive fungal cellobiohydrolases (CBHs) remain challenging due to limited expression hosts, synergy-dependency, and recalcitrant substrates. In particular, GH7 CBHs are a family critically important for the bioeconomy as well as a methodology for improving these and other difficult to engineer enzymes. Here, we target the discovery of highly active natural GH7 CBHs and engineering of variants with improved activity. Using experimentally assayed activities of genome mined CBHs, we applied sequence and structural alignments to top performers to identify key point mutations linked to improved activity. From ∼1500 known GH7 sequences, an evolutionarily diverse subset of 57 GH7 CBH genes was expressed in Trichoderma reesei and screened using a multiplexed activity screening assay. Ten catalytically enhanced natural variants were identified, produced, purified, and tested for efficacy using industrially-relevant conditions and substrates. Three key amino acids in CBHs with performance comparable or superior to Penicillium funiculosum Cel7A were identified and combinatorically engineered into P. funiculosum cel7a, expressed in T. reesei, and assayed on lignocellulosic biomass. The top performer generated using this combined approach of natural diversity genome mining, experimental assays, and computational modeling produced a 41% increase in conversion extent over native P. funiculosum Cel7A, a 55% increase over the current industrial standard T. reesei Cel7A, and 10% improvement over Aspergillus oryzae Cel7C, the best natural GH7 CBH previously identified in our laboratory.

The use of nicotinamide cofactors in cell-free biocatalytic systems is necessitated by the high specificity that these enzymes show for their natural redox mediators. Unfortunately, isolation and use of natural cofactors is costly, which suggests that enhancing their stability is key to enabling their use in industrial processes. This study details NAD+ and NADH stability in three buffer systems (sodium phosphate, HEPES, and Tris) at 19 °C and 25 °C and for up to 43 d. In Tris, both NADH and NAD+ were found to be highly stable. NADH degradation rates of 4 μM/d (19 °C) and 11 μM/d (25 °C) were observed in Tris buffer, corresponding to >90% and 75% remaining after 43 d, respectively. Higher degradation rates (up to 34 μM/d) were observed when sodium phosphate or HEPES buffers were used. The effect of a mild increase in temperature was determined to be significant for long-term stability, and it was shown that degradation under these conditions can be easily monitored via UV–Vis, because the degradation proceeds via the oxidation/de-aromatization of the dihydropyridine ring. Overall, this work emphasizes that the choice of buffer system is consequential for bioreactor systems employing natural nicotinamide cofactors for extended periods of time.

Abstract Background Carbonic anhydrase (CA) enzymes facilitate the reversible hydration of CO2 to bicarbonate ions and protons. Identifying efficient and robust CAs and expressing them in model host cells, such as Escherichia coli, enables more efficient engineering of these enzymes for industrial CO2 capture. However, expression of CAs in E. coli is challenging due to the possible formation of insoluble protein aggregates, or inclusion bodies. This makes the production of soluble and active CA protein a prerequisite for downstream applications. Results In this study, we streamlined the process of CA expression by selecting seven top CA candidates and used two bioinformatic tools to predict their solubility for expression in E. coli. The prediction results place these enzymes in two categories: low and high solubility. Our expression of high solubility score CAs (namely CA5-SspCA, CA6-SazCAtrunc, CA7-PabCA and CA8-PhoCA) led to significantly higher protein yields (5 to 75 mg purified protein per liter) in flask cultures, indicating a strong correlation between the solubility prediction score and protein expression yields. Furthermore, phylogenetic tree analysis demonstrated CA class-specific clustering patterns for protein solubility and production yields. Unexpectedly, we also found that the unique N-terminal, 11-amino acid segment found after the signal sequence (not present in its homologs), was essential for CA6-SazCA activity. Conclusions Overall, this work demonstrated that protein solubility prediction, phylogenetic tree analysis, and experimental validation are potent tools for identifying top CA candidates and then producing soluble, active forms of these enzymes in E. coli. The comprehensive approaches we report here should be extendable to the expression of other heterogeneous proteins in E. coli.

Efforts toward decarbonizing construction materials and industrial processes related to cement and concrete can be aided via multifaceted approaches that target alternative admixtures as well as precision control of fabrication. Chemical admixtures for water reduction have played a crucial role in the development of advanced concrete mixtures. Newer biomass processing techniques developed for aviation fuel production from corn stover biomass produce a more reactive lignin byproduct that is suitable for chemical modifications to mimic the properties of polycarboxylate ether admixtures with a smaller carbon footprint. The present study examines the use of lignin-based water-reducing admixture in cement pastes and mortar mixtures for 3D printing. The experimental program explores the use of different dosages of lignin-based admixture to produce 3D-printed samples with appropriate extrudability and buildability. The rheological characterization was performed to determine the flow curve of various mixtures. Finally, the heat of hydration of cement pastes was monitored via isothermal calorimetry to assess the impact of lignin-based admixtures on the hydration process of cement. The results of this study indicate that the use of biomass by-products, such as lignin-based admixtures have great potential to effectively control the fresh-state properties of cement-based materials.

The field of industrial microbiology has great potential to tackle the most pressing challenges facing society, but today, this entails much more than classical fermentation technology followed by product upgrading. Methods are needed to confer unit-operation-level functionality and whole-system-level robustness, as well as flexibility. As examples of this concept, we focus here on methodologies often deployed at industrial scale as discrete processes—such as biological, chemical, and electrochemical unit operations—that are also capable of substantial complementarity when combined sequentially and operated continuously. A key and relatively new type of conversion step is made possible by catalyst immobilization methods at both the enzymatic and cellular level. Cases drawn from electro-fermentation for acetoin production and syngas-mediated microbial electrosynthesis of ethanol exemplify the synergy of combined operational approaches. Because modular processes can be treated as a series of reactor elements, considerable flexibility is possible. As discussed, however, not all processes are amenable to this intensification.

The decarbonization of the chemical industry and a shift toward circular economies because of high global CO2 emissions make CO2 an attractive feedstock for manufacturing chemicals. Moreover, H2 is a low-cost and carbon-free reductant because technologies such as solar-driven electrolysis and supercritical water (scH2O) gasification enable sustainable production of molecular hydrogen (H2). We review the recent advances in engineering Ralstonia eutropha, the representative species of “Knallgas” bacteria, for utilizing CO2 and H2 to autotrophically produce 2,3-butanediol (2,3-BDO). This assessment is focused on state-of-the-art approaches for splitting H2 to supply energy in the form of ATP and NADH to power cellular reactions and employing the Calvin-Benson-Bassham cycle for CO2 fixation. Major challenges and opportunities for application and future perspectives are discussed in the context of developing other promising CO2 and H2-utilizing microorganisms, exemplified by Zymomonas mobilis.

Hybrid processes, featuring biological conversion of lignocellulose to small molecules followed by chemo-catalytic conversion to larger molecules suitable for difficult-to-electrify transport modes, are a promising route to biomass-derived fuels in demand for climate stabilization.

Abstract Heterologous protein production has been challenging in the hyper-cellulolytic fungus, Trichoderma reesei as the species is known for poor transformation efficiency, low homologous recombination frequency, and marginal screening systems for the identification of successful transformants. We have applied the 2A-peptide multi-gene expression system to co-express four proteins, which include three cellulases: a cellobiohydrolase (CBH1), an endoglucanase (EG1), and a β-D-glucosidase (BGL1), as well as the enhanced green fluorescent protein (eGFP) marker protein. We designed a new chassis vector, pTrEno-4X-2A, for this work. Expression of these cellulase enzymes was confirmed by real-time quantitative reverse transcription PCR and immunoblot analysis. The activity of each cellulase was assessed using chromogenic substrates, which confirmed the functionality of the enzymes. Expression and activity of these enzymes were proportional to the level of eGFP fluorescence, thereby validating the reliability of this screening technique. An 18-fold differencein protein expression was observed between the first and third genes within the 2A-peptide construct. The availability of this new multi-gene expression and screening tool is expected to greatly impact multi-enzyme applications, such as the production of complex commercial enzyme formulations and metabolic pathway enzymes, especially those destined for cell-free applications.

Microbial conversion of biomass relies on a complex combination of enzyme systems promoting synergy to overcome biomass recalcitrance. Some thermophilic bacteria have been shown to exhibit particularly high levels of cellulolytic activity, making them of particular interest for biomass conversion. These bacteria use varying combinations of CAZymes that vary in complexity from a single catalytic domain to large multi-modular and multi-functional architectures to deconstruct biomass. Since the discovery of CelA from Caldicellulosiruptor bescii which was identified as one of the most active cellulase so far identified, the search for efficient multi-modular and multi-functional CAZymes has intensified. One of these candidates, GuxA (previously Acel_0615), was recently shown to exhibit synergy with other CAZymes in C. bescii, leading to a dramatic increase in growth on biomass when expressed in this host. GuxA is a multi-modular and multi-functional enzyme from Acidothermus cellulolyticus whose catalytic domains include a xylanase/endoglucanase GH12 and an exoglucanase GH6, representing a unique combination of these two glycoside hydrolase families in a single CAZyme. These attributes make GuxA of particular interest as a potential candidate for thermophilic industrial enzyme preparations. Here, we present a more complete characterization of GuxA to understand the mechanism of its activity and substrate specificity. In addition, we demonstrate that GuxA exhibits high levels of synergism with E1, a companion endoglucanase from A. cellulolyticus. We also present a crystal structure of one of the GuxA domains and dissect the structural features that might contribute to its thermotolerance.

Background Terrestrial plant biomass is the primary renewable carbon feedstock for enabling transition to a sustainable bioeconomy. Consolidated bioprocessing (CBP) by the cellulolytic thermophile Clostridium thermocellum offers a single step microbial platform for production of biofuels and biochemicals via simultaneous solubilization of carbohydrates from lignocellulosic biomass and conversion to products. Here, solubilization of cell wall cellulosic, hemicellulosic, and pectic polysaccharides in the liquor and solid residues generated during CBP of poplar biomass by C. thermocellum was analyzed. Results The total amount of biomass solubilized in the C. thermocellum DSM1313 fermentation platform was 5.8, 10.3, and 13.7% of milled non-pretreated poplar after 24, 48, and 120 h, respectively. These results demonstrate solubilization of 24% cellulose and 17% non-cellulosic sugars after 120 h, consistent with prior reports. The net solubilization of non-cellulosic sugars by C. thermocellum (after correcting for the uninoculated control fermentations) was 13 to 36% of arabinose (Ara), xylose (Xyl), galactose (Gal), mannose (Man), and glucose (Glc); and 15% and 3% of fucose and glucuronic acid, respectively. No rhamnose was solubilized and 71% of the galacturonic acid (GalA) was solubilized. These results indicate that C. thermocellum may be selective for the types and/or rate of solubilization of the non-cellulosic wall polymers. Xyl, Man, and Glc were found to accumulate in the fermentation liquor at levels greater than in uninoculated control fermentations, whereas Ara and Gal did not accumulate, suggesting that C. thermocellum solubilizes both hemicelluloses and pectins but utilizes them differently. After five days of fermentation, the relative amount of Rha in the solid residues increased 21% indicating that the Rha-containing polymer rhamnogalacturonan I (RG-I) was not effectively solubilized by C. thermocellum CBP, a result confirmed by immunoassays. Comparison of the sugars in the liquor versus solid residue showed that C. thermocellum solubilized hemicellulosic xylan and mannan, but did not fully utilize them, solubilized and appeared to utilize pectic homogalacturonan, and did not solubilize RG-I. Conclusions The significant relative increase in RG-I in poplar solid residues following CBP indicates that C. thermocellum did not solubilize RG-I. These results support the hypothesis that this pectic glycan may be one barrier for efficient solubilization of poplar by C. thermocellum .

Consolidated bioprocessing using oleaginous yeast is a promising modality for the economic conversion of plant biomass to fuels and chemicals. However, yeast are not known to produce effective biomass degrading enzymes naturally and this trait is essential for efficient consolidated bioprocessing. We expressed a chimeric cellobiohydrolase I gene in three different oleaginous, industrially relevant yeast: Yarrowia lipolytica, Lipomyces starkeyi, and Saccharomyces cerevisiae to study the biochemical and catalytic properties and biomass deconstruction potential of these recombinant enzymes. Our results showed differences in glycosylation, surface charge, thermal and proteolytic stability, and efficacy of biomass digestion. L. starkeyi was shown to be an inferior active cellulase producer compared to both the Y. lipolytica and S. cerevisiae enzymes, whereas the cellulase expressed in S. cerevisiae displayed the lowest activity against dilute-acid-pretreated corn stover. Comparatively, the chimeric cellobiohydrolase I enzyme expressed in Y. lipolytica was found to have a lower extent of glycosylation, better protease stability, and higher activity against dilute-acid-pretreated corn stover.

Yarrowia lipolytica is known to be capable of metabolizing glucose and accumulating lipids intracellularly; however, it lacks the cellulolytic enzymes needed to break down cellulosic biomass directly. To develop Y. lipolytica as a consolidated bioprocessing (CBP) microorganism, we previously expressed the heterologous CBH I, CBH II, and EG II cellulase enzymes both individually and collectively in this microorganism. We concluded that the coexpression of these cellulases resulted in a metabolic drain on the host cells leading to reduced cell growth and lipid accumulation. The current study aims to build a new cellulase coexpressing platform to overcome these hinderances by (1) knocking out the sucrose non-fermenting 1 (Snf1) gene that represses the energetically expensive lipid and protein biosynthesis processes, and (2) knocking in the cellulase cassette fused with the recyclable selection marker URA3 gene in the background of a lipid-accumulating Y. lipolytica strain overexpressing ATP citrate lyase (ACL) and diacylglycerol acyltransferase 1 (DGA1) genes. We have achieved a homologous recombination insertion rate of 58% for integrating the cellulases-URA3 construct at the disrupted Snf1 site in the genome of host cells. Importantly, we observed that the disruption of the Snf1 gene promoted cell growth and lipid accumulation and lowered the cellular saturated fatty acid level and the saturated to unsaturated fatty acid ratio significantly in the transformant YL163t that coexpresses cellulases. The result suggests a lower endoplasmic reticulum stress in YL163t, in comparison with its parent strain Po1g ACL-DGA1. Furthermore, transformant YL163t increased in vitro cellulolytic activity by 30%, whereas the “total in vivo newly formed FAME (fatty acid methyl esters)” increased by 16% in comparison with a random integrative cellulase-expressing Y. lipolytica mutant in the same YNB-Avicel medium. The Snf1 disruption platform demonstrated in this study provides a potent tool for the further development of Y. lipolytica as a robust host for the expression of cellulases and other commercially important proteins.

In this study, filtration efficiency of different respirators, face masks, and a 2-ply cotton handkerchief bandana was compared for particles in the size range of 60 nm-4 mu m under a "perfect fit" condition. The filtration efficiency at the most penetrating particle size of 0.3 mu m on average ranged from 83-99% for N95 and KN95 respirators, 42-88% for surgical masks, 16-23% for cloth masks, and 9% for bandana. We also investigated the effects of using double surgical masks or layering a cloth mask over various surgical masks in terms of their filtration characteristics. In most of these combinations, the filtration efficiency improved by -25% for particles 0.3-1 mu m in diameter without any substantial change in the filter quality factor when compared to the highest of the individual mask results. To investigate the reusability of cotton cloth masks, 2-layer cotton fabric sample coupons were machine washed and dried for 52 cycles leading to an increase in inhalation resistance (-20 Pa) without affecting size-resolved filtration efficiency. Scanning electron microscopy revealed that washing and drying led to the gradual deconstruction of cotton fibers at the scale of several micrometers to hundreds of nanometers in the form of delamination of the fiber wall and fibrillation of the nanofiber constituents. Results indicate that cloth masks may be layered over surgical masks for additional benefits, and that cloth masks made out of cotton fabric can be washed and reused numerous times without a significant loss in filtration efficiency.

Plant-biomass-based nanomaterials have attracted great interest recently for their potential to replace petroleum-sourced polymeric materials for sustained economic development. However, challenges associated with sustainable production of lignocellulosic nanoscale polymeric materials (NPMs) need to be addressed. Producing materials from lignocellulosic biomass is a value-added proposition compared with fuel-centric approach. This report focuses on recent progress made in understanding NPMs-specifically lignin nanoparticles (LNPs) and cellulosic nanomaterials (CNMs)-and their sustainable production. Special attention is focused on understanding key issues in nano-level deconstruction of cell walls and utilization of key properties of the resultant NPMs to allow flexibility in production to promote sustainability. Specifically, suitable processes for producing LNPs and their potential for scaled-up production, along with the resultant LNP properties and prospective applications, are discussed. In the case of CNMs, terminologies such as cellulose nanocrystals (CNCs) and cellulose nanofibrils (CNFs) used in the literature are examined. The term cellulose nano-whiskers (CNWs) is used here to describe a class of CNMs that has a morphology similar to CNCs but without specifying its crystallinity, because most applications of CNCs do not need its crystalline characteristic. Additionally, progress in enzymatic processing and drying of NPMs is also summarized. Finally, the report provides some perspective of future research that is likely to result in commercialization of plant-based NPMs.