Zhiguang Zhu

Increasing Cellulose Accessibility Is More Important Than Removing Lignin: A Comparison of Cellulose Solvent-Based Lignocellulose Fractionation and Soaking in Aqueous Ammonia

While many pretreatments attempt to improve the enzymatic digestibility of biomass by removing lignin, this study shows that improving the surface area accessible to cellulase is a more important factor for achieving a high sugar yield. Here we compared the pretreatment of switchgrass by two methods, cellulose solvent‐ and organic solvent‐based lignocellulose fractionation (COSLIF) and soaking in aqueous ammonia (SAA). Following pretreatment, enzymatic hydrolysis was conducted at two cellulase loadings, 15 filter paper units (FPU)/g glucan and 3 FPU/g glucan, with and without BSA blocking of lignin absorption sites. The hydrolysis results showed that the lignin remaining after SAA had a significant negative effect on cellulase performance, despite the high level of delignification achieved with this pretreatment. No negative effect due to lignin was detected for COSLIF‐treated substrate. SEM micrographs, XRD crystallinity measurements, and cellulose accessibility to cellulase (CAC) determinations confirmed that COSLIF fully disrupted the cell wall structure, resulting in a 16‐fold increase in CAC, while SAA caused a 1.4‐fold CAC increase. A surface plot relating the lignin removal, CAC, and digestibility of numerous samples (both pure cellulosic substrates and lignocellulosic materials pretreated by several methods) was also developed to better understand the relative impacts of delignification and CAC on glucan digestibility. Biotechnol. Bioeng. 2011; 108:22–30.

Comparative study of corn stover pretreated by dilute acid and cellulose solvent‐based lignocellulose fractionation: Enzymatic hydrolysis, supramolecular structure, and substrate accessibility

Liberation of fermentable sugars from recalcitrant biomass is among the most costly steps for emerging cellulosic ethanol production. Here we compared two pretreatment methods (dilute acid, DA, and cellulose solvent and organic solvent lignocellulose fractionation, COSLIF) for corn stover. At a high cellulase loading [15 filter paper units (FPUs) or 12.3 mg cellulase per gram of glucan], glucan digestibilities of the corn stover pretreated by DA and COSLIF were 84% at hour 72 and 97% at hour 24, respectively. At a low cellulase loading (5 FPUs per gram of glucan), digestibility remained as high as 93% at hour 24 for the COSLIF‐pretreated corn stover but reached only ∼60% for the DA‐pretreated biomass. Quantitative determinations of total substrate accessibility to cellulase (TSAC), cellulose accessibility to cellulase (CAC), and non‐cellulose accessibility to cellulase (NCAC) based on adsorption of a non‐hydrolytic recombinant protein TGC were measured for the first time. The COSLIF‐pretreated corn stover had a CAC of 11.57 m2/g, nearly twice that of the DA‐pretreated biomass (5.89 m2/g). These results, along with scanning electron microscopy images showing dramatic structural differences between the DA‐ and COSLIF‐pretreated samples, suggest that COSLIF treatment disrupts microfibrillar structures within biomass while DA treatment mainly removes hemicellulose. Under the tested conditions COSLIF treatment breaks down lignocellulose structure more extensively than DA treatment, producing a more enzymatically reactive material with a higher CAC accompanied by faster hydrolysis rates and higher enzymatic digestibility. Biotechnol. Bioeng. 2009;103: 715–724.

A high-energy-density sugar biobattery based on a synthetic enzymatic pathway

High-energy-density, green, safe batteries are highly desirable for meeting the rapidly growing needs of portable electronics. The incomplete oxidation of sugars mediated by one or a few enzymes in enzymatic fuel cells suffers from low energy densities and slow reaction rates. Here we show that nearly 24 electrons per glucose unit of maltodextrin can be produced through a synthetic catabolic pathway that comprises 13 enzymes in an air-breathing enzymatic fuel cell. This enzymatic fuel cell is based on non-immobilized enzymes that exhibit a maximum power output of 0.8 mW cm(-2) and a maximum current density of 6 mA cm(-2), which are far higher than the values for systems based on immobilized enzymes. Enzymatic fuel cells containing a 15% (wt/v) maltodextrin solution have an energy-storage density of 596 Ah kg(-1), which is one order of magnitude higher than that of lithium-ion batteries. Sugar-powered biobatteries could serve as next-generation green power sources, particularly for portable electronics.

Efficient sugar release by the cellulose solvent-based lignocellulose fractionation technology and enzymatic cellulose hydrolysis.

Efficient liberation of fermentable soluble sugars from lignocellulosic biomass waste not only decreases solid waste handling but also produces value-added biofuels and biobased products. Industrial hemp, a special economic crop, is cultivated for its high-quality fibers and high-value seed oil, but its hollow stalk cords (hurds) are a cellulosic waste. The cellulose-solvent-based lignocellulose fractionation (CSLF) technology has been developed to separate lignocellulose components under modest reaction conditions (Zhang, Y.-H. P.; Ding, S.-Y.; Mielenz, J. R.; Elander, R.; Laser, M.; Himmel, M.; McMillan, J. D.; Lynd, L. R. Biotechnol. Bioeng. 2007, 97 (2), 214- 223). Three pretreatment conditions (acid concentration, reaction temperature, and reaction time) were investigated to treat industrial hemp hurds for a maximal sugar release: a combinatorial result of a maximal retention of solid cellulose and a maximal enzymatic cellulose hydrolysis. At the best treatment condition (84.0% H3PO4 at 50 degrees C for 60 min), the glucan digestibility was 96% at hour 24 at a cellulase loading of 15 filter paper units of cellulase per gram of glucan. The scanning electron microscopic images were presented for the CSLF-pretreated biomass for the first time, suggesting that CSLF can completely destruct the plant cell-wall structure, in a good agreement with the highest enzymatic cellulose digestibility and fastest hydrolysis rate. It was found that phosphoric acid only above a critical concentration (83%) with a sufficient reaction time can efficiently disrupt recalcitrant lignocellulose structures.

Evaluations of cellulose accessibilities of lignocelluloses by solute exclusion and protein adsorption techniques

Cellulose accessibilities of a set of hornified lignocellulosic substrates derived by drying the never dried pretreated sample and a set of differently pretreated lodgepople pine substrates, were evaluated using solute exclusion and protein adsorption methods. Direct measurements of cellulase adsorption onto cellulose surface of the set of pretreated substrates were also carried out using an in situ UV–Vis spectrophotometric technique. The cellulose accessibilities measured by the solute exclusion and a cellulose‐binding module (CBM)‐containing green fluorescent protein (TGC) adsorption methods correlate well for both sets of samples. The substrate enzymatic digestibilities (SEDs) of the hornified substrates are proportional to the measured cellulose accessibilities. Approximately over 90% of the SED was contributed by the accessible pore surfaces of the hornified substrates, suggesting that the substrate external surface plays a minor role contributing to cellulose accessibility and SED. The cellulose accessibilities of the pretreated substrates correlated well with the amounts of cellulase adsorbed. The SEDs of these substrates directly correlated with the amounts of adsorbed cellulase. Biotechnol. Bioeng. 2012; 109:381–389.

Biohydrogenation from Biomass Sugar Mediated by In Vitro Synthetic Enzymatic Pathways

Different from NAD(P)H regeneration approaches mediated by a single enzyme or a whole-cell microorganism, we demonstrate high-yield generation of NAD(P)H from a renewable biomass sugar--cellobiose through in vitro synthetic enzymatic pathways consisting of 12 purified enzymes and coenzymes. When the NAD(P)H generation system was coupled with its consumption reaction mediated by xylose reductase, the NADPH yield was as high as 11.4 mol NADPH per cellobiose (i.e., 95% of theoretical yield--12 NADPH per glucose unit) in a batch reaction. Consolidation of endothermic reactions and exothermic reactions in one pot results in a very high energy-retaining efficiency of 99.6% from xylose and cellobiose to xylitol. The combination of this high-yield and projected low-cost biohydrogenation and aqueous phase reforming may be important for the production of sulfur-free liquid jet fuel in the future.

Cellulose Solvent-Based Biomass Pretreatment Breaks Highly Ordered Hydrogen Bonds in Cellulose Fibers of Switchgrass

The switchgrass (SG) samples pretreated by cellulose solvent‐ and organic solvent‐based lignocellulose fractionation were characterized by enzymatic hydrolysis, substrate accessibility assay, scanning electron microscopy, X‐ray diffraction (XRD), cross polarization/magic angle spinning (CP/MAS) 13C nuclear magnetic resonance (NMR), and Fourier transform infrared spectroscopy (FTIR). Glucan digestibility of the pretreated SG was 89% at hour 36 at one filter paper unit of cellulase per gram of glucan. Crystallinity index (CrI) of pure cellulosic materials and SG before and after cellulose solvent‐based pretreatment were determined by XRD and NMR. CrI values varied greatly depending on measurement techniques, calculation approaches, and sample drying conditions, suggesting that the effects of CrI data obtained from dried samples on enzymatic hydrolysis of hydrated cellulosic materials should be interpreted with caution. Fast hydrolysis rates and high glucan digestibilities for pretreated SG were mainly attributed to a 16.3‐fold increase in cellulose accessibility to cellulase from 0.49 to 8.0 m2/g biomass, because the highly ordered hydrogen‐bonding networks in cellulose fibers of biomass were broken through cellulose dissolution in a cellulose solvent, as evidenced by CP/MAS 13C‐NMR and FTIR. Biotechnol. Bioeng. 2011; 108:521–529.

Bamboo saccharification through cellulose solvent-based biomass pretreatment followed by enzymatic hydrolysis at ultra-low cellulase loadings

The modified cellulose solvent- (concentrated phosphoric acid) and organic solvent- (95% ethanol) based lignocellulose fractionation (COSLIF) was applied to a naturally-dry moso bamboo sample. The biomass dissolution conditions were 50 degrees C, 1 atm for 60 min. Glucan digestibility was 88.2% at an ultra-low cellulase loading of one filter paper unit per gram of glucan. The overall glucose and xylose yields were 86.0% and 82.6%, respectively. COSLIF efficiently destructed bamboos fibril structure, resulting in a approximately 33-fold increase in cellulose accessibility to cellulase (CAC) from 0.27 to 9.14 m(2) per gram of biomass. Cost analysis indicated that a 15-fold decrease in use of costly cellulase would be of importance to decrease overall costs of biomass saccharification when cellulase costs are higher than

One-step production of lactate from cellulose as the sole carbon source without any other organic nutrient by recombinant cellulolytic Bacillus subtilis

0.15 per gallon of cellulosic ethanol.

Deep oxidation of glucose in enzymatic fuel cells through a synthetic enzymatic pathway containing a cascade of two thermostable dehydrogenases.

Although intensive efforts have been made to create recombinant cellulolytic microorganisms, real recombinant cellulose-utilizing microorganisms that can produce sufficient secretory active cellulase, hydrolyze cellulose, and utilize released soluble sugars for supporting both cell growth and cellulase synthesis without any other organic nutrient (e.g., yeast extract, peptone, amino acids), are not available. Here we demonstrated that over-expression of Bacillus subtilis endoglucanase BsCel5 enabled B. subtilis to grow on solid cellulosic materials as the sole carbon source for the first time. Furthermore, two-round directed evolution was conducted to increase specific activity of BsCel5 on regenerated amorphous cellulose (RAC) and enhance its expression/secretion level in B. subtilis. To increase lactate yield, the alpha-acetolactate synthase gene (alsS) in the 2,3-butanediol pathway was knocked out. In the chemically defined minimal M9/RAC medium, B. subtilis XZ7(pBscel5-MT2C) strain (ΔalsS), which expressed a BsCel5 mutant MT2C, was able to hydrolyze RAC with cellulose digestibility of 74% and produced about 3.1g/L lactate with a yield of 60% of the theoretical maximum. When 0.1% (w/v) yeast extract was added in the M9/RAC medium, cellulose digestibility and lactate yield were enhanced to 92% and 63% of the theoretical maximum, respectively. The recombinant industrially safe cellulolytic B. subtilis would be a promising consolidated bioprocessing platform for low-cost production of biocommodities from cellulosic materials.