Applied Biochemistry and Biotechnology (v.94, #1)

Oxidative lime pretreatment of high-lignin biomass by Vincent S. Chang; Murlidhar Nagwani; Chul-Ho Kim; Mark T. Holtzapple (1-28).
Lime (Ca[OH]2) and oxygen (O2) were used to enhance the enzymatic digestibility of two kinds of high-lignin biomass: poplar wood and newspaper. The recommended pretreatment conditions for poplar wood are 150°C, 6 h, 0.1 g of Ca(OH)2/g of dry biomass, 9 mL of water/g of dry biomass, 14.0 bar absolute oxygen, and a particle size of −10 mesh. Under these conditions, the 3-d reducing sugar yield of poplar wood using a cellulase loading of 5 filter paper units (FPU)/g of raw dry biomass increased from 62 to 565 mg of eq. glucose/g of raw dry biomass, and the 3-d total sugar (glucose + xylose) conversion increased from 6 to 77% of raw total sugars. At high cellulase loadings (e.g., 75 FPU/g of raw dry biomass), the 3-d total sugar conversion reached 97%. In a trial run with newspaper, using conditions of 140°C, 3 h, 0.3 g of Ca(OH)2/g of dry biomass, 16 mL of water/g of dry biomass, and 7.1 bar absolute oxygen, the 3-d reducing sugar yield using a cellulase loading of 5 FPU/g of raw dry biomass increased from 240 to 565 mg of eq. glucose/g of raw dry biomass. A material balance study on poplar wood shows that oxidative lime pretreatment solubilized 38% of total biomass, including 78% of lignin and 49% of xylan; no glucan was removed. Ash increased because calcium was incorporated into biomass during the pretreatment. After oxidative lime pretreatment, about 21% of added lime could be recovered by CO2 carbonation.
Keywords: High-lignin biomass; pretreatment; lime; oxidative; oxygen; enzymatic digestibility; cellulase; sugar; material balance; delignification

Adsorption of Thermomonospora fusca E5 and Trichoderma reesei cellobiohydrolase I cellulases on synthetic surfaces by Carolyn S. Baker; Worakrit Suvajittanont; Michelle K. Bothwell; Joe McGuire (29-40).
The interfacial behavior of Thermomonospora fusca E5 and Trichoderma reesei cellobiohydrolase I (CBHI) cellulases were studied at synthetic surfaces. For this purpose, colloidal silica and polystyrene particles were used to prepare cellulase-particle suspensions that could be analyzed by solution-phase techniques. Circular dichroism spectroscopy of each cellulase, alone as well as in suspension with silica, was used to determine whether structural changes occurred on adsorption. Changes in spectra were observed for CBHI, but not for E5. Gel-permeation chromatography of the cellulase-particle suspensions showed that neither cellulase binds to silica, suggesting that changes in spectra for CBHI were a result of solution-phase phenomena. Microfiltration of cellulase-polystyrene suspensions showed that both cellulases bind to polystyrene. However, circular dichroism experiments with polysterene proved unworkable, owing to excessive light absorption by the polystyrene. Adsorption kinetics of each cellulase were recorded, in situ, at hydrophilic and silanized, hydrophobic silica surfaces using ellipsometry. Ellipsometric data recorded for each cellulase at hydrophilic silica showed insignificant adsorption. Binding did occur between each cellulase and silanized silica, most likely mediated through hydrophobic associations. Adsorption in this case was irreversible to dilution.
Keywords: Thermomonospora fusca E5 ; Trichoderma reesei CBHI; cellulase adsorption; cellulase binding; hydrophobic effects

Fermentation and costs of fuel ethanol from corn with quick-germ process by Frank Taylor; Andrew J. Mcaloon; James C. Craig Jr.; Ping Yang; Jenny Wahjudi; Steven R. Eckhoff (41-49).
The Quick-Germ process developed at the University of Illinois at Urbana-Champaign is a way to obtain corn oil, but with lower capital costs than the traditional wet-milling process. Quick-Germ has the potential to increase the coproduct credits and profitability of the existing dry-grind fuel ethanol process, but the fermentability of the corn remaining after oil recovery has not been tested. Therefore, a series of pilot scale (50 L) fermentations was carefully controlled and monitored with unique methods for standard inoculation and automatic sampling. It was found that the concentration of suspended solids was significantly reduced in the Quick-Germ fermentations. When compared at the same concentration of fermentable sugars, the fermentation rate and yield were not statistically different from controls. When Quick-Germ was integrated into a state-of-the-art dry-grind fuel ethanol process, computer simulation and cost models indicated savings of approx $0.01/L of ethanol ($0.04/gal) with the Quick-Germ process. Additional savings associated with the lower suspended solids could not be quantified and were not included. However, the savings are sensitive to the price of corn oil.
Keywords: Quick-Fiber; ethyl alcohol; cost estimation; cornstarch; oil; dry-grind process; wet milling; distiller’s dried grains

Metabolic flux analysis of clostridium thermosuccinogenes by Jayanth Sridhar; Mark A. Eiteman (51-69).
Clostridium thermosuccinogenes are anaerobic thermophilic bacteria that ferment various carbohydrates to succinate and acetate as major products and formate, lactate, and ethanol as minor products. Metabolic carbon flux analysis was used to evaluate the effect of pH and redox potential on the batch fermentation of C. thermosuccinogenes. In a first study, the effects of four pH values (6.50, 6.75, 7.00, and 7.25) on intracellular carbon flux at a constant redox potential of −275 mV were compared. The flux of carbon toward succinate and formate increased whereas the flux to lactate decreased significantly with a pH increase from 6.50 to 7.25. Both specific growth rate and specific rate of glucose consumption were unaffected by changes in pH. The fraction of carbon flux at the phosphoenolpyruvate (PEP) node flowing to oxaloacetate increased with an increase in pH. At the pyruvate node, the fraction of flux to formate increased with increasing pH. At the acetyl CoA node, the fraction of flux to acetate increased significantly with an increase in pH. A second study elucidated the effect of four controlled culture redox potentials (−225, −250, −275, and −310 mV) on metabolic carbon flux at a constant pH of 7.25. Lower values of culture redox potential were correlated with increased succinate, acetate, and formate fluxes and decreased ethanol and hydrogen fluxes in C. thermosuccinogenes. Lactate formation was not significantly influenced by redox potential. At the PEP node, the fraction of carbon to oxaloacetate increased with a decrease in redox potential. At the pyruvate node, the fraction of carbon to formate increased, while at the acetyl CoA node, the fraction of carbon flux to acetate increased with reduced redox potential. The presence of hydrogen in the headspace or the addition of nicotinic acid to the growth media resulted in increased hydrogen and ethanol fluxes and decreased succinate, acetate, formate, and lactate fluxes.
Keywords: Clostridium thermosuccinogenes ; anaerobic fermentation; culture redox potential; NADH; metabolic flux; organic acids

Sugar-mediated crosslinking of α-biotinylated-lys to cysteamine-agarose support by Mikhail Linetsky; Roy D. LeGrand; Valeri V. Mossine; B. J. Ortwerth (71-96).
Advanced glycation end products (AGEs) and, specifically, protein-protein AGE crosslinks have long been studied for their potential role in aging, diabetic complications and Alzheimer disease. With few exceptions, the chemical nature of these structures remains unknown. We report here a simple approach that allows the preparation and isolation of milligram quantities of sugar-mediated AGE Lys-Lys-like crosslinks from glycation mixtures. The method is based on a sugar-dependent incorporation of N α -biotinyl-l-Lys into cysteaminyldisulfide Sepharose 6B (AE-S-S-Sepharose 6B). Glycation mixtures with six different sugars showed a time- and sugar-dependent decrease in the concentration of the support-bound primary amino groups and accounted for almost 90% loss of cysteaminyl amino groups at the end of the various incubation periods. 4-Hydroxyazobenzene-2-carboxylic acid-avidin assays indicated the incorporation of N α-biotinyl-l-Lys equal to 8% of the total support amino groups with methylglyoxal after 7d and 1% with fructose and glucose after 1 mo of incubation. Treatment of the washed, sugar-modified supports with 2-mercaptoethanol released the bulk of the bound AGE modifications and the crosslinks. Subsequent fractionation of these preparations over a monomeric avidin column afforded a complete separation of sugar-mediated AGE modifications and the crosslinks. Depending on the sugar employed, micromolar amounts of biotinylated Lys-Lys-like crosslinks were generated by this two-step procedure from 8 mL of the original AE-S-S-Sepharose 6B.
Keywords: Crosslinks; Maillard reactions; advanced glycation end products; 2-aminoethyldisulfide sepharose 6B; Nα-biotinyl lysine