Scale-up and evaluation of high solid ionic liquid pretreatment and enzymatic hydrolysis of switchgrass.

BACKGROUND: Ionic liquid (IL) pretreatment is receiving significant attention as a potential process that enables fractionation of lignocellulosic biomass and produces high yields of fermentable sugars suitable for the production of renewable fuels. However, successful optimization and scale up of IL pretreatment involves challenges, such as high solids loading, biomass handling and transfer, washing of pretreated solids and formation of inhibitors, which are not addressed during the development stages at the small scale in a laboratory environment. As a first in the research community, the Joint BioEnergy Institute, in collaboration with the Advanced Biofuels Process Demonstration Unit, a Department of Energy funded facility that supports academic and industrial entities in scaling their novel biofuels enabling technologies, have performed benchmark studies to identify key challenges associated with IL pretreatment using 1-ethyl-3-methylimidazolium acetate and subsequent enzymatic saccharification beyond bench scale. RESULTS: Using switchgrass as the model feedstock, we have successfully executed 600-fold, relative to the bench scale (6 L vs 0.01 L), scale-up of IL pretreatment at 15% (w/w) biomass loading. Results show that IL pretreatment at 15% biomass generates a product containing 87.5% of glucan, 42.6% of xylan and only 22.8% of lignin relative to the starting material. The pretreated biomass is efficiently converted into monosaccharides during subsequent enzymatic hydrolysis at 10% loading over a 150-fold scale of operations (1.5 L vs 0.01 L) with 99.8% fermentable sugar conversion. The yield of glucose and xylose in the liquid streams were 94.8% and 62.2%, respectively, and the hydrolysate generated contains high titers of fermentable sugars (62.1 g/L of glucose and 5.4 g/L cellobiose). The overall glucan and xylan balance from pretreatment and saccharification were 95.0% and 77.1%, respectively. Enzymatic inhibition by [C2mim][OAc] at high solids loadings requires further process optimization to obtain higher yields of fermentable sugars. CONCLUSION: Results from this initial scale up evaluation indicate that the IL-based conversion technology can be effectively scaled to larger operations and the current study establishes the first scaling parameters for this conversion pathway but several issues must be addressed before a commercially viable technology can be realized, most notably reduction in water consumption and efficient IL recycle.

High accuracy dual lens transmittance measurements.

We show how to determine the transmittance of short focal length lenses (f approximately 19 mm and f approximately 2 5 mm, in this case) with a combined uncertainty of 3 parts in 10(4) or better by measuring transmittances of lens pairs of a set of three or more lenses with the same nominal focal length. Uncertainties are minimized by optimizing the radiometric design of the setup and the measurement procedure. The technique is particularly useful in systems where the detector acceptance angle limits the beam geometry to relatively collimated beams.

Transfection of HepG2 cells with hGSTA4 provides protection against 4-hydroxynonenal-mediated oxidative injury.

4-Hydroxynonenal (4-HNE) is a mutagenic alpha,beta-unsaturated aldehyde produced during oxidative injury that is conjugated by several glutathione S-transferase (GST) isoforms. The alpha class human GSTA4-4 enzyme (hGSTA4-4) has a particularly high catalytic efficiency toward 4-HNE conjugation. However, hGST4-4 expression is low in most human cells and there are other aldehyde metabolizing enzymes that detoxify 4-HNE. In the current study, we determined the effect of over-expression of hGSTA4 mRNA on the sensitivity of HepG2 cells to 4-HNE injury. HepG2 cells transfected with an hGSTA4 vector construct exhibited high steady-state hGSTA4 mRNA, high GST-4-HNE catalytic activities, but lower basal glutathione (GSH) concentrations relative to insert-free vector (control) cells. Exposure to 4-HNE elicited an increase in GSH concentrations in the control and hGSTA4 cells, although the dose-response of GSH induction differed among the two cell types. Specifically, hGSTA4 cells had significantly higher GSH concentrations when exposed to 5-15 microM 4-HNE, but not at 20 microM 4-HNE, suggesting extensive GSH utilization at high concentrations of 4-HNE. The hGSTA4 cells exhibited a significant growth advantage relative to control cells in the absence of 4-HNE, and a trend towards increased growth at low dose exposures to 4-HNE. However, the hGSTA4 cells did not exhibit a growth advantage relative to control cells at higher 4-HNE exposures associated with increased GSH utilization. As expected, the hGSTA4 cells showed resistance to 4-HNE stimulated lipid peroxidation at all 4-HNE doses. In summary, our data indicates that over-expression of hGSTA4 at levels conferring high GST-4-HNE conjugating activity confers a partial growth advantage to HepG2 cells and protects against 4-HNE oxidative injury. However, the loss of proliferative capacity of hGSTA4 cells challenged with levels of 4-HNE associated with severe oxidative stress indicates a role of other aldehyde metabolizing enzymes, and/or GSH-electrophile transporter proteins, in providing full cellular protection against 4-HNE toxicity.

Measurement of liver collagen synthesis by heavy water labeling: effects of profibrotic toxicants and antifibrotic interventions.

Enhanced production of collagen is central to fibrotic disorders such as hepatic cirrhosis and pulmonary fibrosis. We describe a sensitive, quantitative, and high-throughput technique for measuring hepatic collagen synthesis in vivo through metabolic labeling with heavy water ((2)H(2)O). Rats and mice received (2)H(2)O in drinking water for up to 35 days. Deuterium incorporation into collagen-bound amino acids (AA) alanine and hydroxyproline (OHP) was measured by gas chromatography-mass spectrometry. A threefold stimulation of collagen fractional synthesis was observed under the maximum dosage of carbon tetrachloride (CCl(4); 1.67 ml/kg). Deuterium enrichment was systematically 20% higher in AA from monomeric collagen relative to dimeric collagen, consistent with slower turnover of the latter. Administration of 1% griseofulvin to mice resulted in a significant, threefold increase in liver collagen synthesis, observable within 12 days and consistent with predicted interstrain differences (C57/Bl6J > BALB/c). Deuterium enrichments of OHP from total liver proteins correlated well with alanine or OHP from isolated collagen. Fibrogenesis subsided after withdrawal of CCl(4) exposure and was reduced to various degrees by coadministration of interferon-gamma, rosiglitazone, atorvastatin, or enalapril. Changes in isotopically measured collagen synthesis correlated with, but were more sensitive and reproducible than, standard histological staining (trichrome) for fibrosis. In summary, liver collagen synthesis can be measured sensitively and with high precision over a short time period, without radioactivity, thereby providing a relatively high-throughput in vivo strategy for rapidly measuring profibrotic activities of suspected hepatotoxicants and antifibrotic activities of drug candidates.

Comparison of Calibration Methods for Tristimulus Colorimeters.

Uncertainties in source color measurements with a tristimulus colorimeter are estimated for calibration factors determined, based on a known source spectral distribution or on accurate measurements of the spectral responsivities of the colorimeter channels. Application is to the National Institute of Standards and Technology (NIST) colorimeter and an International Commission on Illumination (CIE) Illuminant A calibration. Detector-based calibration factors generally have lower uncertainties than source-based calibration factors. Uncertainties are also estimated for calculations of spectral mismatch factors. Where both spectral responsivities of the colorimeter channels and the spectral power distributions of the calibration and test sources are known, uncertainties are lowest if the colorimeter calibration factors are recalculated for the test source; this process also avoids correlations between the CIE Source A calibration factors and the spectral mismatch factors.

Several glutathione S-transferase isozymes that protect against oxidative injury are expressed in human liver mitochondria.

The mitochondrial environment is rich in reactive oxygen species (ROS) that may ultimately peroxidize membrane proteins and generate unsaturated aldehydes such as 4-hydroxy-2-nonenal (4HNE). We had previously demonstrated the presence of hGSTA4-4, an efficient catalyst of 4HNE detoxification, in human liver mitochondria to the exclusion of the cytosol. In the present study, GSH-affinity chromatography was used in conjunction with biochemical and proteomic analysis to determine the presence of additional cytosolic glutathione S-transferases (GSTs) in human hepatic mitochondria. HPLC-subunit analysis of GSH affinity-purified liver mitochondrial proteins indicated the presence of several potential mitochondrial GST isoforms. Electrospray ionization-mass spectrometry analysis of eluted mitochondrial GST subunits yielded molecular masses similar to those of hGSTP1, hGSTA1 and hGSTA2. Octagonal matrix-assisted laser desorption/ionization time of flight mass spectrometry and proteomics analysis using MS-FIT confirmed the presence of these three GST subunits in mitochondria, and HPLC analysis indicated that the relative contents of the mitochondrial GST subunits were hGSTA1>hGSTA2>hGSTP1. The mitochondrial localization of the alpha and pi class GST subunits was consistent with immunoblotting analysis of purified mitochondrial GST. Enzymatic studies using GSH-purified mitochondrial GST fractions demonstrated the presence of significant GST activity using the nonspecific GST substrate 1-chloro-2,4-dinitrobenzene (CDNB), as well as 4HNE, delta(5)-androstene-3,17-dione (ADI), and cumene hydroperoxide (CuOOH). Interestingly, the specific mitochondrial GST activities toward 4HNE, a highly toxic alpha,beta-unsaturated aldehyde produced during the breakdown of membrane lipids, exceeded that observed in liver cytosol. These observations are suggestive of a role of GST in protecting against mitochondrial injury during the secondary phase of oxidative stress, or modulation of 4HNE-mediated mitochondrial signaling pathways. However, other properties of mitochondrial GST, such as conjugation of environmental chemicals and binding of lipophilic non-substrate xenobiotics and endogenous compounds, remain to be investigated.

Measurement of protein turnover rates by heavy water labeling of nonessential amino acids.

In vivo measurements of protein synthesis using isotope-labeled amino acids (AAs) are hampered by the heterogeneity of AA pools and, for slow turnover proteins, the difficulty and expense of long-term labeling. Continuous oral heavy water (2H2O) labeling can safely maintain stable body water 2H enrichments for weeks or months. 2H is metabolically incorporated into C-H bonds of nonessential AAs (NEAAs) and hence into proteins. No posttranslational label exchange occurs, so 2H incorporation into protein NEAAs, in principle, reports on protein synthesis. Here, we show by mass isotopomer distribution analysis (MIDA) of 2H2O-labeled rodent tissue proteins that metabolic 2H flux into C-H bonds of Ala, Gly, or Gln used for protein synthesis is nearly complete. By 2H2O labeling of rodents, turnover of bone and muscle mixed proteins was quantified and stimulation of liver collagen synthesis by CCl4 was detected. Kinetics of several human serum proteins were also measured, reproducing published t1/2 estimates. Plateau enrichments in Ala varied among different proteins. Moderate amounts of protein, isolated chromatographically or electrophoretically, sufficed for kinetic analyses. In conclusion, 2H2O labeling permits sensitive, quantitative, operationally simple measurements of protein turnover in vivo by the rise-to-plateau approach, especially for proteins with slow constitutive turnover.

Uncertainty Propagation for NIST Visible Spectral Standards.

Uncertainties in the NIST spectral standards for detectors and sources in the visible wavelength range are propagated from the high accuracy cryogenic radiometer measurements, taking correlations into account at every stage. Partial correlations between spectral values at different wavelengths, important for subsequent radiometric calculations, are estimated. Uncertainty propagation through fitting and through transfer spectral measurements is described in detail. Detector uncertainties are propagated through the spectral comparator facility for external calibrations and for internal photometric quantities. Uncertainties in spectral irradiance are derived for the detector-based temperature determination, then propagated through working standards to calibrated artifacts. Spectral irradiance calibrations are generally provided at a limited number of wavelengths. Interpolation, rather than fitting, is recommended for the interpolation of NIST-provided spectral irradiance values.

Ontogenic differences in human liver 4-hydroxynonenal detoxification are associated with in vitro injury to fetal hematopoietic stem cells.

4-hydroxynonenal (4HNE) is a highly mutagenic and cytotoxic alpha,beta-unsaturated aldehyde that can be produced in utero during transplacental exposure to prooxidant compounds. Cellular protection against 4HNE injury is provided by alcohol dehydrogenases (ADH), aldehyde reductases (ALRD), aldehyde dehydrogenases (ALDH), and glutathione S-transferases (GST). In the present study, we examined the comparative detoxification of 4HNE by aldehyde-metabolizing enzymes in a panel of adult and second-trimester prenatal liver tissues and report the toxicological ramifications of ontogenic 4HNE detoxification in vitro. The initial rates of 4HNE oxidation and reduction were two- to fivefold lower in prenatal liver subcellular fractions as compared to adult liver, and the rates of GST conjugation of 4HNE were not detectable in either prenatal or adult cytosolic fractions. GSH-affinity purification of hepatic cytosol yielded detectable and roughly equivalent rates of GST-4HNE conjugation for the two age groups. Consistent with the inefficient oxidative and reductive metabolism of 4HNE in prenatal liver, cytosolic fractions prepared from prenatal liver exhibited a decreased ability to protect against 4HNE-protein adduct formation relative to adults. Prenatal liver hematopoietic stem cells (HSC), which constitute a significant percentage of prenatal liver cell populations, exhibited ALDH activities toward 4HNE, but little reductive or conjugative capacity toward 4HNE through ALRD, ADH, and GST. Cultured HSC exposed to 5 microM 4HNE exhibited a loss in viability and readily formed one or more high molecular weight 4HNE-protein adduct(s). Collectively, our results indicate that second trimester prenatal liver has a lower ability to detoxify 4HNE relative to adults, and that the inefficient detoxification of 4HNE underlies an increased susceptibility to 4HNE injury in sensitive prenatal hepatic cell targets.

Uncertainties in Interpolated Spectral Data.

Interpolation is often used to improve the accuracy of integrals over spectral data convolved with various response functions or power distributions. Formulae are developed for propagation of uncertainties through the interpolation process, specifically for Lagrangian interpolation increasing a regular data set by factors of 5 and 2, and for cubic-spline interpolation. The interpolated data are correlated; these correlations must be considered when combining the interpolated values, as in integration. Examples are given using a common spectral integral in photometry. Correlation coefficients are developed for Lagrangian interpolation where the input data are uncorrelated. It is demonstrated that in practical cases, uncertainties for the integral formed using interpolated data can be reliably estimated using the original data.

Conjugation of 4-hydroxynonenal by largemouth bass (Micropterus salmoides) glutathione S-transferases.

The glutathione S-transferases (GST) are a major group of conjugative enzymes involved in the detoxification of electrophilic compounds and products of oxidative stress. We have previously described the kinetics of hepatic GST conjugation in largemouth bass using a variety of synthetic GST reference substrates. In the present study, we investigated the ability of largemouth bass hepatic GSTs to conjugate 4-hydroxynon-2-enal (4HNE), a mutagenic and cytotoxic alpha-beta-unsaturated aldehyde produced during oxidative injury. Hepatic cytosolic fractions from largemouth bass rapidly catalyzed GSH-dependent 4HNE conjugation, with the rate of GST-4HNE conjugation in bass liver exceeding those of several other mammalian and aquatic species. No apparent sex-related differences in GST-4HNE activity were observed among adult bass. SDS-PAGE and Western blotting analysis of GSH affinity-purified bass liver cytosolic GST revealed the presence of two major GST subunits of approximately 30 and 27 KDa that exhibited slight cross-reactivity when probed with a rat alpha class GST antibody, but not to rat mu, pi or theta class GST. The rapid conjugation of 4HNE by hepatic GST suggests an important role for GSTs in protecting against peroxidation of polyunsaturated fatty acids in bass liver.

Comparative expression of two alpha class glutathione S-transferases in human adult and prenatal liver tissues.

The ability of the fetus to detoxify transplacental drugs and chemicals can be a critical determinant of teratogenesis and developmental toxicity. Developmentally regulated expression of alpha class glutathione S-transferases (GSTs) is of particular interest, since these isozymes have high activity toward peroxidative byproducts of oxidative injury that are linked to teratogenesis. The present study was initiated to examine the expression and catalytic activities of alpha class GST isozymes in human prenatal liver. Northern analysis demonstrated the presence of hGSTA1 and/or A2 (hGSTA1/2) and hGSTA4 steady-state mRNAs in second trimester prenatal livers. Western blotting of prenatal liver proteins provided corroborating evidence via detection of an hGSTA1/2-reactive protein in both cytosol and mitochondria and of hGSTA4-4-reactive protein in mitochondria alone. Catalytic studies demonstrated that prenatal liver cytosolic GSTs were active toward 1-chloro-2,4-dinitrobenzene (a general GST reference substrate), delta5-androstene-3,17-dione (relatively specific for hGSTA1-1), and 4-hydroxynonenal, a highly mutagenic alpha,beta-unsaturated aldehyde produced during oxidative damage and a substrate for hGSTA4-4. Total GSH-peroxidase and GST-dependent peroxidase activities were 9- and 18-fold higher, respectively, in adult liver than in prenatal liver. Multiple tissue array analyses demonstrated considerable tissue-specific and developmental variation in GST mRNA expression. In summary, our results demonstrate the presence of two important alpha class GSTs in second trimester human prenatal tissues, and indicate that mitochondrial targeting of GST may represent an important pathway for removal of cytotoxic products in prenatal liver. Furthermore, the relatively inefficient prenatal reduction of hydroperoxides may underlie an increased susceptibility to maternally transferred pro-oxidant drugs and chemicals.