Quantifying Actual and Theoretical Ethanol Yields for Switchgrass Strains Using NIRS Analyses

Quantifying actual and theoretical ethanol yields from biomass conversion processes such as simultanteous saccharification and fermentation (SSF) requires expensive, complex fermentation assays, and extensive compositional analyses of the biomass sample. Near-infrared reflectance spectroscopy (NIRS) is a non-destructive technology that can be used to obtain rapid, low-cost, high-throughput, and accurate estimates of agricultural product composition. In this study, broad-based NIRS calibrations were developed for switchgrass biomass that can be used to estimate over 20 components including cell wall and soluble sugars and also ethanol production and pentose sugars released as measured using a laboratory SSF procedure. With this information, an additional 13 complex feedstock traits can be determined including theoretical and actual ethanol yields from hexose fermentation. The NIRS calibrations were used to estimate feedstock composition and conversion information for biomass samples from a multi-year switchgrass (Panicum virgatum L.) biomass cultivar evaluation trial. There were significant differences among switchgrass strains for all biomass conversion and composition traits including actual ethanol yields, ETOHL (L Mg^?1) and theoretical ethanol yields, ETOHTL (L Mg^?1), based on cell wall and non-cell wall composition NIRS analyses. ETOHL means ranged from 98 to 115 L Mg^?1 while ETOHTL means ranged from 203 to 222 L Mg^?1. Because of differences in both biomass yields and conversion efficiency, there were significant differences among strains for both actual (2,534?C3,720 L ha^?1) and theoretical (4,878?C7,888 L ha^?1) ethanol production per hectare. It should be feasible to improve ethanol yields per hectare by improving both biomass yield and conversion efficiency by using NIRS analyses to quantify differences among cultivars and management practices.     

Production of acetic acid by Dekkera/Brettanomyces yeasts under conditions of constant pH

Sixty yeast strains were previously screened for their ability to produce acetic acid, in shaken flask batch culture, from either glucose or ethanol. Seven of the strains belonging to the Brettanomyces and Dekkera genera, from the ARS Culture Collection, Peoria, IL, were further evaluated for acetic acid production in bioreactor batch culture at 28 °C, constant aeration (0.75 v/v/m) and pH (6.5). The medium contained either 100 g glucose/l or 35 g ethanol/l as the carbon/energy source. Dekkera intermedia NRRL YB-4553 produced 42.8 and 14.9 g acetic acid/l from the two carbon sources, respectively, after 64.5 h. The optimal pH was determined to be 5.5. When the initial glucose concentration was 150 or 200 g/l, the yeast produced 57.5 and 65.1 g acetic acid/l, respectively.     

Genetic Characterization of the Carotenoid Biosynthetic Pathway in Methylobacterium extorquens AM1 and Isolation of a Colorless Mutant

Genomic searches were used to reconstruct the putative carotenoid biosynthesis pathway in the pink-pigmented facultative methylotroph Methylobacterium extorquens AM1. Four genes for putative phytoene desaturases were identified. A colorless mutant was obtained by transposon mutagenesis, and the insertion was shown to be in one of the putative phytoene desaturase genes. Mutations in the other three did not affect color. The tetracycline marker was removed from the original transposon mutant, resulting in a pigment-free strain with wild-type growth properties useful as a tool for future experiments.     

Augmentation of anti-tumor responses of adoptively transferred CD8+T cells in the lymphopenic setting by HSV amplicon transduction

Treatment of cancer with cytotoxic agents may induce lymphopenia. Adoptively transferred T cells have been reported to display enhanced anti-tumor efficacy in the lymphopenic setting. We reasoned that the anti-tumor effects of adoptively transferred cells in the lymphopenic host could be further augmented through local provision of an innate stimulus in the tumor bed. Utilizing a model in which mice were irradiated to induce lymphopenia, with limited shielding to allow tumor growth, we demonstrate that “triple” therapy consisting of radiation-induced lymphopenia, adoptive transfer of naïve CD8+ T cells, and intra-tumoral HSV amplicon injection resulted in reduced tumor growth compared to the combination of any two of the aforementioned interventions. To gain insight into the mechanism underlying this effect we studied the effects of HSV amplicon transduction into tumors on cytokine expression and on anti-tumor specific T cells. HSV amplicon transduction specifically induced several cytokine mRNAs including IFN-γ, and IP-10. Adoptively transferred transgenic OT-1 T cells directed against Ovalbumin were more effective against Ovalbumin-expressing tumors when combined with intra-tumoral HSV amplicon injections in the lymphopenic host. Following intra-tumoral HSV-amplicon injections, anti-tumor T cells secreted higher levels of interferon-γ in response to in-vitro re-stimulation with tumor cells, implying that HSV amplicon injection provided a strong signal for T cell activation. Combining adoptive transfer of naïve T cells in the lymphopenic setting with local T cell stimulation may facilitate expansion and activation of anti-tumor T cell populations in vivo, resulting in enhanced anti-tumor responses without the need to resort to prolonged in vitro T cell culture and/or manipulation.     

Composition of corn dry-grind ethanol by-products: DDGS, wet cake, and thin stillage

DDGS and wet distillers' grains are the major co-products of the dry grind ethanol facilities. As they are mainly used as animal feed, a typical compositional analysis of the DDGS and wet distillers' grains mainly focuses on defining the feedstock's nutritional characteristics. With an increasing demand for fuel ethanol, the DDGS and wet distillers' grains are viewed as a potential bridge feedstock for ethanol production from other cellulosic biomass. The introduction of DDGS or wet distillers' grains as an additional feed to the existing dry grind plants for increased ethanol yield requires a different approach to the compositional analysis of the material. Rather than focusing on its nutritional value, this new approach aims at determining more detailed chemical composition, especially on polymeric sugars such as cellulose, starch and xylan, which release fermentable sugars upon enzymatic hydrolysis. In this paper we present a detailed and complete compositional analysis procedure suggested for DDGS and wet distillers' grains, as well as the resulting compositions completed by three different research groups. Polymeric sugars, crude protein, crude oil and ash contents of DDGS and wet distillers' grains were accurately and reproducibly determined by the compositional analysis procedure described in this paper.     

Butanol production by Clostridium beijerinckii. Part I: use of acid and enzyme hydrolyzed corn fiber

Fermentation of sulfuric acid treated corn fiber hydrolysate (SACFH) inhibited cell growth and butanol production (1.7 ± 0.2 g/L acetone butanol ethanol or ABE) by Clostridium beijerinckii BA101. Treatment of SACFH with XAD-4 resin removed some of the inhibitors resulting in the production of 9.3 ± 0.5 g/L ABE and a yield of 0.39 ± 0.015. Fermentation of enzyme treated corn fiber hydrolysate (ETCFH) did not reveal any cell inhibition and resulted in the production of 8.6 ± 1.0 g/L ABE and used 24.6 g/L total sugars. ABE production from fermentation of 25 g/L glucose and 25 g/L xylose was 9.9 ± 0.4 and 9.6 ± 0.4 g/L, respectively, suggesting that the culture was able to utilize xylose as efficiently as glucose. Production of only 9.3 ± 0.5 g/L ABE (compared with 17.7 g/L ABE from fermentation of 55 g/L glucose-control) from the XAD-4 treated SACFH suggested that some fermentation inhibitors may still be present following treatment. It is suggested that inhibitory components be completely removed from the SACFH prior to fermentation with C. beijerinckii BA101. In our fermentations, an ABE yield ranging from 0.35 to 0.39 was obtained, which is higher than reported by the other investigators.     

When good organs go to bad people

A number of philosophers have argued that alcoholics should receive lower priority for liver transplantations because they are morally responsible for their medical conditions. In this paper, I argue that this conclusion is false. Moral responsibility should not be used as a criterion for the allocation of medical resources. The reason I advance goes further than the technical problem of assessing moral responsibility. The deeper problem is that using moral responsibility as an allocation criterion undermines the functioning of medicine.     

Quantification of central metabolic fluxes in the facultative methylotroph methylobacterium extorquens AM1 using 13C-label tracing and mass spectrometry

The metabolic fluxes of central carbon metabolism were measured in chemostat-grown cultures of Methylobacterium extorquens AM1 with methanol as the sole organic carbon and energy source and growth-limiting substrate. Label tracing experiments were carried out using 70% (13)C-methanol in the feed, and the steady-state mass isotopomer distributions of amino acids derived from total cell protein were measured by gas chromatography coupled to mass spectrometry. Fluxes were calculated from the isotopomer distribution data using an isotopomer balance model and evolutionary error minimization algorithm. The combination of labeled methanol with unlabeled CO(2), which enters central metabolism in two different reactions, provided the discriminatory power necessary to allow quantification of the unknown fluxes within a reasonably small confidence interval. In wild-type M. extorquens AM1, no measurable flux was detected through pyruvate dehydrogenase or malic enzyme, and very little flux through alpha-ketoglutarate dehydrogenase (1.4% of total carbon). In contrast, the alpha-ketoglutarate dehydrogenase flux was 25.5% of total carbon in the regulatory mutant strain phaR, while the pyruvate dehydrogenase and malic enzyme fluxes remained insignificant. The success of this technique with growth on C(1) compounds suggests that it can be applied to help characterize the effects of other regulatory mutations, and serve as a diagnostic tool in the metabolic engineering of methylotrophic bacteria.     

Genetic characterization of the carotenoid biosynthetic pathway in Methylobacterium extorquens AM1 and isolation of a colorless mutant

Genomic searches were used to reconstruct the putative carotenoid biosynthesis pathway in the pink-pigmented facultative methylotroph Methylobacterium extorquens AM1. Four genes for putative phytoene desaturases were identified. A colorless mutant was obtained by transposon mutagenesis, and the insertion was shown to be in one of the putative phytoene desaturase genes. Mutations in the other three did not affect color. The tetracycline marker was removed from the original transposon mutant, resulting in a pigment-free strain with wild-type growth properties useful as a tool for future experiments.     

Determination of metabolic flux changes during fed-batch cultivation from measurements of intracellular amino acids by LC-MS/MS

Metabolic flux analysis using (13)C-labeled substrates is a well-developed method for investigating cellular behavior in steady-state culture condition. To extend its application, in particular to typical industrial conditions, such as batch and fed-batch cultivations, a novel method of (13)C metabolic flux analysis is proposed. An isotopomer balancing model was developed to elucidate flux distributions in the central metabolism and all amino acids synthetic pathways. A lysine-producing strain of Escherichia coli was cultivated by fed-batch mode in a growth medium containing yeast extract. Mass distribution data was derived from both intracellular free amino acids and proteinogenic amino acids measured by LC-MS/MS, and a correction parameter for the protein turnover effect on the mass distributions of intracellular amino acids was introduced. Metabolic flux distributions were determined in both exponential and stationary phases. Using this new approach, a culture phase-dependent metabolic shift was detected in the fed-batch culture. The approach presented here has great potential for investigating cellular behavior in industrial processes, independent of cultivation modes, metabolic phase and growth medium.