Homology-Based Functional Proteomics By Mass Spectrometry and Advanced Informatic Methods

Functional characterization of biochemically-isolated proteins is a central task in the biochemical and genetic description of the biology of cells and tissues. Protein identification by mass spectrometry consists of associating an isolated protein with a specific gene or protein sequence in silico, thus inferring its specific biochemical function based upon previous characterizations of that protein or a similar protein having that sequence identity. By performing this analysis on a large scale in conjunction with biochemical experiments, novel biological knowledge can be developed. The study presented here focuses on mass spectrometry-based proteomics of organisms with unsequenced genomes and corresponding developments in biological sequence database searching with mass spectrometry data. Conventional methods to identify proteins by mass spectrometry analysis have employed proteolytic digestion, fragmentation of resultant peptides, and the correlation of acquired tandem mass spectra with database sequences, relying upon exact matching algorithms; i.e. the analyzed peptide had to previously exist in a database in silico to be identified. One existing sequence-similarity protein identification method was applied (MS BLAST, Shevchenko 2001) and one alternative novel method was developed (MultiTag), for searching protein and EST databases, to enable the recognition of proteins that are generally unrecognizable by conventional softwares but share significant sequence similarity with database entries (~60-90%). These techniques and available database sequences enabled the characterization of the Xenopus laevis microtubule-associated proteome and the Dunaliella salina soluble salt-induced proteome, both organisms with unsequenced genomes and minimal database sequence resources. These sequence-similarity methods extended protein identification capabilities by more than two-fold compared to conventional methods, making existing methods virtually superfluous. The proteomics of Dunaliella salina demonstrated the utility of MS BLAST as an indispensable method for characterization of proteins in organisms with unsequenced genomes, and produced insight into Dunaliella?s inherent resilience to high salinity. The Xenopus study was the first proteomics project to simultaneously use all three central methods of representation for peptide tandem mass spectra for protein identification: sequence tags, amino acids sequences, and mass lists; and it is the largest proteomics study in Xenopus laevis yet completed, which indicated a potential relationship between the mitotic spindle of dividing cells and the protein synthesis machinery. At the beginning of these experiments, the identification of proteins was conceptualized as using ?conventional? versus ?sequence-similarity? techniques, but through the course of experiments, a conceptual shift in understanding occurred along with the techniques developed and employed to encompass variations in mass spectrometry instrumentation, alternative mass spectrum representation forms, and the complexities of database resources, producing a more systematic description and utilization of available resources for the characterization of proteomes by mass spectrometry and advanced informatic approaches. The experiments demonstrated that proteomics technologies are only as powerful in the field of biology as the biochemical experiments are precise and meaningful

Life Cycle Assessment of Greenhouse Gas Emissions from Ethanol and Biopolymers

Conclusions • Regulatory LCA is not likely to be used for non‐fuel chemicals alone in the near future • Significant GHG emission credits for corn‐ethanol can be obtained by using only roughly 6‐9% of initial starch for production of biopolymers based on previous LCA theory • Pay close attention to values in calculating credits per kg—these have to stand up in litigation to ensure the credit • Credits are proportional to the mass of polymer produced • Many theoretical issues are uncertain and credits will only be determined in conjunction with EPA • Indirect emissions are uncertain and are a dominant factor in determining total life cycle GHG emissions and the importance of potential co‐product credits from biopolymer

Securing Foreign Oil: A Case for Including Military Operations in the Climate Change Impact of Fuels

Military operations are major industrial activities that use massive amounts of fuel and materials that significantly contribute to climate change. In this article, we assert that military activity to protect international oil trade is a direct production component for importing foreign oil— as necessary for imports as are pipelines and supertankers—and therefore the greenhouse gas (GHG) emissions from that military activity are relevant to U.S. fuel policies related to climate change. Military security for protection of global maritime petroleum distribution is part of the acquisition process, but in addition, recent Middle Eastern wars may also be related to securing petroleum reserves. A component of U.S. motor fuel policy has been to encourage the development of biofuels as substitutes for petroleum, both to reduce dependence on foreign oil and to reduce GHG emissions. To qualify for this substitution under the U.S. Energy Independence and Security Act of 2007 (EISA), specific biofuel types must reduce GHG emissions by set amounts from 20 to 60 percent compared with gasoline. The EISA legislation demands evaluation of not only direct life cycle emissions from biofuels, but also all potentially significant indirect emissions. Yet the gasoline emissions against which this is compared consist only of direct life cycle emissions, which to this point have not included emissions due to the military component of transporting foreign oil to the United States. These military emissions are analyzed here to determine their contribution to the life cycle GHG emissions from gasoline production. This analysis builds on a recent estimate that emissions from military security raised the GHG intensity of U.S. gasoline derived from Middle Eastern imports by twofold compared with direct emissions

Indirect Land Use Emissions in the Life Cycle of Biofuels: Regulations vs. Science

Recent legislative mandates have been enacted at state and federal levels with the purpose of reducing life cycle greenhouse gas (GHG) emissions from transportation fuels. This legislation encourages the substitution of fossil fuels with “low-carbon” fuels. The burden is put on regulatory agencies to determine the GHG-intensity of various fuels, and those agencies naturally look to science for guidance. Even though much progress has been made in determining the direct life cycle emissions from the production of biofuels, the science underpinning the estimation of potentially significant emissions from indirect land use change (ILUC) is in its infancy. As legislation requires inclusion of ILUC emissions in the biofuel life cycle, regulators are in a quandary over accurate implementation. In this article, we review these circumstances and offer some suggestions for how to proceed with the science of indirect effects and regulation in the face of uncertain science. Besides investigating indirect deforestation and grassland conversion alone, a more comprehensive assessment of the total GHG emissions implications of substituting biofuels for petroleum needs to be completed before indirect effects can be accurately determined. This review finds that indirect emissions from livestock and military security are particularly important, and deserve further research

Securing Foreign Oil: A Case for Including Military Operations in the Climate Change Impact of Fuels

Military operations are major industrial activities that use massive amounts of fuel and materials that significantly contribute to climate change. In this article, we assert that military activity to protect international oil trade is a direct production component for importing foreign oil— as necessary for imports as are pipelines and supertankers—and therefore the greenhouse gas (GHG) emissions from that military activity are relevant to U.S. fuel policies related to climate change. Military security for protection of global maritime petroleum distribution is part of the acquisition process, but in addition, recent Middle Eastern wars may also be related to securing petroleum reserves. A component of U.S. motor fuel policy has been to encourage the development of biofuels as substitutes for petroleum, both to reduce dependence on foreign oil and to reduce GHG emissions. To qualify for this substitution under the U.S. Energy Independence and Security Act of 2007 (EISA), specific biofuel types must reduce GHG emissions by set amounts from 20 to 60 percent compared with gasoline. The EISA legislation demands evaluation of not only direct life cycle emissions from biofuels, but also all potentially significant indirect emissions. Yet the gasoline emissions against which this is compared consist only of direct life cycle emissions, which to this point have not included emissions due to the military component of transporting foreign oil to the United States. These military emissions are analyzed here to determine their contribution to the life cycle GHG emissions from gasoline production. This analysis builds on a recent estimate that emissions from military security raised the GHG intensity of U.S. gasoline derived from Middle Eastern imports by twofold compared with direct emissions

Life Cycle Assessment of Greenhouse Gas Emissions from Ethanol and Biopolymers

Conclusions • Regulatory LCA is not likely to be used for non‐fuel chemicals alone in the near future • Significant GHG emission credits for corn‐ethanol can be obtained by using only roughly 6‐9% of initial starch for production of biopolymers based on previous LCA theory • Pay close attention to values in calculating credits per kg—these have to stand up in litigation to ensure the credit • Credits are proportional to the mass of polymer produced • Many theoretical issues are uncertain and credits will only be determined in conjunction with EPA • Indirect emissions are uncertain and are a dominant factor in determining total life cycle GHG emissions and the importance of potential co‐product credits from biopolymer

Opportunities for Nebraska in Future Carbon Markets: Final Technical Report for NCESR Project 3-#303

This study was funded to explore potential opportunities for Nebraska in future carbon markets, most explicitly those opportunities related to the possibility of replacing fossil fuels with biomass at Nebraska corn ethanol plants. The most direct and significant finding is that biomass-fired CHP (combined heat and power) technology is not economically viable for Nebraska corn ethanol plants under current conditions. We estimate in the study that corn stover price would have to be at least $50 per ton of dry matter for the requisite amounts to be delivered to any of the three ethanol plant locations considered (Adams, Norfolk and Wood River). At this price, adoption of CHP would reduce ethanol plant fuel expenditures from about$0.16 per gallon for fossil fuels to about $0.10 per gallon for corn stover, and in addition could add nearly$0.04 per gallon in receipts from sale of surplus electricity to the grid, for a net operating cost reduction of about $0.095 per gallon. However, retrofitting a plant for CHP would require large capital investments with an amortized cost of about$0.24 per gallon, substantially greater than the fuel savings. Potential carbon markets could add only marginal improvements to the prospects for CHP feasibility, adding revenues of about $0.02 per gallon from carbon offsets and perhaps another$0.014 from renewable energy credits. This would bring net operating cost savings to about $0.13 per gallon, still far from paying for the$0.24 per gallon capital cost. CHP technology could become feasible if the capital cost for retrofitting a plant were to fall by 50%, or if natural gas and electricity prices were to rise considerably - at least 60% relative to 2009 prices. Another consideration is the impact of BCAP, USDA\u27s Biomass Crop Assistance Program. This program offers producers a matching payment for whatever price they receive for biomass from an authorized biomass-using facility. The practical effect of this would be to cut in half the price that biomass facilities must pay for delivered biomass, except that the matching payments are limited to two years. Ethanol plants would not be able to invest the capital for retrofitting to biomass based on lower prices for biomass that are limited to only two years, so BCAP will have little impact on CHP feasibility. It is possible that CHP-based ethanol could have a higher market value because of a lower carbon footprint, in California or states that adopt similar policies. We have not made an estimate of this value, because current California regulations do not include soil carbon losses within the boundary of the LCA (life cycle analysis) for the carbon content of biofuels. Our estimates are that conversion to stover-fired CHP would reduce the GHG intensity of the ethanol by 13.3 gCO2e MJ-1. However, the reduction of Midwest corn ethanol\u27s footprint by that amount would provide a fuel with a GHG reduction of only 11% relative to gasoline, which would result in a minimal carbon premium in California even if their regulations were changed to recognize it. An important contribution of this project has been the estimation of supply curves for various amounts of corn stover or switchgrass to be delivered at one of the three delivery points in the study. Biomass in large quantities may be used for other purposes, such as for co-firing with coal in electrical generating plants, or as a feedstock for cellulosic ethanol. The relationship between delivered price and quantity is important information in the evaluation of any such project. One significant finding of the study is that corn stover price would need to be at least $50 per ton of dry matter to have small amounts of less than 100,000 tons per year delivered, or$55-$62 per ton to have a million tons per year delivered, depending on the location in Nebraska. A second significant finding is the lack of competitiveness of switchgrass as a source of biomass in the area of the study. Given current switchgrass technology, prices would have to be$70-$75 per ton of dry matter for delivery of 100,000 to one million tons per year. The project conducted several background studies to be able to address the above issues, results of which are summarized in the report. We reviewed the history and status of climate change initiatives in the U.S. and internationally, from which we were able to identify carbon credits as possible benefits in the future, and renewable energy credits and BCAP benefits available currently and the near future. We also reviewed and summarized the literature on ethanol\u27s carbon footprint attributable to Indirect Land Use Change (ILUC), and though we did not attempt any original research on this issue, a thesis study was in progress at the close of the project examining the potential effects of corn stover revenues on the expansion of cropland into pasture and hay lands in Nebraska. Finally, we examined the relationships between prices of energy sources in Nebraska (natural gas, electricity, and diesel) to aid in understanding how changing energy prices would affect financial feasibility of retrofitting to CHP

Life Cycle Greenhouse Gas Emissions in Maize No-Till Agroecosystems in Southern Brazil Based on a Long-Term Experiment

Brazilian agriculture is constantly questioned concerning its environmental impacts, particularly greenhouse gas (GHG) emissions. This research study used data from a 34-year field experiment to estimate the life cycle GHG emissions intensity of maize production for grain in farming systems under no-tillage (NT) and conventional tillage (CT) combined with Gramineae (oat) and legume (vetch) cover crops in southern Brazil. We applied the Feedstock Carbon Intensity Calculator for modeling the “field-to-farm gate” emissions with measured annual soil N2O and CH4 emissions data. For net CO2 emissions, increases in soil organic C (SOC) were applied as a proxy, where the CT combined with oat was a reference. The life cycle GHG emissions intensity for maize was negative under NT farming systems with Gramineae and legume cover crops, −0.7 and −0.1 kg CO2e kg−1 of maize, respectively. CT with oats as a cover crop had a GHG intensity of 1.0 kg CO2e kg−1 of maize and 2.2 Mg CO2e ha−1. NT with cover crops increased SOC (0.7 C Mg ha−1 yr−1, 0–100 cm) and contributed to the mitigation of life cycle GHG emissions of maize production. This research shows that NT with cover crops is a sustainable solution for farming in southern Brazil