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All sourcesNASA NTRSDOE OSTI
DOE OSTI2015

Process Improvements to Biomass Pretreatment of Fuels and Chemicals

MBI, a 501c(3) company focusing on de-risking and scaling up bio-based technologies, has teamed with Michigan State University and the Idaho National Laboratory to develop and demonstrate process improvements to the ammonia fiber expansion (AFEX) pretreatment process. The logistical hurdles of biomass handling are well known, and the regional depot concept - in which small, distributed bioprocessing operations collect, preprocess, and densify biomass before shipping to a centralized refinery - is a promising alternative to centralized collection. AFEXTM (AFEX is a trademark of MBI) has unique features among pretreatments that would make it desirable as a pretreatment prior to densification at the depot scale. MBI has developed a novel design, using a packed bed reactor for the AFEX process that can be scaled down economically to the depot scale at a lower capital cost as compared to the traditional design (Pandia type reactor). Thus, the purpose of this project was to develop, scale-up, demonstrate, and improve this novel design The key challenges are the recovery of ammonia, consistent and complete pretreatment performance, and the overall throughput of the reactor. In this project an engineering scale packed bed AFEX system with 1-ton per day capacity was installed at MBI’s building. The system has been operational since mid-2013. During that time, MBI has demonstrated the robustness, reliability, and consistency of the process. To date, nearly 500 runs have been performed in the reactors. There have been no incidences of plugging (i.e., inability to remove ammonia from biomass after the treatment), nor has there been any instance of a major ammonia release into the atmosphere. Likewise, the sugar released via enzyme hydrolysis has remained consistent throughout these runs. Our economic model shows a 46% reduction in AFEX capital cost at the 100 ton/day scale compared to the traditional design of AFEX (Pandia type reactor). The key performance factors were demonstrated; >94% ammonia recovery, >75% sugar yields at high solid loading, and complete utilization of the sugars for ethanol production at the 2500 liter scale. Fermentation tests were performed using Zymomonas mobilis 8b and densified AFEX-treated corn stover at >20% solid loading. The obtained titer (~60g/l), productivity (2.5 g/L-h), and yield (330 L/tonne of biomass) exceeded the performance targets set out by NREL. The key findings from these efforts are: no contamination was observed, no cleanup of the sugar stream was required, and no major nutrient addition was required. Our economic model shows that using a packed bed design for the AFEX process and pelleted AFEX-treated biomass reduces the ethanol production cost by 24% when compared to using the traditional AFEX design.

09 BIOMASS FUELS↗
DOE OSTI2013

Novel biofuel formulations for enhanced vehicle performance

This interdisciplinary research program at Michigan State University, in collaboration with Ford Motor Company, has explored the application of tailored or designed biofuels for enhanced vehicle performance and reduced emissions. The project has included a broad range of experimental research, from chemical and biological formation of advanced biofuel components to multicylinder engine testing of blended biofuels to determine engine performance parameters. In addition, the project included computation modeling of biofuel physical and combustion properties, and simulation of advanced combustion modes in model engines and in single cylinder engines. Formation of advanced biofuel components included the fermentation of five-carbon and six-carbon sugars to n-butanol and to butyric acid, two four-carbon building blocks. Chemical transformations include the esterification of the butyric acid produced to make butyrate esters, and the esterification of succinic acid with n-butanol to make dibutyl succinate (DBS) as attractive biofuel components. The conversion of standard biodiesel, made from canola or soy oil, from the methyl ester to the butyl ester (which has better fuel properties), and the ozonolysis of biodiesel and the raw oil to produce nonanoate fuel components were also examined in detail. Physical and combustion properties of these advanced biofuel components were determined during the project. Physical properties such as vapor pressure, heat of evaporation, density, and surface tension, and low temperature properties of cloud point and cold filter plugging point were examined for pure components and for blends of components with biodiesel and standard petroleum diesel. Combustion properties, particularly emission delay that is the key parameter in compression ignition engines, was measured in the MSU Rapid Compression Machine (RCM), an apparatus that was designed and constructed during the project simulating the compression stroke of an internal combustion engine under highly instrumented conditions. Simulation of and experimentation on combustion in single and multicylinder engines was carried out in detail throughout the project. The combustion behavior of biofuel blends neat and in petroleum were characterized in the MSU optical engine, in part to validate results obtained in the RCM and to provide data for comparison with simulations. Simulation of in- cylinder, low-temperature combustion included development of an extensive fuel injection model that included fuel spray breakup, evaporation, and ignition, along with prediction of cylinder temperature, pressure, and work produced. Single cylinder and multicylinder engine tests under advanced low-temperature combustion conditions conducted at Ford Motor Company validated experimental and simulation results obtained in the MSU engine and in MSU simulations. Single cylinder engine tests of an advanced biofuel containing biodiesel and dibutyl succinate, carried out under low-temperature combustion conditions, showed similar power generation and gas-phase emissions (CO, HC, NOx), but a reduction in particulates of as much as 60% relative to neat biodiesel and 95% relative to petroleum diesel at the same operating conditions. This remarkable finding suggests that biofuels may be able to play a role in eliminating the need for particulate removal systems in diesel vehicles. The multicylinder engine tests at Ford, carried out using butyl nonanoate as an advanced biofuel, also gave promising results, showing a strong decline in particulate emissions and simultaneously a modest decrease in NOx emissions relative to standard petroleum diesel at the same conditions. In summary, this project has shown that advanced biofuels and their blends are capable of maintaining performance while reducing emissions, particularly particulates (soot), in 3 compression ignition engines. The interdisciplinary nature of biofuel production and testing has identified fuel properties that are capable of producing such performance, thus providing direction for the implementation of renewable fuels for U.S. transportation. The testing and simulation studies have deepened our understanding of combustion 1) by advancing the rigor with which simulations can be carried out and 2) by illustrating that differences in biofuel and petroleum fuel properties can be used to predict differences in combustion behavior in engines. The future viability of biofuels for compression ignition (diesel) engines is now subject to economic (cost) uncertainty more so than to technical barriers, as the advanced biofuel blends developed here can improve cold-weather fuel properties, provide similar engine performance, and reduce emissions.

33 ADVANCED PROPULSION SYSTEMS↗
DOE OSTI2016

High Pressure Biomass Gasification

According to the Billion Ton Report, the U.S. has a large supply of biomass available that can supplement fossil fuels for producing chemicals and transportation fuels. Agricultural waste, forest residue, and energy crops offer potential benefits: renewable feedstock, zero to low CO 2 emissions depending on the specific source, and domestic supply availability. Biomass can be converted into chemicals and fuels using one of several approaches: (i) biological platform converts corn into ethanol by using depolymerization of cellulose to form sugars followed by fermentation, (ii) low-temperature pyrolysis to obtain bio-oils which must be treated to reduce oxygen content via HDO hydrodeoxygenation), and (iii) high temperature pyrolysis to produce syngas (CO + H 2 ). This last approach consists of producing syngas using the thermal platform which can be used to produce a variety of chemicals and fuels. The goal of this project was to develop an improved understanding of the gasification of biomass at high pressure conditions and how various gasification parameters might affect the gasification behavior. Since most downstream applications of synags conversion (e.g., alcohol synthesis, Fischer-Tropsch synthesis etc) involve utilizing high pressure catalytic processes, there is an interest in carrying out the biomass gasification at high pressure which can potentially reduce the gasifier size and subsequent downstream cleaning processes. It is traditionally accepted that high pressure should increase the gasification rates (kinetic effect). There is also precedence from coal gasification literature from the 1970s that high pressure gasification would be a beneficial route to consider. Traditional approach of using thermogravimetric analyzer (TGA) or high-pressure themogravimetric analyzer (PTGA) worked well in understanding the gasification kinetics of coal gasification which was useful in designing high pressure coal gasification processes. However, similar approach for biomass gasification was not very useful and was the impetus for this study. Specifically, we aimed this study at three broad objectives: (i) defining operating conditions at which C 2 -C 4 hydrocarbons are formed since these represent loss of carbon efficiency, (ii) understanding the formation of tar species which create downstream processing difficulties in addition of carbon efficiency loss, and (iii) kinetics of biomass gasification where it would be possible to understand the effect of operating conditions and gas phase composition.

09 BIOMASS FUELS↗
DOE OSTI2014

Self-deconstructing algae biomass as feedstock for transportation fuels

The potential for producing biofuels from algae has generated much excitement based on projections of large oil yields with relatively little land use. However, numerous technical challenges remain for achieving market parity with conventional non-renewable liquid fuel sources. Among these challenges, the energy intensive requirements of traditional cell rupture, lipid extraction, and residuals fractioning of microalgae biomass have posed significant challenges to the nascent field of algal biotechnology. Our novel approach to address these problems was to employ low cost solution-state methods and biochemical engineering to eliminate the need for extensive hardware and energy intensive methods for cell rupture, carbohydrate and protein solubilization and hydrolysis, and fuel product recovery using consolidated bioprocessing strategies. The outcome of the biochemical deconstruction and conversion process consists of an emulsion of algal lipids and mixed alcohol products from carbohydrate and protein fermentation for co-extraction or in situ transesterification.

09 BIOMASS FUELS↗
DOE OSTI2012

Bio-energy Alliance High-Tonnage Bio-energy Crop Production and Conversion into Conventional Fuels

Maintaining a predictable and sustainable supply of feedstock for bioenergy conversion is a major goal to facilitate the efficient transition to cellulosic biofuels. Our work provides insight into the complex interactions among agronomic, edaphic, and climatic factors that affect the sustainability of bioenergy crop yields. Our results provide science-based agronomic response measures that document how to better manage bioenergy sorghum production from planting to harvest. We show that harvest aids provide no significant benefit as a means to decrease harvest moisture or improve bioenergy yields. Our efforts to identify optimal seeding rates under varied edaphic and climatological conditions reinforce previous findings that sorghum is a resilient plant that can efficiently adapt to changing population pressures by decreasing or increasing the numbers of additional shoots or tillers – where optimal seeding rates for high biomass photoperiod sensitive sorghum is 60,000 to 70,000 seeds per acre and 100,000 to 120,000 seeds per acre for sweet varieties. Our varietal adaptability trials revealed that high biomass photoperiod sensitive energy sorghum consistently outperforms conventional photoperiod insensitive sweet sorghum and high biomass forage sorghum as the preferred bioenergy sorghum type, with combined theoretical yields of both cellulosic and fermentable water-soluble sugars producing an average yield of 1,035 gallons of EtOH per acre. Our nitrogen trials reveal that sweet sorghums produce ample amounts of water-soluble sugars with minimal increases in nitrogen inputs, and that excess nitrogen can affect minor increases in biomass yields and cellulosic sugars but decrease bioenergy quality by decreasing water-soluble sugar concentrations and increasing ash content, specifically when plant tissue nitrogen concentrations exceed 0.6 %, dry weight basis. Finally, through our growth and re-growth trials, we show that single-cut high biomass sorghum bioenergy yields significantly exceed those of multiple-cut high biomass sorghum systems. Our agronomic yield and quality data will be uploaded to the Bioenergy KDF Website before the end of February 2013, with a date for public access to be determined pending peer-reviewed publication of our findings.

09 BIOMASS FUELS↗
DOE OSTI2016

Energy-Efficient Purification of Bio-Fuels and Bio-Chemicals Using a Novel Advanced Polymeric Membrane System: CRADA Final Report

The team collaborated to develop, demonstrate and move toward the commercialization of a pervaporation membrane system, which reduces the energy required for product isolation from dilute fermentation broths. Avoiding the energy consumption associated with the latent heat of vaporization for water was the key benefit. Essentially, this work isolated high boilers from dilute feeds without evaporating water. These activities lead to the development, fabrication, and demonstration of proprietary, advanced polymer membrane cartridges that dramatically reduce the cost of bio-chemical and bio-fuel manufacture.

09 BIOMASS FUELS↗
DOE OSTI2016

Enhanced Mixed Feedstock Processing Using Ionic Liquids

Biomass pretreatment using certain ionic liquids (ILs) is very efficient, generally producing a substrate that is amenable to saccharification with fermentable sugar yields approaching theoretical limits. Although promising, several challenges must be addressed before IL pretreatment technology becomes commercially viable. Once of the most significant challenges is the affordable and scalable recovery and recycle or the IL itself. Pervaporation is a highly selective and scalable membrane separation process for quantitatively recovering volatile solutes or solvents directly from non-volatile solvents that could prove more versatile for IL dehydration than traditional solvent extraction processes, as well as efficient and energetically more advantageous than standard evaporative techniques. In this study we evaluated a commercially available pervaporation system for IL dehydration and recycling as part of an integrated IL pretreatment process using 1-ethyl-3-methylimidazolium acetate ([C2C1Im][OAc]) that has been proven to be very effective as a biomass pretreatment solvent. We demonstrate that >99.9 wt% [C2C1Im][OAc] can be recovered from aqueous solution and recycled at least five times. A preliminary techno-economic analysis validated the promising role of pervaporation in improving overall biorefinery process economics, especially in the case where other IL recovery technologies might lead to significant losses. These findings establish the foundation for further development of pervaporation as an effective method of recovering and recycling ILs using a commercially viable process technology.

09 BIOMASS FUELS↗
DOE OSTI2017

Computational Modeling of Fluctuations in Energy and Metabolic Pathways of Methanogenic Archaea

The methanogenic archaea, anaerobic microbes that convert CO2 and H2 and/or other small organic fermentation products into methane, play an unusually large role in the global carbon cycle. As they perform the final step in the anaerobic breakdown of biomass, methanogens are a biogenic source of an estimated one billion tons methane each year. Depending on the location, produced methane can be considered as either a greenhouse gas (agricultural byproduct), sequestered carbon storage (methane hydrate deposits), or a potential energy source (organic wastewater treatment). These microbes therefore represent an important target for biotechnology applications. Computational models of methanogens with predictive power are useful aids in the adaptation of methanogenic systems, but need to connect processes of wide-ranging time and length scales. In this project, we developed several computational methodologies for modeling the dynamic behavior of entire cells that connects stochastic reaction-diffusion dynamics of individual biochemical pathways with genome-scale modeling of metabolic networks. While each of these techniques were in the realm of well-defined computational methods, here we integrated them to develop several entirely new approaches to systems biology. The first scientific aim of the project was to model how noise in a biochemical pathway propagates into cellular phenotypes. Genetic circuits have been optimized by evolution to regulate molecular processes despite stochastic noise, but the effect of such noise on a cellular biochemical networks is currently unknown. An integrated stochastic/systems model of Escherichia coli species was created to analyze how noise in protein expression gives—and therefore noise in metabolic fluxes—gives rise to multiple cellular phenotype in isogenic population. After the initial work developing and validating methods that allow characterization of the heterogeneity in the model organism E. coli, the project shifted toward investigations of the methanogen Methanosarcina acetivorans. By integrating an unprecedented transcriptomics dataset for growth of the methanogen on many substrates with an in silico model, heterogeneity in metabolic pathway usage and methane production were examined. This lent insight into the physiological requirements of the organism under different environmental conditions and uncovered the unique regulatory role that mRNA half-life has in shaping metabolic flux distributions in this organism.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2014

Value Added Products from Renewable Biofuels

Cellulosic ethanol is an emerging biofuel that will make strong contributions to American domestic energy needs. In the US midwest the standard method for pretreatment of biomass uses hot acid to deconstruct lignocellulose. While other methods work, they are not in common use. Therefore it is necessary to work within this context to achieve process improvements and reductions in biofuel cost. Technology underlying this process could supplement and even replace commodity enzymes with engineered microbes to convert biomass-derived lignocellulose feedstocks into biofuels and valueadded chemicals. The approach that was used here was based on consolidated bioprocessing. Thermoacidophilic microbes belonging to the Domain Archaea were evaluated and modfied to promote deconvolution and saccharification of lignocellulose. Biomass pretreatment (hot acid) was combined with fermentation using an extremely thermoacidophilic microbial platform. The identity and fate of released sugars was controlled using metabolic blocks combined with added biochemical traits where needed. LC/MS analysis supported through the newly established Nebraska Bioenergy Facility provided general support for bioenergy researchers at the University of Nebraska. The primary project strategy was to use microbes that naturally flourish in hot acid (thermoacidophiles) with conventional biomass pretreatment that uses hot acid. The specific objectives were: to screen thermoacidophilic taxa for the ability to deconvolute lignocellulose and depolymerize associated carbohydrates; evaluate and respond to formation of “inhibitors” that arose during incubation of lignocellulose under heated acidic conditions; identify and engineer “sugar flux channeling and catabolic blocks” that redirect metabolic pathways to maximize sugar concentrations; expand the hydrolytic capacity of extremely thermoacidophilic microbes through the addition of deconvolution traits; and establish the Nebraska Bioenergy Facility (NBF) at the University of Nebraska-Lincoln.

09 BIOMASS FUELS↗
DOE OSTI2013

Sweet Sorghum Alternative Fuel and Feed Pilot Project

The University of Arizona undertook a “pilot” project to grow sweet sorghum on a field scale (rather than a plot scale), produce juice from the sweet sorghum, deliver the juice to a bio-refinery and process it to fuel-grade ethanol. We also evaluated the bagasse for suitability as a livestock feed and as a fuel. In addition to these objectives we evaluated methods of juice preservation, ligno-cellulosic conversion of the bagasse to fermentable sugars and alternative methods of juice extraction.

09 BIOMASS FUELS↗
DOE OSTI2012

Advancing the Renewable Industry in Minnesota

This report deals with seven topics: 1. "Utilizing Ash Wastes as a Nutrient Source for Corn": As forms of gasification and combustion grow in the area of renewable energy in Minnesota the question arises regarding the utilization/application of the ash co product produced from these processes. Currently there are four facilities in Minnesota producing an ash co product (three ethanol facilities and one combusting biomass to produce energy). These ash wastes are generated from using ethanol by-products as a fuel or heating source for fermentation. Other ash wastes from agricultural sources include turkey litter ash. When applied to agricultural fields, ash wastes can be a source of nutrients for agricultural crops. Chemical analyses of ash wastes vary, but 200 to 300 lb of P 2 O 5 and K 2 O per ton of ash is typical. The value of ash wastes as a fertilizer has increased because commercial fertilizer prices have increased significantly over the last few years. Specifically: Compaction/Agglomeration research- Research included development of an appropriate product for use in current delivery systems by densifying the ash into the form of pellets or briquettes which may reduce fertilizer input cost to farmers. The initiative addresses the use of phosphorus and potassium from co-firing or gasification processes as a fertilizer source. 2. "Use of Glycerol as a Corn Replacement in Calf Starter Diets": Glycerol is a sugar alcohol by-product of bio-diesel production. About 1 gallon of glycerin is produced for every 10 gallons of bio-diesel of which the glycerol content may vary between 63 and almost 100%. There is some uncertainty of the exact energy value of glycerol as an ingredient for animal feed but it has been successfully used as a replacement for corn up to 10% of the diet dry matter for lactating dairy cows. There is a lack of information on incorporating glycerol into diets for pre- and post weaned dairy heifer calves which has the potential to expand the market for this by product. Preliminary work at SROC indicates that there are no palatability problems using glycerol up to 6% of the mix in post weaning pellets for group fed dairy heifers. 3. "Intervention Strategies for Reduction of Food-borne Pathogens in Cattle Fed Ethanol Byproducts": Ethanol plants and cattle producers create a symbiotic relationship: The use of wet and dry distiller’s grains in beef cattle rations provides significant improvement in overall feedlot performance for many producers in Minnesota and the Upper Midwest. Distiller’s grain is the coproduct that remains following the distillation of ethanol. It may be dehydrated to produce dried distiller’s grain (DDG) which is then commonly used as livestock feed. The use of distiller’s grains in livestock rations has been the subject of numerous research projects over the past ten years. Recently published research data conducted at Kansas State University (K-State) has created controversy over the feeding of distiller’s grains in beef cattle rations. The research indicates that cattle fed distiller’s grain may have an increased probability of Escherichia coli O157 in their hindgut. Specifically: This research assessment was a two part project consisting of feeding trials conducted at the University of Minnesota and an intervention study conducted at Kansas State University. This report is the Kansas State University portion. This report is a task-level final technical report. 4. "Impact of Distillers' Grains and Glycerin on Cattle Fecal Shedding of Escherichia coli O157:H7" 5. "Study Assessing the Opportunities and Potential of Soybean Based Products and Technologies": Based on demand/market potential, economic feasibility, stage of development and strength of institutional support, Informa Economics, Inc. ("Informa") narrowed down a list of more than 100 emerging soybean products and to 8 of the most promising for soybean, considered to have the greatest potential to add significant value to Minnesota's soybean commodity production. However, as with the potential of any biobased product or technology, the development of these emerging soybean products and technologies will be heavily reliant on future market price environments (especially for petroleum) and government policies. This report identifies what the contractor believes are the top 8 products and technologies for soybeans at the time of publishing. 6. "Study Assessing the Opportunities and Potential of Corn-Based Products and Technologies": Based on demand/market potential, economic feasibility, stage of development and strength of institutional support, Informa Economics, Inc. ("Informa") narrowed down a list of more than 100 emerging corn products and technologies to 8 of the most promising for corn, considered to have the greatest potential to add significant value to Minnesota's corn commodity production. However, as with the potential of any biobased product or technology, the development of these emerging corn products and technologies will be heavily reliant on future market price environments (especially for petroleum) and government policies. This report identifies what the contractor believes are the top 8 products and technologies for corn at the time of publishing. 7. "Distributed Power Generation Technology (Roseau Gasification)."

09 BIOMASS FUELS↗
DOE OSTI2017

Final Technical Report - Consolidating Biomass Pretreatment with Saccharification by Resolving the Spatial Control Mechanisms of Fungi

Consolidated bioprocessing (CBP) of lignocellulose combines enzymatic sugar release (saccharification) with fermentation, but pretreatments remain separate and costly. In nature, lignocellulose-degrading brown rot fungi consolidate pretreatment and saccharification, likely using spatial gradients to partition these incompatible reactions. With the field of biocatalysis maturing, reaction partitioning is increasingly reproducible for commercial use. Therefore, my goal was to resolve the reaction partitioning mechanisms of brown rot fungi so that they can be applied to bioconversion of lignocellulosic feedstocks. Brown rot fungi consolidate oxidative pretreatments with saccharification and are a focus for biomass refining because 1) they attain >99% sugar yield without destroying lignin, 2) they use a simplified cellulase suite that lacks exoglucanase, and 3) their non-enzymatic pretreatment is facilitative and may be accelerated. Specifically, I hypothesized that during brown rot, oxidative pretreatments occur ahead of enzymatic saccharification, spatially, and the fungus partitions these reactions using gradients in pH, lignin reactivity, and plant cell wall porosity. In fact, we found three key results during these experiments for this work: 1) Brown rot fungi have an inducible cellulase system, unlike previous descriptions of a constitutive mechanism. 2) The induction of cellulases is delayed until there is repression of oxidatively-linked genes, allowing the brown rot fungi to coordinate two incompatible reactions (oxidative pretreatment with enzymatic saccharification, to release wood sugars) in the same pieces of wood. 3) This transition is mediated by the same wood sugar, cellobiose, released by the oxidative pretreatment step. Collectively, these findings have been published in excellent journal outlets and have been presented at conferences around the United States, and they offer clear targets for gene discovery en route to making biofuels and biochemicals affordable, commercially.

09 BIOMASS FUELS↗
DOE OSTI2017

Deconstruction of Macroalgae

The goal of this collaborative project was to explore the feasibility of using macroalgae as a feedstock for biofuel. Specifically to focus on the conversion of macroalgae to biofuels or other valuable co-products which are low carbon, cost-effective, and sustainable. The main objectives were to develop a library of the composition of macroalgae, develop optimal pretreatments, investigate and determine and optimize enzymatic cocktails capable of efficiently hydrolyzing the resultant polysaccharides into monomeric sugars. The initial technical objective was to identify, verify, and develop new modes of pre-treatment and evaluate enzymes for hydrolysis of macroalgae to fermentable sugars.

09 BIOMASS FUELS↗
DOE OSTI2017

Spider silk MASP1 and MASP2 proteins as carbon fiber precursors

The objective of this project is to develop an unconventional non-petroleum based carbon fiber precursor which has the potential to be produced in high yield and quantities. Methods will be developed to produce pilot-scale quantities of fibers from spider silk proteins with mechanical properties at least 75% that of the natural dragline silk fibers in tensile strength and elongations of less than 5%. The precursor fibers will be converted to carbon fibers, with a goal of >250Ksi strength and 1-2% elongation. Cost analysis will be performed and the process optimized. Task 1: Subtask 1. Protein production: We exceeded the go/ no go milestone of 1.0g/L of one of the spider silk protein (MSp2) purified last FY and have now increased from 5L to 500L fermentations. We have made a series of changes to the purification protocol from the initial report last FY. These led to a reduction in the time needed for the purification and reduced the purification costs by nearly 90%. Subtask 2. Fiber spinning: The major focus has been to produce more material to send 24 fiber thread to ONRL. We are still developing the methodology to successfully spin 24 fiber yarns. This involves both the spinning dope solutions as well as the methods to keep the fibers from fusing during the post spin stretch. The second area of focus has been to standardize the spin dopes for making the fibers. We now know that the conductivity (indicative of salt remaining with the protein after purification) is an important factor in successful spinning as is the pH. We now know that we need to be below 600 uS conductivity and that the most effective pH is protein dependent. Subtask 3. Silkworm silk: We have found the transgenic silkworms made using gene replacement at the fibroin light chain instead of heavy chain as we did previously have a higher tensile strength. See figures below showing the curve for the top end of the cocoon fibers. This tensile strength is the same as the average for spider dragline silk. Task 2. Carbonization: The major accomplishment in the latter part of the work is that the ONRL group has successfully heated the spider silk protein fibers all the way up to 1700°C and produced a very competitive carbon fiber based on mechanical properties. Several important factors were discovered during these initial trials: 1) the ramp speed for increasing the temperature is critical; 2) maintaining tension on the fiber during the heating process because as it is heated it tends to expand; and 3) narrow temperature window in which stretching the fiber during heating leads to much better final materials. Task 3. Techno-Economic Methods: The techno-economic analysis was expanded to determine the relative cost of production with the bacterial production system compared to the transgenic alfalfa and goat production systems. The comparisons show two important things. For all systems the key factor in the final price is the amount of spider silk protein produced for whatever measure of volume or weight is used. Second alfalfa can be the cheapest but is subject to the possible regulatory control unless the US develops a more comprehensive approach to GMOs. The silkworm analysis was not completed due to a variety of confounding factors. The primary one was that if the production were shifted overseas then the cost would likely be nearly equivalent to current silk prices of $5-15/kg. However if concerns about the location of production is important then it would need to be done in the US and initial costs would be much higher but if the later scenario is utilized then the cost would be lowered but it was not possible to calculate exact costs.

36 MATERIALS SCIENCE↗
DOE OSTI2017

Surface characterization of pretreated and microbial-treated populus cross-sections

The first objective of this thesis is to illustrate the advantages of surface characterization in biomass utilization studies. The second objective is to gain insight into the workings of potential consolidated bioprocessing microorganisms on the surface of poplar samples. The third objective is to determine the impact biomass recalcitrance has on enzymatic hydrolysis and microbial fermentation in relation to the surface chemistry.

09 BIOMASS FUELS↗
DOE OSTI2017

Decision-support for Digester-Algae IntegRation for Improved Environmental and Economic Sustainability (DAIRIEES)

Manure management is a major concern for dairy farms, as manure emits significant quantities of greenhouse gases and contains concentrated nutrients, especially nitrogen and phosphorus. Current manure management practices consist of spreading minimally processed manure on agricultural fields, which releases greenhouse gases directly to the atmosphere and often leads to nutrient overloading on fields and runoff to surface and groundwater. A novel manure treatment system has been proposed that mitigates many of the current environmental concerns and creates value added products from the manure including bioplastics, electricity, fertilizer, and animal bedding. DAIRIEES, an Excel based model, allows users to enter characteristics about a dairy farm’s manure, manure management plan, and regional market. Based on these inputs, the five main processes of the integrated system—fermenter, anaerobic digester, bioplastics reactor, algae cultivation, and hydrothermal liquefaction or fast pyrolysis system—are analyzed in detail using data from laboratory scale experiments supplemented by information on full-scale processes from the literature. The model can be used to estimate performance of the integrated manure treatment system, including: 1) carbon and nutrient sequestration, 2) quantities and market value of end products, and 3) the system’s overall economic viability. The DAIRIEES model outlines the major economic considerations for construction and operation of a full scale integrated treatment system. This information can be used to inform a more detailed pro forma analysis of the deployed system.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2017

Novel Biocatalytic Platform for Ethanol Production from Lignocellulosic Feedstock

The goals of the CRADA were achieved by illustrating the scalability of immobilized yeast technology, demonstrating lignocellulosic feedstock consumption by the immobilized cells, and confirming Microvi’s proprietary polymer matrix ethanol toxicity tolerance. We conducted fermentations at 2L and 300L scales. For carbon source, we performed pretreatment and saccharification at 100L scale to produce lignocellulosic sugars with glucose and xylose.

09 BIOMASS FUELS↗
DOE OSTI2018

Pyramiding genes and alleles for improving energy cane biomass yield- Final Report

The overall goal of this project is to identify genes and gene interaction networks contributed to the extreme segregants with 30 folds biomass yield difference in sugarcane F2 populations. Towards achieving this goal, yield trials of 108 F2 extreme segregants from S. officinarum LA Purple and S. robustum MOL5829 (LM population) were carried out in two locations in three years. A yield trial of the second F2 population from S. officinarum LA Purple and S. spontaneum US56-14-4 (LU population) was installed in the summer of 2014 and the first set of yield component data was collected. For genotyping, transcriptomes from leaves and stalks of 70 extreme segregants of the LM F2 population and 119 individuals of the LU F2 populations were sequenced. The genomes of 91 F1 individuals from the LM populations are being sequenced to construct ultra-high density genetic maps for each of the two parents for both assisting the LA Purple genome assembling and for testing a hypothesis of female restitution. The genomes of 110 F2 individuals from single F1 in the LU population, a different set from the 119 F2 individuals used for transcriptome sequencing, are being sequenced for mapping genes and QTLs affecting biomass yield and for testing a hypothesis of female restitution. Gene expression analysis between extreme segregants of high and low biomass yield showed up-regulation of cellulose synthase, cellulose, and xylan synthase in high biomass yield segregants among 3,274 genes differentially expressed between the two extremes. Our transcriptome results revealed not only the increment of cell wall biosynthesis pathway is essential, but the rapid turnover of certain cell wall polymers as well as carbohydrate partitioning are also important for recycling and energy conservation during rapid cell growth in high biomass sugarcane. Seventeen differentially expressed genes in auxin, one in ethylene and one in gibberellin related signaling and biosynthesis pathways were identified, which could potentially regulate biomass yield. Differentially expressed genes, PIF3 and EIL5, involved in gibberellin and ethylene pathway could play an important role in biomass accumulation. Differential gene expression analysis was also carried out on the LU population. High-biomass yield was mainly determined by assimilation of carbon in source tissues. The high-level expression of fermentative genes in the low-biomass group was likely induced by their low-energy status. The haploid (tetraploid) genome of S. spontanium AP85-441 was sequenced with chromosome level assembly and allele defined annotation. This reference genome along with the upcoming S. officinarum genome will allow us to identify genes and alleles contributed to biomass yield.

09 BIOMASS FUELS↗