Recipient Organization
STATE UNIV OF NEW YORK
(N/A)
SYRACUSE,NY 13210
Performing Department
Chemistry
Non Technical Summary
Research efforts will focus on the conversion of lignocellulosic biomass from willow (Salix) to sugars useful for the synthesis of ethanol, by yeast fermentation, and bio-oil from algae grown under heterotrophic conditions. Ethanol represents an important oxygenated blending component for gasoline while algal oil can used as a feedstock for bio-diesel and other hydrocarbon-based fuels. We have previously shown for debarked sugar maple wood chips, that Electron Beam (EB) irradiation, as a pretreatment, significantly increases both the rate and ultimate yield of fermentable sugars produced by cellulase enzymes at a total energy input well below many other pretreatments being proposed for woody biomass. In addition, EB does not generate inhibitors of yeast fermentation and may actually provide a fermentation rate benefit (SUNY RF Patent Disclosure 1925-550). These results have not yet been duplicated for plantation-grown fast-growing willow biomass which has been a focal point for woody feedstock development at SUNY-ESF for over two decades. Post-harvest willow chips typically contain a small fraction of bark on each chip which could possibly impact sugar yield or bio-fuel fermentation productivity. As a result, evaluation of EB treated willow will be a major objective of this proposed project. In addition to producing ethanol from willow biomass, the utility of EB pretreatment in generating both C5 and C6 sugars which can be used by certain microalgae, such as Chlorella protothecoides, to produce bio-oils under heterotrophic growth conditions will also be evaluated in this project. Currently the DOE has a major new initiative in "Targeted Algal Biofuels and Bioproducts" (TABB) aimed at reducing the cost of algal biofuels below $5 per gallon of gasoline equivalent. Using lower cost woody biomass as a feedstock, rather than agricultural commodities, coupled with lower energy input pretreatments (EB), it is envisioned that the work proposed herein will provide preliminary results that will enhance the quality of future funding proposal submissions to TABB and the joint USDA / DOE Biomass R&D Initiative (BRDI).
Animal Health Component
80%
Research Effort Categories
Basic
20%
Applied
80%
Developmental
(N/A)
Goals / Objectives
The overall objective of this project is to increase the potential value of plantation grown woody biomass (willow) as a feedstock for the wood-based biorefinery by establishing that EB pretreatment not only increases the kinetics and ultimate yield of fermentable sugars from biomass, as shown in Figure 1, but also minimizes inhibitory effects in the production of ethanol or algal bio-oil from heterotrophic algae, a major new emphasis of the DOE for 2014 and beyond. Specific objectives are highlighted below for this project which is envisioned to be completed in 2 years: 1. A recent study by Al-Sheikhly (Ref 4) has shown that the effectiveness of EB irradiation on biomass depends on a series of reactions involving free radicals that cause bond scission and also suggests that free radical lifetimes in biomass are related to EB dose and other factors such as moisture content, oxygen in air and the addition of "adjuncts" such as certain halides. In this study, we will systematically evaluate each of these parameters for bark-containing willow chips (0-50% moisture content) obtained from ESF properties and "control" samples of hardwood pulp containing mostly cellulose. EB treatment will be conducted in the Pilot Plant of the IBA Group in Edgewood, NY, a global, industrial supplier of EB equipment with whom we maintain an ongoing collaboration. The composition of these feedstocks before and after pretreatment will be determined using techniques available in Jahn Lab (NMR, MS, NIR).2. Willow chips treated in Objective 1 will be "saccharified" using commercial cellulases and xylanases available from Novozymes (Cellic CTec-3 and HTec-3, respectively).The rate and yield of sugar production will be monitored and compared to unreacted control samples as well as our work with maple and switchgrass.3. Sugar solutions from Objective 3 will be fermented to ethanol using commercial yeast (Fermentis Ethanol Red) and to a bio-oil using the heterotrophic microalgae Chlorella protothecoides under non-photosynthetic conditions that favor oil production. The production rate and ultimate yield of ethanol and bio-oil will be determined to assess the impact of EB conditions and to determine if fermentation "inhibitors" are created by comparison to controls including sugar solutions prepared from pure compounds not containing any "wood byproducts".
Project Methods
Willow Feedstock: Samples of chipped willow biomass (Salix sp.) from ongoing feedstock productivity trials will be obtained from Dr. Tom Volk (SUNY-ESF) in kg quantities. These chips contain both heartwood plus bark estimated to be 12-15% by mass (Volk, personal communication - publication in progress). Compositional analysis of willow biomass will involve techniques already in use in our lab including High-Resolution TGA and Near IR (NIR), which provide the cellulose, hemicellulose and lignin content of wood. Proton NMR can also be used to quantify the specific sugars resulting from enzyme hydrolysis of willow (glucose, xylose, other C6 and C5 sugars, etc. Ref. 12) EB Pretreatment: As discussed above, the work of Al-Sheikhly (Ref. 3) on the mechanisms of radiation-induced degradation of cellulosic substances suggests that free radical lifetime is an important element in determining the effect of EB pretreatment with product yield being a function of radical propagation reaction rates in comparison to termination of the reaction chain. In prior work by the co-PI's, the total effective dose of EB irradiation, 500-1000 kGy, has ben administered over a time span of seconds to minutes. In this proposed study, all samples will be irradiated at IBA Industrial, located in Edgewood NY, using a 3 MeV 90 kilowatt Dynamitron® electron beam accelerator. Wood chip samples will be placed in polyethylene bags, purged with inert gas (nitrogen or argon) to remove oxygen and then sealed. Samples will be irradiated at dose rates of 50, 5, 0.5 and 0.05 kGy/s. In principle, fewer radicals should produce fewer terminations and allow for morechain cleavage which is beneficial for enzyme activity. The dose rate will be fine-tuned to determine the optimum rate based on the ultimate yield of sugars from willow biomass by cellulase enzymes. Some attention will also be focused on the use of halides during irradiation to increase the production of free radicals per unit of EB dose, thereby reducing the total level of EB energy needed (Ref. 3) and, potentially, improving process economics. Saccharification: Conversion of woody biomass to fermentable sugars will be accomplished using commercially available Novozymes Cellic CTec2 and HTec2 preparations which exhibit cellulase and hemicellulase activity, respectively, yielding both C6 (glucose) and C5 (xylose) sugars. These enzymes can be employed simultaneously and are reported to act "synergistically" and they share optimal temperature and pH ranges of activity (50°C, pH = 5; Novozymes Technical Literature for Cellic enzymes). Sugar yield as a function of treatment time will be monitored by NIR methods already in place in the Stipanovic lab for both control and EB pretreated samples. Ethanol Fermentation: As was done previously with sugar maple feedstock, the conversion rate and yield of ethanol production from sugars derived from ground willow will be evaluated for both "control" and EB pretreated samples to identify any inhibiters, possibly from willow bark, or promoters as seen with sugar maple. Ethanol production over time will be monitored by NIR spectroscopy using a chemometric model already developed in the PI's lab Algal Bio-oil Production: A culture of Chlorella protothecoides will be obtained from the University of Texas algal culture collection (UTEX 256) and it will be grown in the basal culture medium described by Chen and Walker (Ref. 13), initially with glucose (30 g /L) and yeast extract (4 g/L). Cultures will be initially incubated in shake flasks in the dark at 28°C at pH 6.8. Additional studies will be conducted with blends of glucose and xylose ranging from 15 to 60 g/L of total sugar to establish an optimum level of xylose and total sugars for both biomass production and bio-oil yield. Sugar utilization over time will be monitored by NIR spectroscopy using a chemometric model and algal cell growth will be monitored by measuring OD at 540 nm. After initial work with "pure" sugars, C5 and C6 sugar containing willow hydrolyzates will be evaluated at the 5L scale (approx. 5:2 glucose:xylose). Depending on the results of the glucose / xylose optimization work above, additional xylose from HWE could be added. Cells will be harvested using centrifugation and the residual biomass will be rinsed with distilled water and freeze dried which will enable a yield (g/L) to be calculated based on dry weight. It is anticipated that existing High Resolution Thermogravimetric Analysis (HR-TGA) methods can be applied to this dried algal biomass to determine its % protein, % carbohydrate, % moisture and % ash values. The % bio-oil (lipids) will be determined by a method previously described (Ref. 13) whereby the oil is isolated from dried algal cells using sequential hexane extraction and solvent evaporation. Potential exists at SUNY-ESF to scale up to 20L bio-reactors and, ultimately, the 1000L fermenter in the SUNY-ESF Biofuels Pilot Plant at the Syracuse CoE. Bio-Oil Characterization: To characterize the lipid distribution found in the produced algal bio-oil, a GC/MS method based on the work of Xu et. al (Ref. 16) will be exploited after converting the fatty acids into methyl esters (FAME's). These methods are available in the Analytical and Technical Services (A&TS) group at ESF on a cost per sample basis (funding included in budget). The heating value of the oil itself (J / kg) can be determined using Differential Scanning Calorimetry (DSC) in air using equipment available in the Chemistry Department.