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ARS Home » Midwest Area » Peoria, Illinois » National Center for Agricultural Utilization Research » Bioenergy Research » Research » Research Project #427438

Research Project: Biochemical Technologies to Enable the Commercial Production of Biofuels from Lignocellulosic Biomass

Location: Bioenergy Research

2015 Annual Report

Objective 1. Develop technologies that enable the commercial production of marketable lipid-based advanced biofuels from lignocellulosic biomass hydrolyzates. Objective 2. In collaboration with industrial biorefiners, develop technologies that enable widespread commercial production of cellulosic ethanol from lignocellulosic biomass.

Goal 1. Develop oleaginous yeast and associated processes for converting hydrolyzates of lignocellulosic biomass to lipids for biodiesel and valuable co-products for other uses. Goal 2. Apply novel patented stress-tolerant yeast strains under commercial conditions to convert hydrolyzates of lignocellulosic biomass to ethanol.

Progress Report
The ARS culture collection, which is among the largest depositories of yeasts in the world, was efficiently mined for oleaginous yeast functioning in hydrolyzates of lignocellulose by conducting the first systematic phylogeny guided screen. This strategy streamlined the exploration of the more than 200 potential oleaginous strains available by allowing research efforts to hone in on relatives of the best lipid-accumulating strains identified by our screening, Lipomyces and the related genus Myxozyma, were particular foci of the study because they are known hyper lipid producers but a few other key genera previously noted in the literature were also checked. The conversion of the hexose and pentose sugars typical of plant biomass was first studied in a synthetic medium at acidic pH. Some of the Lipomyces strains were found to have exceptional intracellular lipid contents compared to previously described oleaginous yeasts and were able to consume glucose and xylose sugar mixtures in an efficient manner. A more selective and higher throughput screening of lipid production by isolates passing the primary synthetic medium screen was accomplished on inhibitory hydrolyzates in aerobic 96-well deep-well plates. After evaluation in corn stover and switchgrass hydrolyzates, a few top candidates for development were selected on the basis of mixed sugar lipid yields, lipid accumulation and productivity, reduced sensitivity to diauxy, lipid fatty acid composition and carotenoid production. Hydrolyzates were prepared at 18-20% solids loading by established ammonia fiber explosion (AFEX) pretreatment and saccharification technologies developed by collaborators in Michigan, as well as by novel ammonia and dilute-acid pretreatment and saccharification technologies developed at the ARS in Peoria, Illinois. The phylogeny of best strains in hydrolyzates was further applied to identify additional close relatives able to perform in hydrolyzates. Using this approach, the screening number was cut by at least 80%. Additionally, our screen may be efficiently extended to check close relatives of high ranking strains that are held in other collections. Applying an advanced two-stage process to manage sugar and nitrogen supplies, top strains were able to use acetic acid and nearly all of the diverse sugars available to rapidly accumulate 50-65% of cell biomass as lipid, which corresponded to economically harvestable concentrations of lipid up to 30 g/L even under acidic pH conditions preferred by industry. The successful identification and process development for top-performing lipid-producing yeast demonstrates lipid productivity and yields in undetoxified hydrolyzates exceeding all other literature reports to date. Additionally, carotenoid production was observed in our process, and further studies are planned as a follow up since carotenoids, as higher value co-products, are expected to greatly enhance the economic feasibility of lipid production for conversion to biodiesel fuel. A rapid lipid measurement method was key to the efficiency of our screen. The sulfo-phospho-vanillin assay and time-domain nuclear magnetic resonance spectroscopy methods were adapted for rapid lipid measurement. A novel technique to allow for monitoring real time lipid production using Nile Red fluorescence was also tested for the purpose of in situ measurement. Research in our laboratory has suggested that appropriate nitrogen (N) delivery to ethanologenic yeast strains may increase yield and productivity in hydrolyzates by at least four fold. Improvement of processes for nitrogen (N) supply at low cost was targeted as a means to facilitate economical bioconversion of lignocellulose to biofuels. In order to deliver amino N more economically to fortify yeast fermentations, various protein sources were screened to determine which had the amino acid profiles needed to support the fermentation of post frost switchgrass hydrolyzates which are low in available amino nitrogen. Low cost agricultural materials (at < $0.25/lb) containing high protein contents (45-60%) such as soyflours, cottonseed flour, and corn steep powder were examined along with processes to support primary amino acid release. Amino acid release kinetics and yield were examined based on standard methods, including enzymatic primary amino nitrogen content (PAN) and high performance liquid chromatographic (HPLC) analysis of the essential amino acids. The yeasts used in this testing were the inhibitor-tolerant S. cerevisiae and best xylose-utilizing strain derived from it, but also the best hydrolyzate tolerant S. stipitis. N source utilization was monitored, including overall PAN, urea, and ammonia. Individual amino acid uptake studies are additionally being pursued. Technical assessment of lower-cost cellulosic ethanol production using ß-glucosidase producing yeast Clavispora NRRL Y-50464 was completed. In this study, ARS scientists also defined parameters for evaluation of strain performance. This comprehensive research provided critical assessment on fundamental technical issues involved in cellulosic ethanol production by simultaneous saccharification and fermentation using an ARS patented strain as an example.

1. Yeast process for lower cost biodiesel fuel from plant biomass. About 1.3 billion tons of plant biomass (lignocellulose) could be harvested each year in the U.S. in the form of energy crops and forest and agricultural residues. Assuming conservative cropping and yield estimates, this biomass could potentially be converted to about 30 billion gallons of biodiesel/year (62% of current U.S. diesel consumption) using microorganisms called “oily” yeasts. With the goal of identifying robust oily yeast for this purpose, Agricultural Research Service scientists in Peoria, Illinois, screened numerous strains from the ARS Culture Collection on two commercially promising hydrolyzates of plant biomass to identify a few robust novel strains able to produce high lipid concentrations. Applying an advanced two-stage process to manage sugar and nitrogen supplies, top strains were able to rapidly accumulate 50-65% of cell biomass as lipid, which corresponded to economically harvestable concentrations of lipid up to 30 g/L even under acidic pH conditions preferred by industry. This new technology is expected to advance the economic feasibility of high quality biodiesel and jet fuels from renewable biomass, reducing our dependence on foreign oil while supporting the rural economy and preserving the environment.

2. Lipomyces sp. yeasts for lower cost, high quality biodiesel fuel from biomass. Twenty-two oleaginous yeasts were screened for lipid production by Agricultural Research Service scientists in Peoria, Illinois, from the ARS Culture Collection. The set included members of the Lipomyces cleave (e.g. family) with a known lipid producing yeast included as control. The yeasts were ranked in terms of the lipid concentration accumulated, intracellular lipid content of yeast (dry weight basis), and rate of lipid production. Each of these strains produced lipids on all the sugars and furthermore exceeded the yield for the control yeast strains. The produced fatty acid profiles of the strains were also analyzed and found favorable for production of biodiesel. The new strains are expected to make the economics of producing renewable fuels more favorable, reducing our dependence on foreign oil while supporting the rural economy and preserving the environment.

3. Technical assessment of lower-cost cellulosic ethanol production using ARS patented strain. Cellulosic ethanol production from agricultural residues and lignocellulosic materials is a complex procedure and involves many factors affecting biocatalyst performance. In addition, strain performances are difficult to compare due to varied processes and cellulosic materials used in the fermentation. Aiming at potential industrial applications, Agricultural Research Service scientists in Peoria, Illinois, designed a fundamental technical assessment based on conditions desired for industrial applications in order to identify practical strains for improved efficiency and cost of cellulosic ethanol production. Applying this method of assessing utility under standard industrial conditions, one strain was confirmed to produce over 40 g/L ethanol from pure cellulose within 72 hr with less added enzymes because the yeast produces the final enzyme needed to break down cellulose to fermentable glucose. This new technology would allow enzyme cost reduction and consolidated process efficiencies providing an estimated savings of ~$0.35/gal in the selling price of ethanol. This accomplishment directly addresses mission goals of ARS research on low-cost cellulosic ethanol production and enhances rural economic development.

Review Publications
Xue, Y.-P., Jin, M., Orjuela, A., Slininger, P.J., Dien, B.S., Dale, B.E., Balan, V. 2015. Microbial lipid production from AFEXTM pretreated corn stover. The Royal Society of Chemistry Advances. 5:28725-28734.
Zhou, H., Lan, T., Dien, B.S., Hector, R.E., Zhu, J.Y. 2014. Comparisons of five Saccharomyces cerevisiae strains for ethanol production from SPORL pretreated lodgepole pine. Biotechnology Progress. 30(5):1076-1083.
Jin, M., Slininger, P.J., Dien, B.S., Waghmode, S.B., Moser, B.R., Orjuela, A., Da Costa Sousa, L., Balan, V. 2015. Microbial lipid based lignocellulosic biorefinery: feasibility and challenges. Trends in Biotechnology. 33(1):43-54.
Nichols, V.A., Miguez, F.E., Jarchow, M.E., Liebman, M.Z., Dien, B.S. 2014. Comparison of cellulosic ethanol yields from midwestern maize and reconstructed tallgrass prairie systems managed for bioenergy. BioEnergy Research. 7:1550-1560.
Moon, J., Liu, Z.L. 2015. Direct enzyme assay evidence confirms aldehyde reductase function of Ydr541cp and Ygl039wp from Saccharomyces cerevisiae. Yeast. 32:399-407.
Liu, Z., Cotta, M.A. 2015. Technical assessment of cellulosic ethanol production using ß-glucosidase producing yeast Clavispora NRRL Y-50464. BioEnergy Research. DOI: 10.1007/s12155-014-9575-9.