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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

2017 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. Objective 3. Develop technologies that serve as co-products for lignocelluloses based refineries and as antibiotic alternatives for use in agriculture and animal production.

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. Goal 3. Develop protective soil bacteria and yeast engineered with bacterial AMP genes for cultivation on biorefinery substrates to supply new antimicrobials for animals and agriculture.

Progress Report
Screening and evaluation tools for lipids production from biomass (Objective 1): A method previously used to monitor lipid production by algal cultures was successfully modified for use with oleaginous yeast. In this method, lipids are transformed into methanol esters in situ and the esters extracted and quantified by gas chromatography (GC). The method is superior to prior art because it allows rapid analysis of individual fatty acids at high sensitivity, which is needed to process numerous small samples from micro- and flask-scale culture screens. Comprehensive data validating its use in quantitation of total lipids and fatty acids in yeasts have been collected. This method will be published along with other tools developed in our laboratory for screening strains and conditions for optimal lipid production using renewable sugar streams from a biorefinery. For example, using these techniques, strains and process conditions were identified which led to the highest lipid accumulation (30 g/L) ever reported from an undetoxified hydrolyzate of lignocellulosic biomass of any kind (switchgrass, in this study). One patent application, six peer-reviewed research papers, numerous presentations at scientific meetings, and several invitations to collaborate on grants with university partners have come from this work. We have recently applied these screening and evaluation techniques to survey a local corn ethanol production facility at various points in the process train. As a result, several new naturally occurring oleaginous yeast were identified and are being further characterized. Biorefinery coproducts as alternative antimicrobials for animal and plant health (Objective 3 added February 2017): The oleaginous yeast Yarrowia is an attractive candidate as a fodder supplement, not just for its beneficial oils but also for its potential to stimulate an animal’s immune system. In a prior survey, members of the Yarrowia clade from the ARS Culture Collection were surveyed on hydrolyzate media to find superior lipid producers. Three of the yeast collection also grew well on crude glycerin, thin stillage, and their combination. Research to express a value-added product in Yarrowia is in progress to develop a new product which could be mass produced as a biorefinery coproduct. In collaboration with the University of Idaho, progress was also made on developing a biological control agent (BCA) for the suppression of dry rot and other maladies of potatoes to replace thiabendazole, an antifungal chemical that is no longer effective due to widespread genetic resistance. The production of BCAs was evaluated on various sugar streams available from a dilute acid pretreated lignocellulosic biomass refinery: xylose-rich, glucose-rich and ~40:60 xylose glucose mixed sugar streams. Parent BCAs (patented technology available) and evolved strains developed for enhanced performance (invention disclosure filed) were evaluated. The strains could be produced from hydrolyzate streams, including xylose-rich sugar conditions. Cell signaling and amino acid availability boost yeast robustness to hydrolyzates (Objective 2): Cell wall integrity signaling pathways in Saccharomyces cerevisiae to detect and respond to cell stress are known, but poorly understood. Comparative quantitative gene expression analysis was applied to dissect gene expression dynamics for an ARS-patented tolerant yeast. A particular sensor gene that has the capability for transmitting signals in response to 2-furaldehyde (furfural) and 5-(hydroxymethyl)-2-furaldehyde (HMF) to maintain a cell wall integrity pathway was discovered. Findings of this research further suggest that S. cerevisiae and perhaps other yeasts could be candidates for further development of next-generation biocatalysts for biofuels. In previous studies, a significant benefit of amino acids to yeast productivity and robustness was observed, notably under conditions of increasing hydrolyzate concentration or with substrate switch from glucose to xylose. In order to deliver amino nitrogen more economically to fortify yeast, various protein sources were screened to identify which had the amino acid profiles needed to support the fermentation of hydrolyzates low in free amino nitrogen. Low cost soyflours (at < $0.25/lb) containing high protein contents (45-60%) have been observed to support yeast performance in harsh hydrolyzates, but efficient amino acid release is required. Amino acid release kinetics and yield, under dilute acid or base hydrolysis conditions, were examined based on standard methods, including an enzymatic assay of ammonia and primary amino nitrogen contents (PAN) and a high performance liquid chromatography (HPLC) assay of individual essential amino acids. The results showed that both dilute acid and base catalyzed hydrolysis of protein could provide similar amino acid yields in reaction times spanning a few minutes to less than an hour. Over six-fold variation in amino acid yield was seen across the various hydrolysis conditions applied, suggesting the sensitivity of this reaction and economics for optimization.

1. New Yarrowia strains with improved robustness to biomass hydrolyzates. Yarrowia yeast strains are widely used for production of diverse lipids for biodiesel; food and healthcare applications; organic acids; and more recently, as proteinaceous feed supplements for the animal and aquaculture industries. ARS researchers in Peoria, Illinois, screened 45 isolates of the Yarrowia clade deposited in the ARS Culture Collection. Strains were evaluated and compared for growth robustness, breadth of sugar metabolism, and lipid production in harsh dilute-acid pretreated switchgrass hydrolyzate media. The top lipid producer was discovered to accumulate over 3-fold higher lipid concentration than a control native Yarrowia strain commonly used for genetic engineering. This discovery of a more robust platform yeast strain is expected to advance research on the conversion of biomass into lipid biofuels and other bioproducts by broadening the genetic diversity available to biotechnology companies venturing into synthetic biology applications.

2. Pathway of xylose utilization of an ARS patented yeast strain. Traditional industrial Saccharomyces yeast strains do not ferment pentose sugars, which comprise about one-third of available sugars from biomass. Efficient xylose conversion is critical to producing ethanol economically from biomass, and although S. cerevisiae strains have been engineered to use xylose, they have been deficient in robustness when cultivated on this sugar. ARS researchers in Peoria, Illinois, discovered and characterized the importance of a gene expression sequence key to allowing an engineered industrial yeast to better utilize xylose for increased ethanol conversion and biosynthesis. The scientists found that expressions of a set of genes led a signature expression of 10 additional genes that were critical for the acquired xylose to ethanol conversion. This first insight into the coordinated signature pathway expression of an industrial yeast engineered to successfully use xylose is a notable advance in the development of robust next-generation yeasts for low-cost biofuels from lignocellulosic materials.

3. A new source of yeast resistance to the metabolic inhibitor furfural. Furfural (2-furaldehyde) is a representative inhibitory compound commonly observed in lignocellulose hydrolysate which interferes with microbial growth and subsequent fermentation. ARS scientists in Peoria, Illinois, previously characterized yeast in situ detoxification by a specific enzyme and discovered the responsible genes. Recently, the scientists found five related but uncharacterized genes in Scheffersomyces (Pichia) stipitis, a naturally occurring pentose sugar utilizing yeast. This finding provides new genetic resources for the research and development community to design robust tolerant strains for lower cost lignocellulose-to-biofuels production.

4. Biological alternative to thiabendazole antifungal chemical for potato disease control. Fusarium dry rot, incited by Fusarium sambucinum, causes greater losses in storage and transit of both seed and commercial potatoes than any other postharvest disease. Chemical control of the causative fungus is no longer possible since 80% of pathogenic strains are resistant to thiabendazole, the only chemical registered for use on food-grade potatoes. As an alternative, new microbial strains antagonistic to F. sambucinum were discovered. Optimal strains capable of superior efficacy in protecting potatoes were developed by ARS scientists, Peoria, Illinois, and subsequently applied to develop dry storage tolerant strains capable of dry rot disease control. Production on low cost renewable substrates and dry storage stable formulations of the biocontrol product are expected to lower costs and expedite application by growers. This new technology is expected to benefit agriculture by providing an antifungal microbial alternative to azole chemicals on the market and by serving as a potential co-product of a renewable lignocellulose biorefinery with the effect of boosting economic feasibility.

Review Publications
Slininger, P.J., Shea-Andersh, M.A., Thompson, S.R., Dien, B.S., Kurtzman, C.P., Sousa, L.D., Balan, V. 2016. Techniques for the evolution of robust pentose-fermenting yeast for bioconversion of lignocellulose to ethanol. Journal of Visualized Experiments. 116:1-15. doi: 10.3791/54227.
Serapiglia, M., Dien, B.S., Boateng, A.A., Casler, M.D. 2017. Impact of harvest time and switchgrass cultivar on sugar release through enzymatic hydrolysis. BioEnergy Research. 10:377-387.
Serapiglia, M., Mullen, C.A., Boateng, A.A., Dien, B.S., Casler, M.D. 2017. Impact of harvest time and cultivar on conversion of switchgrass to bio-oils via fast pyrolysis. BioEnergy Research. 10:388-399.
Qureshi, N., Liu, S., Hughes, S., Palmquist, D., Dien, B., Saha, B. 2016. Cellulosic butanol (ABE) biofuel production from sweet sorghum bagasse (SSB): Impact of hot water pretreatment and solid loadings on fermentation employing Clostridium beijerinckii P260. BioEnergy Research. 9(4):1167-1179. doi: 10.1007/s12155-016-9761-z.
Quarterman, J., Slininger, P.J., Kurtzman, C.P., Thompson, S.R., Dien, B.S. 2017. A survey of yeast from the Yarrowia clade for lipid production in dilute-acid pretreated lignocellulosic biomass hydrolysate. Applied Microbiology and Biotechnology. 101(8):3319-3334. doi: 10.1007/s00253-016-8062-y.
Khalil, S., Ali, T.A., Skory, C., Slininger, P.J., Schisler, D.A. 2016. Evaluation of economically feasible, natural plant extract-based microbiological media for producing biomass of the dry rot biocontrol strain Pseudomonas fluorescens P22Y05 in liquid culture. World Journal of Microbiology and Biotechnology. 32(2):25. doi: 10.1007/s11274-015-1984-1.
Schisler, D.A., Slininger, P.J., Olsen, N.L. 2016. Appraisal of selected osmoprotectants and carriers for formulating Gram-negative biocontrol agents active against Fusarium dry rot on potatoes in storage. Biological Control. 98:1-10. doi: 10.1016/j.biocontrol.2016.03.009.