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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

2019 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
Objective 1: A review of the literature suggested a novel method to directly select for oleaginous yeast in natural environments. To validate this method, soil was sampled at prairie and forestry sites and directly planted on the putative selection medium. From the 100+ colonies that arose, 21 were randomly selected for growth and lipid analysis. Of these 16 produced 1.7 – 4.4 g/l lipids and only 2 were negative. This result confirmed the utility of the method to find lipid producing yeast. Five of these oil producing yeasts were further evaluated on dilute-acid switchgrass at 75% and 90% concentrations. All grew on 75% and 90% concentrated hydrolysates and strains grown on the latter concentration achieved lipid titers of 5.62 – 7.53 g/l. However, these strains were not as good as our elite yeast strains (9-10 g/L lipid) found earlier in the ARS Culture Collection from Peoria, Illinois. Low-moisture ammonia hydroxide (LMA) pretreated switchgrass was used to support oil producing yeast growth. Although over 50% more cell mass was accumulated, slightly less lipid was produced. It was also found that by doubling the inoculum, the lag phase was cut by 10 hr. It is expected that the lower lipid yield is associated with added ammonium which favored yeast biomass over lipid production. A novel ammonia based thermal mechanical process was developed that reduces the ammonia loading over three-fold, and it is hoped to allow operation at lower nitrogen levels to boost higher lipid titers. Evolution procedures for our four elite parent oil producing yeast strains were designed to enrich for variants with shortened lag phase, more rapid growth, and higher lipid accumulation in high solids dilute acid switchgrass hydrolyzates. While early isolates are showing significant levels of improvement, additional isolations from later stages of the evolution process are continuing this summer along with comparative evaluations of isolates to identify most improved variants compared with parent strains. Objective 2: Continued to identify genes and discover renovated pathways underlying the tolerance of the patented industrial yeast Saccharomyces cerevisiae NRRL Y-50049 through transcriptome and proteomic studies. New insight into the characteristics of the adapted industrial yeast opens a window for future more in-depth investigations in understanding mechanisms of the yeast tolerance. Results of this research facilitate development the genetic engineering of the next-generation biocatalysts for low-cost and sustainable production of fuels and chemicals from lignocellulosic materials. Objective 3: Progress continued on developing the Yarrowia yeast for production of antimicrobial peptides (AMPs) for use in poultry finishing diets as a substitute for antimicrobials. In earlier work, three antimicrobial peptides had been targeted for expression from literature, synthetic genes generated, and placed in integration E. coli/Yarrowia shuttle vectors. The chosen host strain was successfully transformed. However, recovery of the AMP failed. A method was planned and an APHIS permit was obtained to allow bioassay and confirmation of AMP activity. Alternative antifungal microbial products are being developed to replace thiabendazole, which is now ineffective against postharvest diseases of potatoes due to resistance of the causative pathogens. Biological control agent (BCA) strains were developed that had increased tolerance of drying and dry storage. This phenotype is desired for more economical and convenient storage, allowing application of BCAs on a large scale. Studies advanced to identify a novel new osmoprotectant to further enhance rehydration and rapid reactivation of cells, thereby boosting efficacy once delivered to post harvest potatoes entering storage.

1. New osmoprotectant to stabilize biocontrol agents during drying and storage. Over 80% of fungal strains that cause potato dry rot are now resistant to thiabendazol (TBZ) and there is growing pressure to develop non-azole alternatives. However, chemical substitutes are limited, especially in postharvest potatoes destined for food use. ARS scientists in Peoria, Illinois, developed bacterial strains that are antifungal biocontrol agents (BCAs) which are active against dry rot, late blight, and pink rot diseases and inhibitory to sprouting. Novel evolved bacteria strains can be grown using switchgrass hydrolysate and show excellent survival during dry storage conditions. Further improved dry storage formulations and production on low cost renewable substrates are expected to lower costs and expedite use of the BCAs by growers. The ARS scientists discovered a new osmoprotective sugar that was found to be more protective of BCA viability during drying and storage than other sugars of similar structure currently used in industry. In earlier research, scientists developed a bioprocess to potentially make this sugar at relatively low cost, which would allow it to be economical for agricultural use. This new technology benefits agriculture by providing an antifungal microbial alternative to azole chemicals and by serving as a potential co-product of a renewable lignocellulose biorefinery with the effect of boosting economic feasibility.

2. New bioreactor technology improved cellulosic ethanol production using ARS patented yeast strain. Current cellulosic ethanol conversion from lignocellulosic materials requires significant extra expenses of hydrolytic enzymes to release fermentable sugars for microbial utilization. Reducing the expense on the digestive enzyme is vital for a sustainable and efficient cellulosic ethanol production from lignocellulosic biomass. An ARS scientist in Peoria, Illinois, developed a new yeast strain Clavispora NRRL Y-50464 that is able to produce beta-glucosidase and produce cellulosic ethanol from corn stover. However, the conventional bioreactor designed for traditional liquid fermentation is not suitable for cellulosic ethanol production using simultaneous saccharification and fermentation (SSF) process. In this research, the ARS scientist, in collaboration with a university, significantly increased the efficiency of cellulosic ethanol production from corn stover through a process engineering approach. A bioreactor was designed with a helical stirring apparatus that provided sufficient mixing power and mass transfer capability during enzymatic hydrolysis for higher levels of cellulosic ethanol production. It significantly reduced the cost of enzyme needed for SSF and the ethanol yield was near the minimum industrial production standard. The outcomes of this study impact renewable bioenergy community in both academic and industrial sectors and provides a reference and guidelines for continued improvement of low-cost cellulosic ethanol production from lignocellulosic materials.

3. Protein expression analysis revealed a fine-tuned mechanism of in situ detoxification pathway for ARS patent tolerant industrial yeast. Overcoming the toxic compounds associated with lignocellulose-to-biofuels conversion poses significant challenges to new strain development for a sustainable bio-based economy. The stress-tolerant industrial yeast Saccharomyces cerevisiae obtained by adaptation can detoxify a major class of inhibitory chemicals derived from lignocellulose-biomass pretreatment. Studies on the yeast tolerance have been carried out extensively; however, limited proteomic evidence is available on the yeast adaptation and mechanisms of the tolerance. Using comparative time-course studies of protein expression profiling in response to synergistic inhibitor challenges to the tolerant yeast strain, ARS scientists revealed a key protein that is required for the in situ detoxification. Outcomes of this research facilitate development of next-generation biocatalysts for sustainable production of fuels and chemicals from lignocellulosic materials. The new proteomic insight into yeast adaptation obtained from this study impacts the research and development communities in both basic and applied scientific investigations.

Review Publications
Geberekidan, M., Zhang, J., Liu, Z.L., Bao, J. 2018. Improved cellulosic ethanol production from corn stover with a low cellulase input using a ß-glucosidase producing yeast. Bioprocess and Biosystems Engineering. 42(2): 297-304.
Nichols, N.N., Hector, R.E., Frazer, S.E. 2019. Genetic transformation of Coniochaeta sp. 2T2.1, key fungal member of a lignocellulose-degrading microbial consortium. Biology Methods and Protocols. 4:1-5.
Dien, B.S., Mitchell, R.B., Bowman, M.J., Jin, V.L., Quarterman, J.C., Schmer, M.R., Singh, V., Slininger, P.J. 2018. Bioconversion of pelletized big bluestem, switchgrass, and low-diversity grass mixtures into sugars and bioethanol. Frontiers in Energy Research. 6:129.
Kim, S.M, Lee, D., Thapa, S., Dien, B.S., Tumbleson, M.E., Rausch, K.D., Singh, V. 2018. Cellulosic ethanol potential of feedstocks grown on marginal land. American Society of Agricultural and Biological Engineers. 61(6):1775-1782.
Quarterman, J.C., Slininger, P.J., Hector, R.E., Dien, B.S. 2018. Engineering Candida phangngensis – an oleaginous yeast from the Yarrowia clade – for enhanced detoxification of lignocellulose-derived inhibitors and lipid overproduction. Federation Of European Microbiological Societies Yeast Research. 18(8):foy102.
Vasconcellos, V.M., Farinas, C.S., Ximenes, E., Slininger, P., Ladisch, M. 2019. Adaptive laboratory evolution of nanocellulose-producing bacterium. Biotechnology and Bioengineering. 116(8):1923-1933.
Slininger, P.J., Dien, B.S., Quarterman, J.C., Thompson, S.R., Kurtzman, C.P. 2019. Screening for oily yeasts able to convert hydrolyzates from biomass to biofuels while maintaining industrial process relevance. Methods in Molecular Biology. 1995:249-283.