Using Green Solvents To Extract Oil From Wet Yeast Cells

As a young boy, I have always had an innate curiosity to understand the world around me. From my time in academia I was able to not only gain a deeper understanding of the world around me but apply what I had learned to make the world a more prosperous place.

I have carried this value-added mindset along with me in my career from my food science undergraduate program learning how to process agricultural inputs into safe and desirable food products to my Master’s project focusing on developing a granulation process to produce a bio-based fertilizer carrier from corn ethanol manufacturing waste to studying the production of single cell oil (SCO) from oleaginous yeast using non-edible feedstocks to utilization of grains to make value-added solutions for the food and pest control industries.

My doctoral research was spent at the Bioprocessing and Industrial Value Added Program (BIVAP) within the Department of Grain Science and Industry at Kansas State University. As an NSF IGERT Fellow, my work focused on an integrated oleaginous yeast platform for the production of SCO as input for biofuels and biobased products. This project entailed looking at various portions of the platform including feedstock utilization, fermentation, lipid analysis and greener solvents for extraction.

SCOs are lipids produced from microbial sources that can serve as input for renewables including biofuels and other biobased products. More specifically SCOs are defined as storage lipids (such as triacylglycerols) produced by oleaginous or oil-rich microorganisms including yeast, mold, and microalgae (Probst et al. 2015). Of these, oleaginous yeast holds much promise because they can produce high oil yields (up to 70%, w/w) and are able to utilize low-value, non-edible feedstocks.

Oil accumulation is a natural phenomenon that oleaginous yeast use to help them survive during times of stress (Ratledge, 2004). Under stress, oil is stored as energy and upon return of favorable conditions the oil can be metabolized for growth and normal cell function. To capitalize on this phenomenon, bioprocess engineering approaches often limit the amount of nitrogen in the culture medium to stress yeast into oil accumulation. This is often performed by using fed-batch fermentation to control the amount of carbon and nitrogen over the course of fermentation.

At the start of fermentation a sufficient amount of nitrogen is provided allowing the cells to replicate and grow then additional carbon (often in the form of sugar) is fed in at later stages which are converted into storage lipid; the more sugar that is provided, the more engorged and fat the oleaginous yeast become. To take advantage of this gluttonous behavior, repeated fed-batch has been used to provide additional sugar to further increase oil production beyond what can be produced using a traditional batch approach (Probst and Vadlani, 2017). Repeated fed-batch is preferred over batch because it can prevent substrate inhibition which can lead to ‘stuck fermentations’ under high sugar loading.

Commercialization of oleaginous yeast SCO remains to be a challenge due to competition from lower cost oilseed crops and high operating costs. Feedstock costs can contribute to upwards of 40% of the total costs (Koutinas, 2014); thus, utilization of low-value substrates will be needed. As mentioned previously, a considerable amount of literature exists demonstrating that low-cost feedstocks can be used for SCO production including the work by Probst and Vadlani (2015) who showed that non-edible hemicellulose-rich bran was utilized by the oleaginous yeast Lipomyces starkeyi to produce SCO.

Downstream processing is also another cost-prohibitive step since oils are produced intracellularly (within the cells) requiring large amounts of energy to lyse or break the cells apart before subsequent oil extraction. Developing novel lysis strategies along with exploring alternative extraction technologies will be important moving SCO technologies forward. Previous work by Probst et al (2107) was able to successfully demonstrate oil extraction from the oleaginous yeast L. starkeyi using an alternative, green solvent called cyclopentyl methyl ether (CPME). Evaluating new, multi-functional solvents such as CPME that can also be used for reaction chemistry may help identify simultaneous extraction and catalytic upgrading processes for oleochemical manufacturing.

While challenges exist, SCO offers significant advantages. In regards to the “Food vs Fuel” debate, SCO can serve as a renewable alternative to oilseed crops for the biofuel market to help relieve pressure on food supply. SCO can also be used to minimize risk in highly volatile markets (e.g. cocoa butter) since shorter production times are used compared to oilseed crops. Other opportunities include utilizing emerging technologies within genetic engineering to produce new products in unexplored markets.

One example includes engineering oleaginous yeast to produce lipids tailored to a specific application; this holds much potential for making designer oils for new, high dollar markets. New developments within biotechnology including rapid screening and selection of new yeast species/strains, genetic transformation (Calvey, 2014), pathway engineering, and lipidomics offer exciting possibilities for improving the economic viability of SCO production.

The research scientist role that I am currently transitioning out of included developing new products within the food ingredient sector mainly focusing on enzymatic bioconversion of starch and other bio-based polysaccharides. This included laboratory exploration and development, pilot scale-up and commercialization. My current food formulation scientist role includes the design of experiment-driven targeted bait technology for the rodenticide industry.

References

• Calvey, C.H., Willis, L.B., Jeffries, T.W., 2014. An optimized transformation protocol for Lipomyces starkeyi. Current Genetics, 60, 223-230.
• Koutinas, A., Chatzifragkou, A., Kopsahelis, N., Papanikolaou, S., Kookos, I., 2014. Design and techno-economic evaluation of microbial oil production as a renewable resource for biodiesel and oleochemical production. Fuel, 116, 566–577.
• Probst, K.V., Wales, M.D., Rezac, M.E., Vadlani, P.V. 2017. Evaluation of green solvents: Oil extraction from oleaginous yeast Lipomyces starkeyi using cyclopentyl methyl ether (CPME). Biotechnology Progress, 33, 1096-1103.
• Probst, K.V. and P.V. Vadlani. 2107. Single cell oil production by Lipomyces starkeyi: Biphasic fed-batch fermentation strategy providing glucose for growth and xylose for oil production. Biochemical Engineering Journal, 121, 49-58.
• Probst, K.V., L.R. Schulte, T.P. Durrett, M. E. Rezac, P.V. Vadlani. 2016. Oleaginous yeast: a value-added platform for renewable oils. Critical Reviews in Biotechnology, 36, 942-955.
• Probst, K.V. & Vadlani, P.V. 2015. Production of single cell oil from Lipomyces starkeyi ATCC 56304 using biorefinery by-products. Bioresource Technology, 198, 268-275.
• Ratledge C. 2004. Fatty acid biosynthesis in microorganisms being used for single cell oil production. Biochimie, 86, 807–815.

This study, Evaluation of green solvents: Oil extraction from oleaginous yeast Lipomyces starkeyi using cyclopentyl methyl ether (CPME) was recently published in the journal Biotechnology Progress.