Biochemical Production of Endophytic Yeast
Ivy Salim
School of Environmental and Forest Sciences, University of Washington
Biofuel: what and why
In the U.S., the debate about biofuel - an alternative transportation fuel coming from currently living organisms - is still contended because the farms are growing corns (first generation)
for fuel production instead of for human and animal consumptions. The future of biofuel relies on lignocellulose from fast growing plants native to the sites and able to tolerate harsher
conditions. Lignocellulose is the main component of plant cell wall – a carbohydrate with
different sorts of sugar types. Cellulose (30-50%) is made up by glucose, while hemicellulose
(20-40%) commonly comprises of glucose (C6), xylose (C5), arabinose (C5), and mannose
(C6).
Methods
The yeast strains would be grown on rich media for two days to reach a high cell density
before starting fermentation process. During fermentation itself, the cells are switched
into a media that mimics the plant hydrolysate. Depending upon the given sugar, it takes
1-5 days to obtain the chemical of interests: bioethanol, xylitol and triglyceride. Fermentation typically has much more sugar concentration than the growing media as the high
C:N ratio triggers switch of metabolic activities in the yeast. While bioethanol and xylitol
use C:N of 20:1 in the absence of oxygen or very limited oxygen level, triglyceride accumulation is more effective at 40:1 with limited oxygen level.
Quantitative measurement was done for the first two products while for the accumulation of lipid a qualitative method was used instead. The first utilized a high performance liquid chromatography (HPLC) instrument where sugar-alcohol concentrations
were read off collected samples. The latter used the aid of fluorescent microscopy after
staining the cells with a green fluorescence dye that emits the wavelength at 505 nm,
while absorbing at 515 nm.
Results
© Marie-Jean Meurs, 2013.
Fossil Fuel (gasoline)
Biofuel (lignocellulosic bioethanol)
From decomposed organic materials
From currently living organic materials
Energy production: 120 MJ/gallon
Energy production: 80 MJ/gal (100%), typical
blend is 10-15% ethanol
Higher greenhouse gases emission: extraction/
drilling, transportation, carbon combustion level
(35% less O2 than ethanol)
Lower greenhouse gases emission: 12-19% less
for E10, plants grown in limited or no fertilizer,
local production of crops, CO2 reincorporation
Degrade lands due to oil sand mining, drilling, excavation of soils
Uses degraded lands
Mixed with hazardous aromatics during processing to ensure more efficient combustion
Uses up more water during processing
Price: dropping since Sept’13 from $2.8 to $2.5
(Nov’13), up again to $2.7 (Dec’13)
Price: $1.6-1.7 (Sept-Oct’13), rising in Nov’13
up to $2.2 (Dec’13)
By-products: chemicals (antibiotics, glycerine),
consumer goods (plastic, paints), agricultural products (fertilizer)
By-products: chemicals (sugar alcohols), consumer goods (paper, compostable plastic based
goods), agricultural products (livestock feeds)
The two graphs above show the various concentration of sugar-alcohol during fermentation processes. They are averaged based on the three trials done, each conducted in triplicates.
The following pictures are from different yeast strains out of 15 screened. Yeast strains
were subjected to three different treatments to test hypothesis: rich media with agitation
at 30C, rich media without agitation at 4C, nitrogen limited media with agitation at 30C.
The upper parts of each photographs set were taken under a reflective light microscope,
showing what the real yeast morphology while the bottom parts were taken under the
fluorescence where the green parts show lipid accumulation. Shown below are of WP1
and AD1 respectively.
•Bioethanol: the fermentation product of plant sugar mainly glucose and xylose, which make up about 70% of the plant sugar composition.
•Biodiesel: a transesterificiation process of triglyceride (vegetable oil/animal fat/algal fat/ yeast oil) with low C alcohol.
Fermentation by endophytic yeast
I focused on the endophytic yeast strains which are able to ferment sugar into various biochemicals. Endophytes are microbes that live inside the plant tissues without causing diseases, but rather help the plants thrive in various environments. The Doty Lab favorite is poplar
on rocks, which happen to be the biofuel crop chosen for the Pacific Northwest. The strains
that I used are: Candida lignohabitans strain AR1, Rhodotorula mucilaginosa strain PTD3
and Rhodotorula graminis strain WP1. The first was isolated from a wild red alder, the second was from a hybrid poplar grown in Boardman, OR as biofuel crop feedstock, while the
last was from a wild poplar by Skykomish river, WA.
Since they are so used to digesting different sugar types,
they are able to utilize the various sugar types found
in plants: glucose, xylose and arabinose. Baker’s yeast
on the other hand is not able to ferment the latter two
due to its lack of appropriate enzymes, namely: Xylose
reductase (XR) and Xylitol Dehydrogenase (XDH). XR
will digest xylose into xylitol, while XDH will digest
xylitol into ethanol. PTD3 was previously screened for
such genes and found to have them although only the
© Applied and Environmental Microbiology, 2011.
former seems to be expressed.
Acknowledgement
This research is made possible by the guidance and support from Prof. Sharon Doty and Dr. Zareen
Khan, continuous support from Agnes H. Anderson, Hugo Winkenwerder and Pack Forest scholarships,
as well as the company and help of Amy Baum, Lisa Hannon, Nick Herschberger, Shyam Kandel, Robert
Tourney, Emilie Viglino, Neil Fleck, Andrew Sher, and Raymond Yap.