This study aims to develop a more efficient small-scale method of converting grass clippings to ethanol using yeast fermentation. The researchers will test how adding different concentrations of magnesium as a cofactor affects the fermentation efficiency of baker's yeast and turbo yeast. Grass clippings will be hydrolyzed and used as the substrate in growth media containing cellulose byproducts. Optical density will be measured to standardize yeast inoculations across conditions with varying magnesium concentrations. Glucose levels and alcohol production will then be analyzed and compared. The researchers hypothesize that more magnesium will increase fermentation rates and yields.

1. RESEARCH POSTER PRESENTATION DESIGN © 2012
www.PosterPresentations.com
There is a societal need for a cost-effective means of
commercial ethanol production that uses non-food
resources as inputs. Current systems are cost-
prohibitive due to inefficiencies when compared to
conventional fossil fuel production. This project
aims to utilize biotechnological methods to create a
system that will facilitate small-scale conversion of
grass clippings to ethanol. The experimentation
focuses on the utilization of the cofactor
magnesium as a means of increasing the efficiency
of glycolysis and alcohol fermentation of substrates
by Saccharomyces cerevisiae. Strains of both
common baker’s yeast and “turbo yeast” (able to
tolerate 40% ethanol) will be grown in media
containing 20% cellulose-derived by-products, and
maintained at 30°C. Optical density will be
employed to ensure consistent inoculum
concentrations and monitor the growth of both
yeast populations. The decrease in glucose
concentrations will be used to correlate alcohol
production. The influence of varied concentrations
of magnesium chloride and/or magnesium sulfate
on growth and fermentation will be monitored.
ABSTRACT
-Enzymatic/chemical hydrolysis of cellulosic
substrates
-Medium preparation
-Yeast culture cultivation
-Optical density (OD) analysis using
spectrophotometer
-Glucose concentration determination using DNS
assay
METHODS
Grass clippings will be hydrolyzed in order to provide
substrates for alcohol fermentation by Saccharomyces
Cerevisiae. Recommendations by Wyman and
associates (2005) regarding hydrolysis of feedstock
will be modified for purposes of scale. H2SO4 will be
applied to the grass, followed by lime, and finally
cellulase and cellobiase extracts.
Two strains of S. Cerevisiae (common baker’s yeast and
“turbo” yeast) will be used in this study. Turbo yeast
has been designed to withstand media with higher-
than-normal alcohol concentrations, reportedly up to
40%. “Instant yeast” samples of the two strains will be
grown in deionized water. Optical density (OD)
analyses will be conducted on dilutions of these
mixtures in order to standardize concentrations of
yeast cells for purposes of inoculation. Prepared
inoculants will then be introduced into various media.
The standard growth medium will be a modification of
that described by Treco and Lundblad (2008);
cellulose-derived by-products will be substituted for
the standard dextrose and will comprise 20% of the
medium (200g/liter).
For each of the two yeast strains, the inoculants of
homogeneous ODs will be introduced into media with
varying concentrations of added magnesium: 0mM
(control), 0.25mM, 0.5mM, and 1mM. Fermentation
batches will be incubated under anaerobic conditions.
Fig. 1: Yeast Growth in Minimal Media. Inoculants of uniform ODs
were introduced into modified minimal medium (Treco and
Lundblad, 2008) increased to 20% dextrose content. OD
readings were taken at indicated time points to measure yeast
growth in the medium under aerobic conditions.
DNS assays will be performed at hours 1, 2.5, 5, 10,
and 20 subsequent to inoculation for each flask, as
per Wang. Reductions in glucose concentrations will
indirectly correlate with alcohol production for each
fermentation batch.
EXPERIMENT PLAN DISCUSSION
It is expected that increased availability of
magnesium will lead to an increase in duration of
fermentative activity, and an overall increased rate
of fermentation (Dombek et al. 1986). These
proposed gains in efficiency may yield higher-
alcohol-content products that require less post-
fermentation processing.
In addition to the current research outlined, several
other aspects of the alcohol fermentation
component of bio-ethanol production require
investigation. These include the potential for
increased fermentative efficiency due to yeast
uptake of the other cofactors that participate in
the holoenzymes involved in glycolysis and alcohol
fermentation. Candidates include zinc, potassium,
and NAD+.
REFERENCES
Bergman, L. 2001. Growth and Maintenance of Yeast.
Methods in Molecular Biology 177: 9-14.
Dombek, K., and Ingram, L. 1986. Magnesium Limitation
and Its Role in Apparent Toxicity of Ethanol during Yeast
Fermentation. Applied and Environmental Microbiology
52: 975-981.
Treco, D., and Lundblad, V. 2008. Basic Techniques of
Yeast Genetics. Current Protocols in Molecular Biology
82: 13.0.1-13.0.4.
Wang, N. Experiment No. 4A: Glucose Assay by
Dinitrosalicylic Colorimetric Method. Nam Sun Wang,
Department of Chemical & Biomolecular Engineering,
University of Maryland.
http://www.eng.umd.edu/~nsw/ench485/lab4a.htm#Method
Wyman, C., et al. 2005. Hydrolysis of Cellulose and
Hemicellulose. In S. Dumitriu (ed.), Polysaccharides:
Structural Diversity and Functional Versatility, Second
Edition, pp. 995-1034. New York: Marcel Dekker.
http://www.microbiologyonline.org.uk/about-
microbiology/introducing-microbes/fungi
http://www.biotek.com/resources/articles/enzymatic-digestion-
of-polysaccharides-2.html
FUNDING
This project is funded in part through the National
Science Foundation (NSF) Transforming
Undergraduate Education in Science (TUES)
Community College Undergraduate Research
Initiative (CCURI), DUE # 1118679.
TOMPKINS CORTLAND COMMUNITY COLLEGE, DRYDEN, NEW YORK
Tim DeMarsh and James R. Jacob
IMPACT OF COFACTORS ON FERMENTATION OF CELLOBIOSE BY YEAST
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0.2
0.4
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1
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1 2.5 5 10 20
OpticalDensityReadings
Hours after inoculation
3/7/13 Baker's yeast + min. med. (#1)
3/7/13 Baker's yeast + min. med. (#2)
3/7/13 Turbo yeast + min. med. (#1)
3/7/13 Turbo yeast + min. med. (#2)