Improving Ethanol Yield From Corn
Fungal physiologist Shelby Freer examines yeasts under high magnification,
projected on monitor, for their ability to make enzymes needed for ethanol
Ethanol to fuel cars early in the 21st century may come from fiber-laden
crop residuesinstead of feed grainsif a vision of agricultural
researchers at Peoria, Illinois, pans out.
But before that vision is fully realized, science and technology will most
likely enable ethanol producers to squeeze a bit more ethanol from
cornthe main source at present.
Through fermentation of starches and sugars found inside the grain, modern
ethanol plants now produce 2.5 gallons of ethyl alcoholethanolfrom
each bushel of corn, says Rodney J. Bothast. Hes an ARS microbiologist at
the National Center for Agricultural Utilization Research (NCAUR) in Peoria,
Bothast and his colleagues have their sights set on making fiber in the
grain's outer layer yield nearly 0.3 additional gallon per bushel.
In 1994, about 1.3 billion gallons of fuel ethanol were produced in the
United States from corn, with more than 60 percent obtained through wet
milling. The wet-milling process involves soaking, or steeping, the corn in
water, grinding it, and separating high-protein germ, oil, and fiber from the
starchy endosperm that is fermented to produce ethanol.
The current practice is to mix the fiber fraction with fermentation solubles
before drying it and forming animal feeds.
Agricultural engineer Michael R. Ladisch and colleagues at Purdue University
in West Lafayette, Indiana, teamed up with the NCAUR scientists to assess ways
to increase ethanol production from corn.
"We estimate that if the fiber were also processed into ethanol, a corn
wet-milling facility that produces 100 million gallons of ethanol per year
could generate an additional $4 to $8 million of annual income," says
microbiologist Robert B. Hespell. He is project leader for ethanol research in
Bothast's Fermentation Biochemistry Research Unit.
Stilling a Criticism
The increased efficiency of corn and ethanol production that has evolved
over the last 10 years may also help to subdue criticisms that petroleum used
to produce corn and process it into ethanol requires more energy than is
released when the ethanol is burned.
According to "Estimating the Net Energy Balance of Corn Ethanol,"
a report published last year by USDA's Economic Research Service (which now
includes the Office of Energy and New Uses), the ethanol energy now produced
from each bushel of corn is 25 percent greater than the amount of energy used
to grow and harvest the corn and distill it into ethanol. This is thanks to
today's higher corn yields, more energy-efficient fertilizer production, and
improved distillation technology.
Unlocking Fiber's Energy Potential
Hespell says his team's research strategy for economically converting fiber
to ethanol is three-pronged. They hope to:
- find better ways to physically and chemically treat the fiber to expedite
its conversion to sugars,
- find enzymes that better convert the fiber to sugars, and
- custom engineer suitable microbes to ferment the sugars D-glucose,
D-xylose, and L-arabinose.
To achieve these goals, the researchers are combining their efforts with
those of other ARS scientists and university cooperators.
At College Station, Texas A&M University researcher Mohammed
Moniruzzaman and chemical engineer Bruce E. Dale, who is now with Michigan
State University, applied a pretreatment called ammonia fiber explosion (AFEX)
to corn fiber to unlock its potential for fermentation.
With it, NCAUR scientists found they could convert some 50 to 60 percent of
the pretreated fibers componentscellulose, hemicellulose, and
starchinto fermentable sugars called monosaccharides, while converting an
additional 20 to 30 percent of the fiber into short sugar polymers.
In the AFEX process, a slurry of water and corn fiber is mixed with highly
pressurized liquid ammonia. Quickly releasing the pressure splits the fiber's
bundles of carbohydrate components that are normally rather inaccessible to
chemicals or microbes because they are so tightly glued together by lignin.
Researchers treated the lignin-freed polymers with mixtures of commercial
enzymes. Some enzymes called amylases and cellulases thoroughly hydrolyzed, or
split apart, chains of starch and cellulose into links of simple fermentable
sugars such as glucose, each with a backbone of six carbon atoms. Today's
ethanol plants typically use bakers' yeastSaccharomyces
cerevisiaeto produce ethanol only from these 6-carbon sugars called
From the corn hemicellulose, or arabinoxylan, xylanase enzymes clipped off
at least 25 percent of the component pentoses or 5-carbon
sugarsmonosaccharides such as arabinose and xylose. That success was
enough to spur a search for xylanases that might be recruited to enhance
"If we can find bacteria that produce more active xylanases, this
ethanol research might also lead to improving the efficiency with which
ruminant livestock such as cattle and sheep digest hemicellulosic crop
residues," says Hespell. He is also involved in research on ruminant
Drawing On Archival Research
Another impetus for the current focus on freeing up pentoses for ethanol
production is recent success by biotechnologists in transforming single
microbial species to subsist on both hexoses and pentoses.
Seeking an alternative pretreatment of the fiber to free up more simple
sugars, the researchers took note of work done by John W. Dunning and Elbert C.
Lathrop at NCAUR in one of the earlier ethanol research programs dating back
more than 50 years.
Recognizing the large amount of sugars in corn cobs and other agricultural
residues, Dunning and Lathrop hydrolyzed hemicellulose with mild sulfuric acid
treatments, forming a solution of mostly pentoses.
But further costly processing, such as deacidifying and removing the toxic
byproduct furfural, was required before microorganisms could use the sugars.
ARS chemist Karel Grohman of Winter Haven, Florida, and Bothast reasoned
that formation of furfural and other chemicals that inhibit fermentation could
be reduced by a two-stage process. First, they quickly hydrolyzed the fiber
with hot, mild acid; then they quickly cooled it and added a mixture of
cellulase and amyloglucosidase enzymes before further hydrolysis.
In the laboratory, Grohman found that the sequential treatment on batches of
low-starch corn fiber resulted in about 85 percent of all polysaccharide
becoming sugars in monomer forms.
Within 2 days, genetically engineered Escherichia coli bacteria
fermented these sugars into solutions of more than 2 percent alcohol.
The hydrolysis is now being scaled up as a continuous process at NCAUR. A
goal is to complete the acid hydrolysis phase within 2 minutes.
After successfully finding ways to break down corn fiber into hexoses and
pentoses, the researchers" next challenge is to identify or develop
strains of microorganisms that will convert both of these sugar types to
ethanol as efficiently as bakers" yeast makes it commercially from
The genetically engineered E. coli strain K011 that Grohman and
Bothast used to produce ethanol from multiple sugars of acid-hydrolyzed fiber
was developed by microbiologist Lonnie O. Ingram at the University of Florida
in Gainesville. The evaluation of K011 was conducted by ARS under a cooperative
Starting with K011, microbiologist Herbert Wyckoff and chemical engineer
Bruce S. Dien at NCAUR worked with Hespell and Bothast to further transform the
microbe. Unlike the older version, the new one does not need antibiotics to
survive in anaerobic fermentation environments like those of commercial ethanol
plants. The scientists are scaling up their laboratory research to the pilot
Another recombinant microbe that can ferment both glucose and xylose is a
strain of S. cerevisiae yeast developed by geneticist Nancy Ho at
Purdue. It was also evaluated under an ARS cooperative agreement.
Because this microbial species has long been used to make ethanol, a
modified version too might someday work well for the industry, says Bothast.
Further research, however, is needed to develop Hos strain into one that
can survive better under industrial conditions, produce ethanol from other
sugars such as arabinose, and quickly produce larger volumes of ethanol.
Fungi, Too, Might Join the Effort
In addition to bacteria and yeasts, genetically transformed filamentous
fungi could become ethanol plant workhorses.
At NCAUR, microbiologists Christopher D. Skory and Shelby N. Freer envision
harnessing industrial and food processing fungi for a "one-pot"
method of producing ethanol. The microbes prodigiously spew out enzymes that
efficiently break down the corn fiber's cellulose and hemicellulose while
producing tiny amounts of ethanol from the resulting sugars. "Through both
mutagenesis and genetic engineering, we hope to increase their ethanol
production," says Skory.
Similar genetic research could lead to one-pot production of lactic acid
that is valuable in food processing and for industrial applications, such as
making biodegradable plastics. Developing such a wet-milling coproduct would
help offset ethanol production costs, since ethanol is fairly low in economic
Considering alternative fermentations of glucose, Freer is screening part of
the ARS Culture Collection located at the NCAUR for Brettanomyces yeasts
that efficiently produce acetic acid from glucose. In earlier screening of the
collection, Freer identified a microbe with a gene responsible for producing a
beta-glucosidase enzyme that breaks down small cellulose polymers into
fermentable sugars. Skory has cloned the gene and inserted it into several
other microbes, including ethanol-producing ones.
In still another screening, microbiologist Badal C. Saha has identified a
yeast that produces a heat-stable beta-glucosidase that works in environments
high in glucose. He is mutating the yeast to try to increase its production of
the enzyme so that it can be used to efficiently convert cellulose to sugars.
In addition to trying to get more ethanol from a bushel of corn, the NCAUR
researchers hope to increase the usefulness of other ethanol fermentation
One abundant low-value product of fermentation is carbon dioxide. NCAUR
plant physiologist Brent Tisserat is evaluating the ability of different CO2
concentrations to speed the growth of plant tissue cultures. He envisions using
such cultures one day to produce food flavorings and high-value
At NCAUR, other scientists are researching potential value-added products
that can be made from wet-milling coproducts. By Ben Hardin, ARS.
Biochemistry Research Unit, National Center for Agricultural Utilization
Research, 1815 N. University St., Peoria, IL 61604; phone (309) 681-6500.
"Improving Ethanol Yield From Corn " was published in the
issue of Agricultural Research magazine.