Where's the Bt? Tracking the Protein's Fate During Ethanol Production
Microbiologist Nancy Nichols and
biochemical engineer Bruce Dien
add yeast to a bioreactor to
begin ethanol fermentation. Bt
and non-Bt corn hybrids were
compared for ethanol yields.
As ethanol's production as a cleaner-burning alternative to petroleum
fuels gains momentum, the research of Bruce S. Dien's team in Peoria,
Illinois, couldn't have come at a better time.
In fermentation experiments at ARS'
National Center for Agricultural Utilization Research, Dien's team charted
the biochemical fate of Bt protein during both wet- and dry-milling
of genetically modified (GM) corn into ethanol and other co-products.
His team also compiled early evidence that corn's ethanol yield is influenced
by how easily its starch breaks down as well as by how much starch it
"In our research with traditional alcohol fermentations, we found
that the digestibility of starch influenced ethanol production efficiency
as much as total starch content did, and that Bt protein in GM
corn was not detectable after fermentation," says Dien, a chemical
engineer in the Fermentation Biotechnology Research Unit (FBRU).
Both topics, he adds, are of interest to seed companies and corn farmers alike as they strive to tap into new, specialty-use markets for the grain crop. Ethanol is deemed a promising product as increasing concern over U.S. dependency on foreign oil has intensified the search for viable alternatives. (See "Bioenergy Today," Agricultural Research, April 2002, p. 4.)
Microbiologist Loren Iten
Bt stands for Bacillus thuringiensis, a soil bacterium
whose genes for making the protein have been copied and spliced into
corn plant DNA as a natural, built-in insecticide. Among the chief targets
of this biotechnological defense is the European corn borer, a moth
whose caterpillar stage costs U.S. farmers an estimated $1 billion annually
Bt corn has gone from less than one-half million acres planted
in 1996the year of its commercial debutto 19.5 million acres
in 2000, the latest year for which the Environmental Protection Agency
has statistics. Yet, despite the good attributed to Bt, such
as cost savings and environmental benefits from reduced use of chemical
insecticides, there's been little research on the protein's fate in
corn during processing into ethanol.
And with ethanol production forecasted to increase from the current
1.7 billion gallons to 4 billion gallons by 2006, the effect of Bt
corn on the biofuel's production presents something of an X-factor,
or unknown variable.
Are Bt ethanol yields the same as those from non-Bt crops?
And if Bt protein shows up during ethanol production, by what
methods could refiners detect and quantify it? These are some of the
questions posed to scientists by industry.
"There isn't a lot of research information out there because the
widespread use of Bt in corn is still relatively new," says
Rodney J. Bothast, a microbiologist at the FBRU. "And since some
Bt corn does enter ethanol production plants, we wanted to see
what happens to it."
So, in 2000, Dien, Bothast, and ARS microbiologist Loren B. Iten teamed
up with two other researchers, Lynda Barrios and Steven R. Eckhoff,
who had already done some preliminary wet- and dry-milling studies at
the University of Illinois Department of Agricultural Engineering in
Together, they designed small-scale, wet- and dry-milling experiments that would allow them to monitor Bt protein of two modified corn hybrids during all stages of ethanol production. They also compared the Bt corns' overall ethanol yield to that of non-Bt versions of the two hybrids. During each stage of the experiment, says Bothast, "we basically repeat in the lab what happens in an actual ethanol plant."
Steeped in Bt?
Wet-milling involves several steps, starting with soaking, or steeping,
corn in water and sulfur dioxide for 24 to 36 hours. During later stages,
the corn is ground up to facilitate separation of its starch, fiber,
germ oil, and protein. Glucose and other simple sugars derived from
the starch fraction are fermented inside giant vats containing live
cultures of Saccharomyces cerevisiae yeast.
"Starch is made up of glucose molecules hooked to one another
by a chemical bond known as an alpha-1,4 linkage," explains Bothast.
"During fermentation, the yeast takes up this glucose molecule,
metabolizes it, and converts it into two products: carbon dioxide and
In dry-milling, ground cornmeal is cooked during a liquefaction stage
with water and an enzyme, alpha-amylase. The enzyme helps breaks down
the starch fraction into smaller parts and keeps it from becoming a
gel. What's left is a mash that's later fermented into ethanol.
For their studies, Dien's team used 300-milliliter flasks or 2.5-liter
or 30-liter bioreactor tanks to ferment corn from two Bt-modified
hybrids and two non-Bt ones.
At each stage they checked for a Bt proteina type known
as CRY1Abusing an antibody-based test known as ELISA, short for
enzyme-linked immunosorbent assay.
Dien says their test results indicate that use of heat during dry-milling
tends to denature, or destroy, Bt protein.
Tests of cornmeal from the two Bt hybrids revealed CRY1Ab protein
concentrations of 196 parts per billion (ppb). But once that meal is
liquefied, "the Bt disappears in less than 15 minutes,"
the scientists report. And there was no detectable trace of it in either
the mash or the resulting ethanol.
A slightly different story unfolded for the wet-milled corn. While
samples of whole kernels, gluten, germ oil, and fiber contained Bt
protein at concentrations of 170 to 453 ppb, no trace could be found
in the starch or steep liquor fractions. Nor should it appear later
on in the ethanol, they say. That's because the Bt protein gets
separated from the starch with the other proteins. (The researchers
note that use of high temperatures to dry corn gluten meal and corn
gluten feed may eliminate any protein that survives the wet-milling
Bt and non-Bt corn hybrids yielded about the same amount of ethanol, and the yields were comparable to those achieved in industrial production. On average, a bushel of corn (56 pounds wet weight) yields about 2.7 gallons of ethanol via dry-milling versus 2.5 for wet-milled corn, notes Dien. The team will publish the findings in an upcoming issue of the journal Cereal Chemistry.
Heavy on Starch
In that same paper, they'll also report preliminary results of a follow-up
study that challenges the common assumption that corn hybrids with high
starch contents yield the most ethanol.
"The take-home message here is that not all starches are created
equal," says Bothast.
In the study, scientists fermented corn samples from five hybrids containing
between 68 and 72 percent starch. Using a standard method, they measured
the hybrids' fermentation efficiency (CE). This refers to how much of
the total starch actually breaks down into the glucose sugar that gives
rise to ethanol.
Representative of the study's findings are the ethanol yields and CE
rating for hybrid A and hybrid C. Hybrid A, with a starch content of
68 percent, yielded 2.73 gallons of ethanol per bushel, with a CE rating
of 92 percent. Hybrid C contained 72 percent starch, and yielded 2.83
gallons of ethanol per bushel. But its lower CE rating, 90 percent,
meant that less of that starch actually converted into sugar, the researchers
Bothast considers the findings preliminary since the five hybrids they
tested are a small sampling of the hundreds now grown commercially.
They also want to further verify the methods by which they obtained
their results, as the research eventually could provide commercial seed
companies with a protocol they could use in their corn breeding programs.
Farmers, too, are keeping close tabs on the scientists' work, since
a hybrid packing lots of easily digested starch can translate into greater
returns on their crop for the ethanol market. Likewise, "the seed
companies will want to be able to show, this is our best hybrid for
making ethanol," says Bothast.
But what actually makes a hybrid's starch readily break down into simple
sugars for easier fermentation remains a mystery.
"That's the million-dollar question," says Bothast. "It could be due to the environment in which the corn is grown, or the DNA comprising its genes and their subsequent effect on the composition of the corn itself."
Perhaps the unsung heroes in this push for peak ethanol production
are the Saccharomyces yeast organisms.
"They've been the workhorses of the industry and are absolutely
our best friends," the microbiologist says. In fact, a chief emphasis
of the Peoria team all along has been maximizing the yeast's ability
to efficiently convert carbohydrates to new, value-added co-products.
"Our goal is to have a fermentation organism that uses all kinds
of sugarsnot just those from starch, but also from the fiber,"
says Bothast. "Theoretically, you could get a 10-percent increase
in ethanol production" from using these other sugars.By Jan
Suszkiw, Agricultural Research Service Information Staff.
This research is part of Quality and Utilization of Agricultural
Products, an ARS National Program (#306) described on the World Wide
Web at http://www.nps.ars.usda.gov.
Bruce S. Dien, Rodney
J. Bothast, and Loren
B. Iten are in the USDA-ARS Fermentation
Biotechnology Research Unit, National Center for Agricultural Utilization
Research, 1815 N. University St., Peoria, IL 61604; phone (309) 681-6270
[Dien], -6566 [Bothast], -6210 [Iten], fax (309) 681-6427.
"Where's the Bt? Tracking the Protein's Fate During Ethanol Production" was published in the July 2002 issue of Agricultural Research magazine.