294 • Impacts of Applied Genetics— Micro-Organisms, Plants, and Animals 
tent of quantifying the potential impact of genetic im- 
provement on each one. It is interesting to note that 
each type of organism has its substrate restrictions, 
and only the anaerobic bacteria such as Clostridium 
thermosaccharolyticum and C. thermohydrosulfori- 
cum can utilize all of the available substrate. 
Substrate pretreatment 
Pretreatment refers to the processing that is re- 
quired to convert a raw material such as sugarcane, 
starch, or cellulosic biomass to a product that is 
fermentable to ethanol. In most cases, the pretreat- 
ment is either extraction of a sugar or hydrolysis of a 
polysaccharide to yield a mono- or disaccharide. 
EXTRACTION OF SUGAR 
Sugar crops such as sugarcane, sugar beets, or 
sweet sorghum are highly desirable raw materials 
for producing ethanol. These crops contain high 
amounts of sugars as sucrose. In addition, the yield 
of fermentable material per acre is high; sugarcane 
and sugar beets yield 7.5 and 4.1 dry tons of biomass 
per acre, respectively.’ 
Sugar is extracted from cane or beets with hot 
water and then recrystallized. The resulting sugars 
are utilized directly by organisms having invertase 
activity (to split sucrose to glucose plus fructose). 
Molasses, a sugary byproduct of the crystallization of 
sucrose, may also contain sucrose although in most 
cases it is inverted with acid. 
The primary use for sugar crops is food sugar. 
Sugar sells for over 20 cents/lb. Molasses, which cur- 
rently sells for about $100/ton (about 10 cents/lb 
sugar) is used extensively as an animal feed. Substan- 
tial amounts of both sugar and molasses are im- 
ported into the United States for food uses and are 
therefore unavailable for ethanol production. There 
are proposals to increase sugar production for use as 
an energy crop; however, this will require the 
development of new land for sugar production. 
STARCH 
The primary raw material for ethanol fermenta- 
tion in the United States is cornstarch. Corn proc- 
essed by wet milling, yields about 36 lb of starch 
from each 56 lb bu; this amount of starch will pro- 
duce 2.5 gal of absolute ethanol. Corn yields are 
typically 80 to 120 bu/acre so that 200 to 300 gal of 
ethanol can be derived per acre of corn per year. 
Pretreatment of starch is initiated by a gelatiniza- 
tion step whereby a starch slurry is heated for 5 min 
at 105° C. After cooling to 98° C, a-amylase is added 
'Paul B. Weisz and John F. Marshall, Science 206:24. 1979. 
to break down the starch to about 15DE (dextrose 
equivalents). This process of liquefaction reduces the 
viscosity such that the solution can be easily mixed. 
After further cooling to 30° C, glucoamviase is added 
along with a starting culture of yeast so that saccha- 
rification and fermentation proceed simultaneousiv. 
The resulting fermentation, to produce typically 8 to 
10 percent ethanol (v'olume per volume), requires 42 
to 48 hr for completion. This compares with a 16- to 
20-hr fermentation if sugar as molasses or cane juice 
is used as the substrate. Thus, the use of starch re- 
quires the addition of enzymes prior to and during 
fermentation, as well as large fermenter capacity as a 
consequence of the slower fermentation time com- 
pared with sugar substrates. 
Improvement in the economy of ethanol fermenta- 
tion based on starch is possible by developing a 
micro-organism that can produce a-amylase and 
glucoamylase and thus eliminate the need to add 
these enzymes. Since the rate of fermentation de- 
pends on the rate of starch hydrolysis, increased lev - 
els of glucoamylase may enhance the rate of starch 
hydrolysis and thus increase the rate of ethanol [)ro- 
duction. This would lower the capital re(iuirements 
as well as the cost of enzyme addition. 
CELLULOSIC BIOMASS 
Processes for the utilization of cellulosic biomass 
to produce liquid fuels all have three features in com- 
mon; 
1. They employ some means of |)retreatment to at 
least effect some initial size rc'duction and. moi'c 
often, cause a disassociation of lignin and cellu- 
lose; 
2. they involve either acid or enzymatic hydrolysis 
of the cellulose and hemicellulose to |)roduce 
mono- and disaccharides; and 
3. they employ fermentation to |)roduce (Mhanol or 
some other chemical. 
A wide variety of process schemes have been pro- 
posed for the conversion of cellulosic biomass to 
liquid fuels; a summary of the major steps in two 
acid hydrolysis and three enzymatic hydrolysis 
schemes in shown in figures I-I)-2 and l-l)-3. The iti- 
itial size reduction is re{]uired to increase the 
amount of biomass surface area that can he con- 
tacted with acid, solvent, steam, enzymes, or 
chemicals that might he used to di.sassociate the 
cellulose and hemicellulose from the lignm 
Pretreatments that have been investigated to 
facilitate the process are summarized in table 
I-D-1. The problems with pretreatment are that thev 
require energy, eciuipment, and often chemu als. 
they result in an irretrievable loss of sugar, and in 
undesirable side-reactions and byproduct lorm.i 
