S t a r c h H y d r o l y s i s f o r Ethanol Production

Gerald B. Borglum

Miles Laboratories, Inc.

I n d u s t r i a l Products Group
P.O. Box 932
E l k h a r t , I n d i a n a 46515

Ethanol and e t h a n o l - g a s o l i n e m i x t u r e s have been considered f o r use as f u e l

since the e a r l y days o f t h e automobile. The abundant and l e s s expensive p e t r o -
leum supply precluded e x t e n s i v e use o f ethanol as f u e l and o n l y i n t h e l a s t few
years has t h e general p u b l i c become aware o f and concerned about t h e d w i n d l i n g
and i n c r e a s i n g l y expensive petroleum s u p p l i e s . I n t e r e s t i n extending gasoline
supplies w i t h e t h a n o l - g a s o l i n e m i x t u r e s has increased g r e a t l y .

Mankind since e a r l y recorded h i s t o r y has produced ethanol from simple

sugars by anaerobic y e a s t f e r m e n t a t i o n . I w i l l discuss t h e enzymatic p r o d u c t i o n
o f t h e simple sugar, glucose from s t a r c h f o r conversion by y e a s t i n t o ethanol
and t h e c o n t r i b u t i o n o f enzyme c o s t i n producing ethanol.

R e f e r r i n g t o F i g u r e 1, s t a r c h i s a polymer o f glucose. The glucose u n i t s

are j o i n e d i n hemiacetal bonds between carbon one and carbon f o u r f o r n e a r l y a l l
t h e bonds and between carbon one and carbon s i x f o r a small number o f bonds
which are branch p o i n t s i n amylopectin, one o f t h e t w o types o f s t a r c h polymers.
The hemiacetal bonds a r e a l l alpha c o n f i g u r a t i o n , t h a t i s , i n t h e Haworth s t r u c -
t u r e shown t h e carbon one bond t o another glucose u n i t i s below t h e p l a n e o f t h e
molecule. Cereal g r a i n s t a r c h i s n o r m a l l y a m i x t u r e o f two types o f polymers:
amylose, a l i n e a r glucose polymer, and amylopectin, a branched polymer.

The process steps i n c o n v e r t i n g s t a r c h t o dextrose a r e g e l a t i n i z a t i o n ,

l i q u e f a c t i o n and s a c c h a r i f i c a t i o n . S t a r c h i s found i n n a t u r e as i n s o l u b l e ,
n o n - d i s p e r s i b l e granules r e s i s t a n t t o enzymic breakdown. S t a r c h - b e a r i n g g r a i n s
such as c o r n , wheat, r y e and sorghum must be ground t o a f i n e meal, a t l e a s t
12-16 mesh, t o expose t h e s t a r c h granules t o t h e s l u r r y i n g water. G e l a t i n i z a -
t i o n i s t h e s w e l l i n g o f t h e s t a r c h g r a n u l e i n t h e presence o f heat and water.
The s t a r c h loses i t s c r y s t a l l i n i t y and becomes an amorphous g e l t h a t can be
attacked by enzymes. A t t h i s p o i n t , t h e s t a r c h o r ground g r a i n s l u r r y thickens
considerably and would be d i f f i c u l t t o process i f an alpha-amylase w e r e n o t
added t o p a r t i a l l y h y d r o l y z e t h e s t a r c h t o d e x t r i n s . The d e x t r i n s o l u t i o n i s
much more f l u i d ; t h u s , we say t h e s t a r c h gel i s l i q u e f i e d . The alpha-amylase
serves t o reduce t h e v i s c o s i t y o f t h e s o l u t i o n and a l s o t o produce a lower
molecular s i z e s u b s t r a t e . T h i s s m a l l e r s u b s t r a t e molecule i s needed f o r t h e
e f f i c i e n t a c t i o n o f glucoamylase which hydrolyzes t h e d e x t r i n s t o glucose.

As described above, t h e h y d r o l y s i s o f s t a r c h t o glucose r e q u i r e s two types

of enzymes. The alpha-amylase i s a b a c t e r i a l thermostable endo-amylase. I t hy-
d r o l y z e s w 1 , 4 bonds a t random p o i n t s i n t h e s t a r c h molecule t o r a p i d l y reduce
t h e v i s c o s i t y of g e l a t i n i z e d s t a r c h s o l u t i o n s . T h i s enzyme i s a metal i o n -
c o n t a i n i n g p r o t e i n and r e q u i r e s a small amount o f calcium i o n d u r i n g use f o r
maximum a c t i v i t y and s t a b i l i t y . The a c t i o n o f alpha-amylase on amylopectin i s
i l l u s t r a t e d i n Figure 2; t h e a c t i o n on amylose i s i d e n t i c a l . The enzyme cannot
hydrolyze w 1 , 6 bonds b u t can by-pass these branch p o i n t s i n amylopectin. The
p r o d u c t of t h e r e a c t i o n i s d e x t r i n s - s h o r t glucose chains, and small amounts o f
glucose and maltose.

Glucoamylase, produced by f u n g i , i s an exo-amylase. It hydrolyzes t h e

maltose and d e x t r i n s from t h e non-reducing end o f t h e molecule. Glucoamylase
hydrolyzes b o t h a-1,4 and w 1 , 6 bonds t o completely degrade t h e d e x t r i n s t o
glucose. The enzyme i s o p t i m a l l y a c t i v e a t pH 3.5-4.5 so pH adjustment a f t e r

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 s a c c h a r i f i c a t i o n i s n o t needed f o r t h e y e a s t f e r m e n t a t i o n . The y e a s t fermenta-
t i o n takes p l a c e a t pH 3.7 - 4.

The most p l e n t i f u l and most commonly used c e r e a l g r a i n as source o f s t a r c h

i s corn. The composition o f t h e c o r n k e r n e l i s g i v e n i n Table 1; the m o i s t u r e
c o n t e n t can v a r y considerably. W i t h 16% w/w m o i s t u r e t h e s t a r c h i s t y p i c a l l y
61% W/W o f t h e c o r n k e r n e l . On a d r y b a s i s t h e s t a r c h c o n s t i t u t e s 72% w/w o f
t h e c o r n composition. There a r e t h r e e cooking procedures used f o r s t a r c h gela-
t i n i z a t i o n and 1 i q u e f a c t i o n : atmospheric batch, pressure batch and continuous
liquefaction. The mash c o n c e n t r a t i o n t o use depends on t h e s u b s t r a t e , process-
i n g c o n d i t i o n s and equipment. T h i s step r e q u i r e s h i g h temperature and s o l i d s
c o n c e n t r a t i o n should be as h i g h as can be handled t o minimize t h e energy c o s t .
I T y p i c a l mash c o n c e n t r a t i o n s a r e 20-30% w/w d r y s o l i d s f o r ground whole c o r n and
2 5 3 5 % W/W d r y s o l i d s f o r corn s t a r c h as s u b s t r a t e .

TABLE 1
Approximate Composition of t h e Corn Kernel

Percent, As-Is Percent, DSB

Moisture 16
Starch 61 72
Protein 9 11
O i l (Fat) 3.8 5
Fiber 2 2
Pentosans 5.3 6
Sugars 1.6 2
M i n e r a l s (Ash) 1.3 2

The s u b s t r a t e s l u r r y , under continuous a g i t a t i o n , i s a d j u s t e d t o pH 6-6.5

w i t h l i m e s l u r r y and 0.02% - 0.15% w/w Taka-Therm on d r y s t a r c h b a s i s (DSB) i s
added t o t h e s l u r r y depending on t h e cooking process as i n d i c a t e d i n Table 2. A
6OoC h o l d p e r i o d i s recommended f o r ground whole c o r n t o ensure thorough hydra-
t i o n . Starch g e l a t i o n begins a t 66OC and maximum v i s c o s i t y i s reached a t 72'C.
I n the atmospheric and pressure batch processes t h e temperature r i s e may need t o
be slowed o r , i f necessary, t h e temperature h e l d t o a l l o w t h e enzyme t i m e t o
a t t a c k t h e s t a r c h and reduce t h e v i s c o s i t y f o r e f f i c i e n t s t i r r i n g . I n the
atmospheric b a t c h process (ABL) t h e s u b s t r a t e i s h e l d a t 9OoC-95OC u n t i l l i q u e -
f a c t i o n i s complete. T h i s t y p i c a l l y r e q u i r e s 30-90 minutes. The s u b s t r a t e
should have a dextrose e q u i v a l e n t (DE) o f a t l e a s t 10-14 and should n o t g i v e a
s t a r c h - i o d i n e b l u e c o l o r . The s u b s t r a t e i n t h e pressure b a t c h process (PBL) i s
h e l d a t 140C-1630C f o r 15 minutes and then f l a s h cooled t o 9Oo-95OC. An addi-
t i o n a l 0.1% w/w, DSB, enzyme i s added and l i q u e f a c t i o n completed as w i t h t h e
atmospheric batch process. I n t h e continuous cooking process (CL) t h e s u b s t r a t e
s l u r r y i s jet-cooked t o 140-1630C, h e l d f o r f i v e minutes a t t h i s temperature
and f l a s h cooled t o 90-95'. A t a t p o i n t 0.13% w/w, DSB, enzyme i s added and
l i q u e f a c t i o n completed as f o r t h e atmospheric b a t c h process.

TABLE 2
S t a r c h L i q u e f a c t ion

ABL
c
PEL
- CL
-
Taka-TherM: Taka-Therm: Ta ka-Therm:

0.15% w/w, DSB 0.05% w/w, DSB 0.02% w/w, DSB

Heat t o 9Oo-95OC Heat t o 140-1630C J e t cook t o 140-1630C

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H o l d a t 9Oo-95'C,
30-90 minutes
F l a s h cool t o 9Oo-95OC,
add 0. I% w/w, DSB
Taka-Therm@

Hold a t 9O-9S0C,
30-90 minutes
Flash cool t o 9Do-95OC,
add 0.13% w/w, DSB
Taka-Therm

Hold a t 9Oo-95OC.
30-90 minutes

There are advantages and disadvantages f o r t h e t h r e e processes. The a t -

1
mospheric b a t c h process does n o t r e q u i r e pressure equipment and h i g h pressure
steam which a r e c o s t advantages. On t h e o t h e r hand, complete g e l a t i n i z a t i o n and
s o l u b i l i z a t i o n o f h i g h c o r n s t a r c h c o n c e n t r a t i o n s i s d i f f i c u l t t o accomplish i n
a s h o r t processing t i m e a t t h e l o w e r atmospheric batch process temperature.
Incomplete l i q u e f a c t i o n g i v e s a lower ethanol y i e l d p e r u n i t mass o f s u b s t r a t e
o r w i l l r e q u i r e a l o n g e r y e a s t f e r m e n t a t i o n t i m e , which a r e c o s t disadvantages.

Diazyme@ L-100, a glucoamylase, i s used t o hydrolyze t h e d e x t r i n s t o dex-

t r o s e f o r t h e y e a s t f e r m e n t a t i o n . The s a c c h a r i f i c a t i o n can be completed b e f o r e
t h e y e a s t f e r m e n t a t i o n o r a continuous s a c c h a r i f i c a t i o n d u r i n g t h e y e a s t fermen-
t a t i o n can be used (Table 3). The f i r s t method i s e s s e n t i a l l y t h a t used i n
d e x t r o s e p r o d u c t i o n . The l i q u e f i e d s t a r c h i s cooled t o 6OoC, t i t r a t e d t o pH
4-4.5 and glucoamylase added a t 0.22% v/w, DSB. The enzyme l e v e l used i s 100
DiazymeB u n i t s p e r pound o f d r y s t a r c h . The m i x t u r e i s h e l d a t 6OoC u n t i l a
r e d u c i n g sugar l e v e l o f 95 DE o r g r e a t e r i s reached; t h i s takes 36-72 hours.
I n s o l u b l e m a t e r i a l s such as p r o t e i n , f i b e r and f a t can be removed a t t h i s stage
i f desired by c e n t r i f u g a t i o n o r f i l t r a t i o n . The syrup i s d i l u t e d t o 19% w/w
s o l i d s w i t h water and cooled t o 3OoC. Yeast i s added f o r t h e fermentation.

TABLE 3
S a c c h a r i f i c a t i o n and Fermentation

L i q u e f i e d Starch: 6OoC
pH 4-4.5
0.22% v/w, DSB, DiazymeB L-100

Compl e t e Continuous

Hold t o 95 + DE D i l u t e t o 19% w/w s o l i d s ,

(36-72 hours) Hold 1-2 hours

D i l u t e t o 19% w/w s o l i d s

Cool t o 3OoC and ferment Cool t o 3OoC and ferment

I n the c o n c u r r e n t o r continuous s a c c h a r i f i c a t i o n process t h e l i q u e f i e d

s t a r c h i s cooled, t i t r a t e d t o pH 4-4.5 and enzyme added as i n t h e complete
s a c c h a r i f i c a t i o n process. The m i x t u r e i s d i l u t e d and h e l d a t 6OoC f o r o n l y one
t o two hours t o produce enough dextrose f o r t h e s t a r t o f t h e fermentation.
D e x t r i n h y d r o l y s i s t o 40 DE i s s u f f i c i e n t t o g i v e t h e y e a s t an i n i t i a l r a p i d
fermentation rate. The syrup i s cooled t o 3OoC and y e a s t i s added f o r t h e
ethanol fermentation. The glucoamylase a c t i o n i s c o n s i d e r a b l y slower a t 3OoC
b u t t h e enzyme a c t i v i t y i s g r e a t enough t o keep t h e y e a s t s u p p l i e d w i t h dex-
t r o s e . DiazymeB L-100 a l s o c o n t a i n s an alpha-amylase which a i d s i n h y d r o l y z i n g
t h e d e x t r i n s and s t a r c h which may have s u r v i v e d t h e l i q u e f a c t i o n step.

To c a l c u l a t e t h e e f f i c i e n c y o f t h e f e r m e n t a t i o n we need t o c a l c u l a t e t h e
t h e o r e t i c a l ethanol y i e l d f r o m s t a r c h . R e f e r r i n g t o Table 4, i n t h e h y d r o l y s i s
of s t a r c h a water molecule i s added across each g l y c o s i d i c bond so one gram
s t a r c h completely hydrolyzed would g i v e 1.11 g glucose. From Gay-Lussac's
e q u a t i o n t h e 1.11 g glucose would t h e o r e t i c a l l y y i e l d 0.567 g ethanol. Using

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t h e d e n s i t y of ethanol we c a l c u l a t e t h e p r o d u c t i o n o f one g a l l o n 100% ethanol
from 11.59 pounds s t a r c h . I n terms of c o r n as s u b s t r a t e and t a k i n g the w e i g h t
o f a bushel of c o r n t o be 56 pounds c o n t a i n i n g 6% w/w s t a r c h , a bushel o f c o r n
would Y i e l d 2.95 g a l l o n s o f ethanol.

TABLE 4

1.0 g starch + H20 -

Theoret i c m n o l Y i e l d

4
1.11 g glucose

2 C2HsOH + 2 Cop
C6H1206
1.11 g

Bushel c o r n
61% w/w s t a r c h
- 0.567 g

2.95 g a l l o n s
Ethanol

This t h e o r e t i c a l y i e l d does n o t t a k e i n t o account ethanol l o s s due t o

carbohydrate used f o r y e a s t growth and t o carbohydrate used i n t h e formation of
small amounts o f non-ethanol p r o d u c t s by t h e y e a s t . A s i m p l i f i e d b i o s y n t h e t i c
pathway from glucose t o ethanol i s shown i n Table 5. T h i s i s t h e Embden-
Meyerhof-Parnas scheme f o r g l y c o l y s i s . G l y c e r o l and l a c t i c a c i d a r e formed i n
small amounts compared t o ethanol s y n t h e s i s b u t c o n t r i b u t e t o g i v e a y i e l d l e s s
t h a n s t o i c h i o m e t r i c f o r m a t i o n o f ethanol from glucose. A l l o w i n g f o r t h e growth
o f y e a s t c e l l s and t h e f o r m a t i o n o f f e r m e n t a t i o n by-products t h e maximum fermen-
t a t i o n e f f i c i e n c y i s about 95% o f s t o i c h m e t r i c y i e l d .

TABLE 5
Ethanol B i o s y n t h e s i s

G1 ucose
J.
J.
Glyceraldehyde-3-P04++Glycerol
J.
J.
Pyruvic Acid 3 Lactic Acid
J.
J.
Ethanol

A f e r m e n t a t i o n w i l l t y p i c a l l y y i e l d 2.5 g a l l o n s ethanol p e r bushel corn, an

85% f e r m e n t a t i o n e f f i c i e n c y . I n c a l c u l a t i n g t h e enzyme c o s t i n producing a
g a l l o n o f ethanol, I used t h e p r i c e s f o r standard packages o f our enzyme p r o -
ducts, a 55 g a l l o n drum f o r Taka-Therm and a 200 l i t e r drum f o r Diazyrne@ L-100.
(Table 6) T h e o r e t i c a l ethanol y i e l d i s one g a l l o n from 11.59 pounds s t a r c h .
The recommended enzyme use l e v e l s a r e 0.15% w/w, DSB f o r t h e a-amylase and 100
Diazyme@ u n i t s o r one m i l l i l i t e r Diazyme@ L-100 p e r pound d r y s t a r c h . The c o s t
f o r t h e t h e o r e t i c a l y i e l d i s 5.4 c e n t s p e r g a l l o n . For a y i e l d a t 85% fermenta-
t i o n e f f i c i e n c y t h e enzyme c o s t i s 6.4 cents p e r g a l l o n . The enzyme c o s t can be
reduced by u s i n g l e s s e r amounts o f enzyme. One must t a k e i n t o c o n s i d e r a t i o n
t h a t an a-amylase l e v e l t h a t i s t o o low w i l l g i v e incomplete s t a r c h s o l u b i l i z a -
t i o n and concomitant l o s s o f ethanol y i e l d . A glucoamylase l e v e l t h a t i s t o o
low w i l l extend t h e f e r m e n t a t i o n t i m e f o r the c o n c u r r e n t s a c c h a r i f i c a t i o n -
fermentation process because dextrose i s n o t produced as r a p i d l y as t h e y e a s t
can c o n v e r t i t t o ethanol.

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 TABLE 6
Enzymest

Standard Pkg.

Ta ka-ThermB 500 l b . drum $1.35/lb.

D iazymed L- 100 200 l i t e r drum $2.625/1 i t e r

One g a l l o n ethanol from 11.59 l b . s t a r c h .

Ta ka-Therm:
11.59 l b . x 0.15% x $1.35/lb. enzyme $0.0235
Diazymed L-100:
11.59 l b . x U l b x $2.625/2, enzyme 0.0304
Total $0.054
Fermentation a t 85% e f f i c i e n c y $0.064

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 FIGURE 1
STRUCTURE OF STARCH

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