Alcohol Fermentation of Sweet Potato.

Membrane
Reactor in Enzymatic Hydrolysis
A. AZHAR and M. K. HAMDY, Department of Biology, Georgia
State University, Atlanta, Georgia, and Department of Food Science,
University of Georgia, Athens, Georgia 30602
Summary
Use of ultrafiltration membrane systems in stirred cell and in thin-channel systems for
immobilizing enzyme (sweet potato intrinsic and crystalline @-amylase) in hydrolysis of
sweet potato through a continuous operation mode were studied. Both the filtration rate
and reducing sugars, produced as the result of enzymatic hydrolysis, decreased with the
filtration time. The immobilized enzymes in the thin-channel system showed a much better
performance compared to that in the stirred cell system. Addition of crystalline sweet potato
P-amylase to the sweet potato increased both the filtration rate and reducing-sugars content.
Alcoholic fermentation of the filtrate resulted in an alcohol content of 4.2%. This reprcsezted
fermentation of 95% of the sugars with an efficiency of 88%.
INTRODUCTION
Ultrafiltration cells have been used for the localization of enzymes in
order to carry out a continuous catalytic reaction.' - 3 Semipermeable
membranes used on these cells retain the appropriate enzyme and sub-
strate molecules until the latter are sufficiently degraded and eventually
pass through the filter. Depending on the molecular size of the enzyme
and its catalytic nature, degradation of high molecular weight substrates
can be controlled to give different molecular weight products based on
the cutoff limit of the semipermeable membrane used. Marshall and Whe-
lan4 showed that glucoamylase digestion of starch in an ultrafiltration cell
brought about almost complete hydrolysis to glucose.
Enzymatic hydrolysis of starch produces sugars (glucose, maltose, etc.)
which are directly useful as substrates for all living microorganisms. Many
organic products could also be derived partly by enzymatic or microbial
transformation of these sugars. Such transformations have been used' for
the manufacture of various essential amino acids, vitamins, glycerol, fats,
organic acids, and many other useful end products. Sugars produced
during the enzymatic hydrolysis of starch can subsequently be fermented
to alcohol by yeast. Miller6 listed the raw materials used in alcoholic
fermentation as cereal grains (mainly corn), sugar cane, sugar beets, fruit
Biotechnology and Bioengineering, Vol. XXI I I , Pp. 1297-1307 (1981)
0 1981 J ohn Wiley & Sons, Inc. CCC OOO6-3592/81/061297- 1 1$01.10
1298 AZHAR AND HAMDY
product wastes, potatoes, rice, sulfite liquors, and high cellulose-con-
taining materials. Humphrey7 summarized some of the most important
uses of alcohol as follows: solvents, beverages, food and feed (via single
cell protein), petrochemical synthesis, and motor fuel. Ethyl alcohol can
also be used as the sole carbon source by a wide variety of microorga-
nisms' for the production of some amino acids, vitamins, and other com-
pounds. This study was undertaken to examine the technical feasibility
of using different ultrafiltration membrane devices to confine P-amylase
enzyme and sweet potato powder in a continuous operational mode.
Sweet potato (Zpomoea batatas) was chosen as a source of starch which
can be converted to fermentable end products since it has a higher starch
yield per unit land cultivated than grains (corn). I t also contains appre-
ciable amounts of highly active amylases responsible for converting starch
to reducing sugars upon incubation at elevated temperature^.^^'^
MATERIALS AND METHODS
Preparation of Freeze-Dried Sweet Potato Powder
Fresh (uncured) Georgia jet sweet potatoes were sliced to a thickness
of 0.4 cm and freeze-dried for 18 hr under a reduced pressure of 50-200
km at a shelf temperature of 0-4C. The moisture content of the freeze-
dried sweet potato, determined by drying samples in a vacuum oven at
80C to a constant weight," was found to be 8%. The freeze-dried sweet
potato was ground in a Wiley mill and the resulting powder was allowed
to pass through a No. 40 wire mesh screen and stored at 2C until use.
The starch content of SPP is 64.4% based on dry weight.
Membrane Reactor
Two membrane reactors were used in this investigation: (A) stirred cell
system and (B) thin-channel system. The schematic diagram of both sys-
tems is shown in Figures 1 (A) and (B). System (A) consisted of an Amicon
Stirred Cell model 52 equipped with a Diaflo ultrafiltration membrane
(Amicon Corp., Lexington, MA) type PM 30 with 30,000 molecular weight
cutoff. System (B) was made of Amicon High Performance Thin-Channel
System model TCFlO and exhibited a 10,000 molecular weight cutoff.
Slurry of freeze-dried sweet potato (1%) in sodium acetate buffer (pH
4.8, 0.016M) was used as substrate for the sweet potato P-amylase (a-1,
4-glucan maltohydrolyase, EC.3.2.1.2, United States Biochemical Corp.,
Cleveland, OH). The reactor vessel was placed in a water bath and kept
at a constant temperature of 50 k 1C which was established by Giri"
to be optimal for the intrinsic P-amylase activity of the sweet potato. The
substrate was constantly mixed by means of a magnetic stirrer in order
to keep the sweet potato powder in suspension. The substrate was then
ALCOHOLIC FERMENTATION OF SWEET POTATO 1299
*;
- 5
- I
I
Fig. 1 . Schematic diagrams of the arrangements used in the stirred cell system (A) and
in the thin-channel system (B). The systems consist of nitrogen gas tank for pressure ( 1) ;
pressure regulator (2); substrate reservoir (3): magnetic stirring bar (4); magnetic stirrer (5);
cell (6); water bath (7); spiral flow channel (8): filter and support (9); peristaltic pump (10);
filtrate outlet (11); sample outlet (12): and fraction collector (13).
introduced at a level of 1% (at To) into the reactor vessel under 15 psi
pressure using nitrogen gas. The filtrate was collected in small glass vials
having a capacity of 12 ml by means of a Gilson Fraction Collector (Mid-
dletown, WI). The retained slurry in the cell (retentate) was circulated
at a rate of 50 ml/min in the thin-channel system.
Retention of Enzyme Activity in the Membrane Reactor
Measurements of the retention of enzyme activities were carried out
in an Amicon High Performance Thin-Channel System model TCFlO,
equipped with a Diaflo ultrafiltration membrane (Amicon Corp., Lexing-
ton, MA) TP30 with 30,000 molecular weight cutoff. Potato starch (1%),
obtained from the Baker Chemical Co. (Phillipsburg, NJ), in sodium ace-
tate buffer (pH 5.8, 0.016M) was used as substrate, and sweet potato (3-
amylase (United States Biochemical Corp., Cleveland, OH) was utilized
at a concentration of 87 p.g/ml. The operating conditions were as those
previously described under Membrane Reactor. At intervals during fil-
tration, 2-ml aliquots were removed from the cell and the specific activities
measured and are reported as p M maltose/min-mg protein. The protein
concentration was determined by Folin phenol reagenti3 and using bovine
serum albumin as standard.
1300 AZHAR AND HAMDY
Carbohydrate Analysis
Reducing sugar determination
The reducing sugars produced during the enzymatic hydrolysis of
freeze-dried sweet potato were determined by the BernfeldI4 method
using maltose as a standard. This method is based on the ability of the
reducing sugars to reduce 3,5-dinitrosalicylic acid.
Total carbohydrate determination
Filtrate collected from an ultrafiltration cell was checked for total car-
bohydrate content using Anthrone reagent and glucose as a standard.
Sugar standards (glucose, maltose), obtained from Fisher Scientific Co.
(Pittsburgh, PA), were dried overnight under vacuum at 65C before being
used.
Alcoholic Fermentation
The filtrate collected from the thin-channel membrane reactor was con-
centrated to a total carbohydrate content of 77.01 mg of glucose equiv-
alent/ml, using a rotavapor (model VESOGD, Rincon Instrument Com-
pany, Inc., Greenville, IL). An aliquot of this concentration (250 mi) was
then used as a substrate for alcoholic fermentation (pH 6.7) conducted
at room temperature after supplementation with 1.68 g of Bacto yeast
extract and 1 .O g of ammonium phosphate monobasic. The medium was
inoculated (5%) with a 24-hr-old culture of Saccharomyces cerevisiae
(Stock culture, Food Science Department, University of Georgia).
Alcohol Determination
An aliquot (25 ml) was removed aseptically from the fermented liquor
and diluted with 25 ml of distilled water, adjusted to pH 7.0-8.0 with
O.1N sodium hydroxide, and distilled using a Leibig condenser. The al-
cohol content of distillate collected (25 mi) was then determined by re-
fractive index measurements, using a Brick-Pheonix Differential Refrac-
tometer (Pheonix Precision Instrument Co., Philadelphia, PA). Absolute
ethyl alcohol (U. S. Industrial Chemical Co., Louisville, KY) was used
as a standard.
RESULTS AND DISCUSSION
Stirred Cel l System
The filtration rates during continuous enzymatic hydrolysis of sweet
potato slurry in a cell reactor under continuous magnetic stirring with and
without added enzyme, p-amylase, are shown in Figure 2. A steady state
was not reached in the reactor (with or without added enzyme) as evi-
ALCOHOLIC FERMENTATION OF SWEET POTATO
1301
2 24
5
E
-
W
a:
z
0
4
a:
2 18
F 12
5
G 6
220
\
W u)
0
2 140
W -I
0
5
5
60
0 25 50 75
TIME ( h )
Fig. 2. The effect of adding crystalline sweet potato p-amylase (87 pg proteinhl reaction
mixture) on the filtration rate (ml/hr) and on the reducing sugar content (measured as maltose)
in the filtrate at 50C, IS psi, in the stirred cell system. Enzyme added ( 0) ; no enzyme added
(J ).
denced by the continuous decline in the filtration rate during the entire
experiment. However, the filtration rate was slower in the absence of
added enzyme, reaching a level of 7.8 ml/hr (33% of initial value) after
36 hr. On the other hand, the addition of p-amylase to the reactor cell
under the same conditions resulted in a much slower change in the fil-
tration rate and a value of 16 ml/hr (72% of initial value) after 66 hr of
continuous filtration was evident. This higher filtration rate, when p-
amylase was added, was due to the acceleration in the hydrolysis of starch
leading to the maltose and limit dextrins. The latter may cause the for-
mation of a gel layer at the membrane surface. The decline in filtration
rate with time in membrane reactors has been reported by Butterworth
et a1.,I6 Closset et al.,I7 and Tachauer et a1. Closset et al.,I7 studying the
continuous hydrolysis of starch by the enzyme P-amylase (isolated from
sweet potato) at different starch and enzyme levels, reported that the
decline in filtration may be due to the formation of a concentrated gel
layer of limit dextrins at the membrane surface caused by the effect of
concentration polarization and to some degree by the aging of the starch
solution. Tachauer et al. studied the continuous starch hydrolysis in
membrane reactor by a pure commercial preparation of a- and p-amylase
mixture, and reported that the permeation rate decreased with time and
appeared to level off after 50-60 hr. These authors stated that the mixture
of a- and P-amylase had a more pronounced effect on permeation rate,
possibly due to the random degradation of the starch molecules by the
mixture of enzymes as compared to P-amylase alone which leads to end-
wise step hydrolysis of starch. Again Tachauer et al. stated that by
creating a mixture of small dextrin molecules, gel formation was inhibited
I302 AZHAR AND HAMDY
in direct proportion to the p-amylase level in the enzyme mixture. Recent
studies by Madgavkar et al.3 showed that glucoamylase enzyme used for
continuous hydrolysis of starch in a membrane reactor reduced the for-
mation of the gel layer and offered higher and more stable performances.
These authors also reported that the highly active glucoamylase (EC.3.2.1.3)
successfully degraded the limit dextrins to glucose and prevented decline
of the permeation rate with time.
Use of sweet potato powder as a substrate could have compounded the
gel layer formation due to the presence of other polysaccharides such as
cellulose and hemicellulose that could not be hydrolyzed by enzyme a-
and p-amylases. Selection of sweet potato powder over a purified starch
obtained from sweet potato for enzymatic hydrolysis was based on the
following considerations: 1) cost in extracting starch from sweet potato,
and 2) presence of highly active saccharifying enzyme @-amylase in sweet
potato tubers.
Figure 2 also illustrates the reducing-sugar content .of the filtrate, meas-
ured as maltose, in the reactor cell with and without added enzyme p-
amylase. When no enzyme was added, the maltose formed was attributed
to the intrinsic p-amylase presence in sweet potato. In this case a rapid
decline in maltose content with time was noted. Addition of enzyme
resulted in higher maltose content in the filtrate which also finally de-
creased with time. This allowed the filtration to be carried out for a much
longer time (70 hr) with only 19% decrease in maltose content.
It should be emphasized that even though the addition of the enzyme,
p-amylase, increased both the filtration rate and the maltose content in
the filtrate, a steady state was not reached. This was mainly due to the
amylopectin component of starch in sweet potato. The P-amylase hy-
drolysis of amylopectin finally results in the formation of limit dextrins
which accumulate at the membrane surface, causing the decrease in both
the filtration rate and maltose content in the filtrate. This is in agreement
with other studies on continuous hydrolysis of starch by P-amylase in a
membrane When glycoamylase, capable of hydrolyzing limit
dextrins to glucose, was used under similar experimental conditions to
those of Closset et a1.I7 and Tachauer et al., however, steady-state values
of the filtration rate and pseudo-steady-state values of the performance
index were ~btai ned. ~
Maltose content increased in the filtrate at the early state of filtration
(Fig, 2). This may be due to the accumulation of maltose in the cell as
the result of enzymatic hydrolysis of starch. However, such an increase
finally ceased when the filtration rate diminished.
Thin-Channel System
Filtration rates obtained during the continuous enzymatic hydrolysis
of sweet potato slurry in a thin-channel reactor are presented in Figure
ALCOHOLIC FERMENTATION OF SWEET POTATO I303
0 7 14 21
TIME (h)
The effect of adding crystalline sweet potato P-amylase (87 pg protein/ml reaction
mixture) on the filtration rate (mVhr) and on the reducing-sugar content (measured as mal-
tose) in the filtrate at 50C, I5 psi, in the thin-channel system. Enzyme added ( 0) ; no enzyme
added ( g) .
Fig. 3.
3. A rapid decline in the filtration rate was observed during early stages
in both systems (i.e., with and without added enzyme @-amylase). This
was followed by a slow decline upon further filtration. Such an initial
decrease in filtration rate (29.8 and 13.9 ml/hr.hr with and without added
enzyme, respectively) is attributed to the formation of a concentrated
layer at the surface of the cell membrane consisting of starch and limit
dextrins plus other nonhydrolyzable polysaccharides. The thickness of
this layer increased with longer filtration time and seemed to be affected
by the pressure applied to the system, level of substrate, enzyme, and
temperature. Filtration for a longer time (14 hr), however, resulted in a
relatively slow decrease in filtration rate (2.7 and 2.9 ml/hr.hr with and
without added enzyme, respectively). Such a slow decrease in filtration
is probably due to the agitation of the reactor cell contents caused by the
pump preventing further rapid increase in the concentrated layer. Faster
hydrolysis of starch, in the presence of added enzyme and the conse-
quently higher rate of limit dextrins accumulation at the membrane sur-
face, resulted in the sharp decline in filtration rate in this case.
It is postulated that the filtration rate is affected by three main types
of particles:
1 ) large size particles such as starch, cellulose, etc., which accumulate
during filtration, decreasing the filtration rate;
2) intermediate size particles which are partially hydrolyzed starch
molecules by the enzymes a- and @-amylase and have little effect on the
filtration rate;
I304 AZHAR AND HAMDY
3) limit dextrins which cannot be hydrolyzed any further by the enzyme
amylases and are small enough to penetrate into the membrane pores,
thus effectively reducing filtration.
In the absence of added enzyme, the large size particles were the main
obstacles to filtration, particularly at the early stages. However, starch
hydrolysis by the intrinsic amylase enzymes present in sweet potato re-
sulted in the formation and accumulation of limit dextrins, causing a gel
layer. Addition of the P-amylase enhanced the degradation of large size
particles (starch) into intermediate sizes which in turn exert little effect
on filtration, resulting in a higher filtration rate especially at the early
stages. The data obtained on filtration rate measurements seemed to fit
best into an exponential equation [eq. (l)] with a constant filtration rate
at infinite time.
Y = b ep Kr + a
(1)
where a is the constant filtration rate (filtration rate at t-); b is the (fil-
tration rate at t = 0) - a; K is the time constant; t is the time; Y is the
filtration rate.
Several constant filtration rates were assumed and corresponding val-
ues for intercept, slope, and correlation coefficient values were calculated
(Table I) using eq. (2):
In(Y - a) = In b - Kt
(2)
Filtration rates of 60 and 40 ml/hr (with and without added enzyme,
respectively) with the highest correlation coefficient values were chosen
and corresponding straight lines of eq. (2) are shown in Figure 4(a). The
higher constant filtration rate in the presence of added enzyme shows
that the filtration rate remained higher in such a system even upon pro-
longed filtration. Thus, the behavior of the thin-channel membrane reactor
can be postulated as follows:
1) Filtration rate declines rapidly during the early stages of filtration
as the result of a concentrated layer formation at the cell membrane
surface.
2) A rather slow decline in filtration rate then follows which is due to
partial removal of the concentrated layer at the cell membrane surface
by the circulating pump.
3) An equilibrium state may finally be reached in which the concentrated
layer does not grow any further due to circulation of the cell contents by
the pump.
4) Presence of added enzyme @-amylase hinders the formation of this
concentrated layer, resulting in a higher filtration rate which continues
even upon prolonged filtration.
Figure 3 also summarizes the data obtained on the reducing carbohy-
drate content of filtrate, during the continuous enzymatic hydrolysis of
ALCOHOLIC FERMENTATION OF SWEET POTATO 1305
TABLE 1
Calculations for the Intercept, Slope, and Correlation Coefficient of the
Straight Line [ MY - a ) =I nb - Kt ] , Assuming Different u Values, in the
Thin-Channel System with and without Added Sweet Potato @-Amylase
Assumed
a Intercept
Correlation
Slope coefficient
Enzyme added 59 5.1730
60 5.1785
61 5.1859
No enzyme added 39 4.8055
40 4.8047
41 4.8047
-0.1633 0.99907
-0.1684 0.99917
- 0.1740 0.99909
-0.1107 0.99136
-0.1139 0.99143
-0.1173 0.99140
sweet potato slurry, using the thin-channel membrane reactor. Reducing
carbohydrate content of the filtrate declined steadily upon filtration in
both systems (i.e., with and without added enzyme P-amylase). This was
due to a decrease in filtration rate which, as mentioned earlier, is attrib-
uted to formation of the concentrated layer, consisting of starch, non-
hydrolyzable polysaccharides, and limit dextrins at the cell membrane
surface. In the absence of added enzyme, reducing carbohydrates were
formed by the action of intrinsic amylases of sweet potato upon starch,
6
-
0
$ 4
1
2
z
14
0
P a
I ?
$12
\ c
._
a
w 10
= 0 10 2 0 30
9 ' 1, , , I (b) 1 -I 0
TIME ( h)
Fig. 4. (a) The linear relationship between the values of the filtration rate minus the
calculated constant filtration rate [In(y - a) ] and time (hr) in the thin-channel system with
and without added sweet potato @-amylase. Calculated constant filtration rates (a) are 60
and 40 mVhr with and without added enzyme, respectively. Enzyme added ( 0) ; no enzyme
added ( 0) . (b) The effect of filtration time (hr) on the retention of sweet potato P-amylase
(87 kg protein/ml reaction mixture) specific activity (reported as mmol maltose/min.mg
protein) in the thin-channel system.
1306 AZHAR AND HAMDY
whereas the presence of added enzyme as expected resulted in an additive
effect leading to a higher value during filtration.
Retention of Enzyme Activity in Membrane Reactor
Figure 4(b) illustrates the data obtained for the specific activities of
enzyme P-amylase (reported as mmol maltose/min.mg protein) during
continuous enzymatic hydrolysis of potato starch in a thin-channel sys-
tem. Specific activities declined gradually, reaching a level of 1.2 mmol
maltose/min.mg protein (90% of the initial value) after 30 hr of continuous
filtration. This decline is possibly due to the decay of enzyme P-amylase
molecules during the course of filtration. Use of sweet potato as substrate
may compensate for such loss in enzymatic activities due to the presence
of the intrinsic and highly active P-amylase of sweet potato. The calcu-
lated biological half-life for enzyme P-amylase [Fig. 4(b)] was 157 hr.
Enzyme leakage through the membrane during filtration was reported
by Butterworth et a1.I6 These authors, using a-amylase (MW = 50,000)
for starch hydrolysis in a membrane reactor with a molecular weight
cutoff of 10,000, reported that about 35% of the original enzyme concen-
tration passed through the membrane during four days of continuous
filtration. These authors also observed similar behavior when the enzyme
glucoamylase was used under the same conditions.
The possibility of enzyme passage through the membrane in this study
using thin-channel membrane reactors is considered to be trivial due to
the large difference in the molecular weight cutoff of the membrane
(10,000) and the molecular weight of the enzyme @-amylase (197,000 &
6000).
Alcoholic Fermentation
Data collected on ethyl alcohol produced upon the fermentation of
filtrate showed a rapid rate of alcohol production during the first four
days of fermentation, resulting in an alcohol level of 3.5%. Fermentation
for longer time resulted only in a slow increase in alcohol content which
leveled off at 4.2%. The amount of sugar fermented was determined and
the efficiency of the fermentate calculated and the results showed that
95% of sugar present was fermented by the yeast to a final ethyl alcohol
content of 4.2% with an efficiency of 88.4%.
CONCLUSION
A thin-channel system showed a superior performance compared with
a stirred cell system for immobilizing the sweet potato intrinsic and crys-
talline P-amylase during hydrolysis of sweet potato slurry. Enzymatic
activity of @-amylase retained in a thin-channel system reached 90% of
initial value after 30 hr of continuous filtration which corresponds to a
ALCOHOLIC FERMENTATI ON OF SWEET POTATO
1307
biological half-life of 157 hr. Alcoholic fermentation of the filtrate resulted
in 4.2% alcohol. This represents a fermentation of 95% of sugars present
with an efficiency of 88%.
The authors thank Dr. S. J . Harmon, Horticulture Department, University of Georgia at
Tifton, Georgia, for supplying them with sweet potato and Dr. R. Toledo, Department of
Food Science, University of Georgia at Athens, Georgia, for assisting with mathematical
interpretation of the results.
References
I . S. P. ONeill, J . R. Wykes, P. Dunnill, and M. D. Lilly, Biotechnol. Bioeng. , 13, 319
2. E. Tachauer, J . T. Cobb, and Y . T. Shah, Biotechnol. Bioeng., 16, 545 (1974).
3. A. M. Madgavkar, Y. T. Shah, and J . T. Cobb, Biorechnol. Bioeng., 19, 1719 (1977).
4. J. J. Marshall and W. J . Whelan, Chem. Ind. , 25, 701 (1971).
5 . V. H. Edwards, Biotechnol. Bioeng. Syrnp. , 5, 321 (1975).
6. D. L. Miller, Biotechnol. Bioeng. Symp. , 5, 345 (1975).
7. A. E. Humphrey, Biotechno/. Bioeng. Sy mp . , 5, 49 (1975).
8. C. Ratledge, Ann. Rep. Ferment. Proc. , 1, 49 (1977).
9. M. W. Hoover and S . J . Harmon, Food Techno/., 21, 1529 (1967).
10. H. J. Deobald, V. C. Hasling, E. A. Catalano, and T. A. McLemore, Food Techno/. .
1 I . AOAC, Official Methods of Analysis (Assoc. Official Anal. Chem., Washington,
12. K. V. Giri, J . Ind. Chem. Soc. , 11, 339 (1934).
13. 0. L. Lowry, N. J . Rosebrough, A. L. Farr, and R. J . Randall, J . Biol. Chem. , 193,
14. P. Bernfeld, Amylases a and p, in Methods in Enzymology, S . P. Colowick and
15. R. Dreywood, Ind. Eng. Chem. , 18, 499 (1946).
16. T. A. Butterworth, D. I. C. Wang, and A. J . Sinskey, BiotechnoL Bioeng. , 12, 615
17. G. P. Closset, J . T. Cobb, and Y . T. Shah, Biotechnol. Bioeng., 16, 345 (1974).
( I97 1).
23, 826 (1969).
D.C., 1975), 12th ed.
265 (1951).
N. 0. Kaplan, Eds. (Academic, New York, 1955), Vol. I , p. 149.
( 1970).
Accepted for Publication October 27, 1980