ENZYMATIC CONVERSION OF CORN COBS TO GLUCOSE WITH

TRiCHODERMA VIRIDE FUNGUS AND THE EFFECT ON

NUTRITIONAL VALUE OF THE CORN COBS 1,~,3
J. w. Ward 4 and T. W. Perry
Purdue University, West Lafayette, IN 47907

Summary

Three experiments were conducted with a

cellulase-producing fungus, Trichoderma viride,
on corn cobs. A microbial plating technique
was used to determine the capacity of Trichoderma viride to exert cellulase activity on various levels of noble agar (1 and 2%) and corn
cob substrate concentrations (2, 3, 4 and 5%).
A significant decrease in cellulase activity was
observed when noble agar was increased from
1 to 2%. Varied corn cob substrate concentrations had no effect on degree of clearing around
wells (P<.05), indicating that cellulase permeability is not affected by corn cob concentrations at the levels used in this study. A randomized factorial design measured the capacity
of Trichoderma viride to degrade corn cobs as
indicated by glucose production in in vitro fermentation. The five factors studied were:
(1) two temperatures, 23 and 37 C; (2) two
particle sizes of corn cobs, coarse ground
through a 12-mm screen (as feed milled) and
fine ground through a 1-mm screen; (3) two
ratios of corn cobs to water (1:4 and 1:6 by
weight); (4) nine intervals (1 to 9 weeks); and
(5) inoculation of cobs with Trichoderma
viride vs no inoculation. Glucose production
was maximized (P<.01) in wk 4 with a combination of finely "ground corn cobs inoculated
with Trichoderma viride, a temperature of 23
C and a 1:6 ratio of corn cobs to water. One rat
growth trial and one sheep digestibility trial
were conducted to investigate nutritional

effects of feeding corn cobs treated with the

cellulase-producing fungus, Trichoderma viride.
In the rat growth trial, untreated or treated
ground corn cobs replaced 0, 60, 80 or 100%
of the glucose of the control diet. At each of
the substitution rates, use of treated corn cobs
improved (P<.05) gain over that obtained when
untreated corn cobs were used. In the lamb
digestion trial, treated and untreated cobs were
compared in a diet co_ntaining 40% ground corn
cobs. Use of treated corn cobs resulted in increases (P<.01) in digestibility of dry matter
(4.8%), N-free extract (9%) and total digestible
nutrients (18%), decreases (P<.01) in digestibility of crude fiber (41%) and ether extract
(8.8%), and no significant change in digestibility of crude protein.
(Key Words: Trichoderma Viride, Rats, Lambs,
Digestion Trial, Corn Cobs, Glucose.)
Introduction

Nutrients in a diet are available for utilization by animals on the basis of the availability
of enzymes necessary for digestion. The animal's ability to utilize a given feedstuff is reflected in animal performance (Church and
Johnson, 1972). An organism has been identified which produces the cellulase enzyme
(Brandt et al., 1972; Sternberg, 1975; Reese,
1976). This organism, Trichoderma viride (TV),
is capable of producing relatively high levels of
cellulase (Mandels et al., 1971, 1974; Mandels,
1975).
Utilization of a fungal organism like TV in
predigestion of cellulosic agricultural residues
would be of great economic benefit to the
1Journal Paper No. 8445, Purdue Agr. Exp. Sta.,
agricultural community. Beneficial effects of
Dept. ofAnim. Sci.
2From the M.S. thesis of the senior author.
the TV organism could increase greatly the
3Appreciation is expressed to Mrs. Alice Bales efficient utilization of agricultural residues in a
Crum for laboratory assistance in the conduct of the
complete diet. Effects of TV on cellulosic resiresearch.
dues like corn cobs have not been previously
4Present address: Dept. of Dairy Sci., Oklahoma
State Univ., Stillwater 74074.
studied.
609
JOURNAL OF ANIMAL SCIENCE, Vol. 54, No. 3, 1982

610

WARD AND PERRY

Objectives of this study were (1) to investigate the ability of TV to produce the cellulase
enzyme complex when corn cobs are utilized
as the substrate, (2) to determine glucose production from such enzymatic breakdown of
corn cobs, and (3) to determine the feeding
value of corn cobs treated with TV.
Experimental Procedure

Phase I. Enzymatic conversion o f cellulose.

The TV QM 9123 s organism was cultured and
maintained at 23 C with Sabourand Dextran
sugar (acid pH). All materials used were sterilized to prevent possible microbial contamination.
In Exp. 1 and 2, cellulase production was
verified by using a microbiological plating
technique (G. W. Rose, personal communication). A plate glass pan resembling a square
petri dish was used. The plates were filled with
an assay medium of various concentrations of
a noble agar-corn cob mixture, and 18 wells
(holes) per plate were made in the agar. The
wells were then filled with a liquid solution
from a culture of TV. Seven days after filling,

s Obtained from the U.S. Army Quartermaster

Corps.

cleared areas around the wells were measured in

centimeters for cellulase activity.
Corn cobs were powdered in a Waring blender and ground through a 200-/~m screen.
In Exp. 1, the cellulase substrate, corn cobs,
was added at 2% by weight to a 1 or 2% noble
agar solution. In Exp. 2, corn cobs were added
at 2, 3, 4 or 5% by weight to a 2% solution of
noble agar.
In Exp. 1 and 2, the plate wells were filled
with .1 ml of TV liquor in varied dilutions of
liquor to sterilized water (undiluted, 1:1, 1:4,
1:16, 1:32 and 1:64). The TV liquor was prepared from a culture grown on a mixture of
200 g of corn cobs and 1,200 g of sterilized
water. This mixture was sterilized and inoculated with TV spores and cultured at 23 C for 4
wk, after which the mixture was strained
through cheesecloth. The liquid portion
(liquor) contained the cellulase enzyme in
u n k n o w n concentrations.
In Exp. 3, corn cob degradation by TV was
measured by glucose production. Pilot research
indicated that cellulosic breakdown was affected by temperature, substrate particle size
and time exposed to TV. A factorial designed
experiment was used to measure glucose production by TV. Five factors were studied: (1)
two temperatures, 23 and 37 C; (2) two particle sizes of corn cobs, coarse ground through a

TABLE 1. COMPOSITION OF RAT DIETS (EXP. 4)

Ingredient

Control

60U

Glucose replacement with cobs (U and T)a, %

60T
80U
80T
IOOU

100T

%
Supplementb
Glucose
Corn cobs c (untreated)
Corn cobs (treated)

30.5
69.5

Dry matter
Crude protein
Crude fiber
Ether extract
N-free extract

92.0
14.8
6.3
10.8
65.3

30.5

27.5

30.5
27.5

42.0

30.5
13.5
56.0

42.0

30.5
13.5

30.5

30.5

69.5
56.0

69.5

Chemical analysisd
92.1
16.4
19.1
5.9
55.2

93.4
16.1

17.7
5.7
57.1

91.8
17.1
24.2
6.1
48.7

93.5
16.4
20.2
5.7
54.3

91.5
16.1
29.9
5.9
44.5

94.1
16.0
25.1
5.5
50.5

au = untreated corn cobs; T = corn cobs previously inoculated with Trichoderma viride.
bsupplement: isolated soybean protein, 49.1%; corn soil, 16.4%; alphacel, 16.4%; CaCO3, 1%; rnonosodium
phosphate, 1.6%; lysine, .4%; methionine, 1.4%; vitamin fortification, 7.2%; and mineral salt, 6.5%.
CIFN 1-02-783.
dDry matter basis.

ENZYMATIC CONVERSION OF CORN COBS

12-mm screen and fine ground through a 1-mm
screen; (3) two ratios of corn cobs to water
(1:4 and 1:6, by weight); (4) nine time periods
by weeks; and (5) inoculation of corn cobs with
TV vs no inoculation.
A total of 288 Erlenmeyer flasks (500 ml)
were used in Exp. 3, and the various concentrations of water and corn cob particle sizes were
added to the treatments. The flasks were then
sterilized at 120 C for 20 rain and cooled to 23
C. After cooling, one-half of the flasks were
inoculated with TV spores and incubated at
their respective temperatures.
Four additional Erlenmeyer flasks with
treatment combinations of corn cobs and water
were autoclaved and dried to a constant weight.
A complete analysis for glucose concentration
was conducted (table 5; AOAC, 1970). This
analysis served as a base line for glucose production. All treatment flasks were analyzed for
glucose according to procedures outlined by
Whistler et al. (1963) and Southgate (1976).
Statistical analysis of the experimental data
was conducted according to procedures o f
Hicks (1973) and Anderson and McLean (1974)
with significant differences between means
determined by the Newman-Keuls Sequential
Range Test (Keuls, 1952).
Phase II. Feeding study. The feeding value of

611

ground corn cobs treated with TV fungus or

not treated was studied in a growth trial with
weanling rats and in a digestion trial with
lambs.
Corn cobs were ground through a 1-mm
screen and autoclaved for 20 min at 120 C. The
ground corn cobs were placed in plastic-lined
208qiter drums. Sterilized water was added to
the corn cobs to achieve a 1 to 6 ratio of corn
cobs to water (by weight). TV spores were
mixed t h r o u g h o u t the corn cobs in the drums
and the drums were sealed at 23 C. After 4
wk, the inoculated corn cobs were dried at 55 C
to a constant weight. Untreated corn cobs were
treated in the same way except there was no
inoculation with TV spores.
Fifty-six 3-wk-old white male rats were
used in Exp. 4. Isolated soybean proteinglucose (table 1), fortified, served as the control
di.et, which was included to serve as a positive
marker for normal growth performance, in
addition, untreated and treated corn cobs replaced 60, 80 and 100% of the glucose to make
up seven experimental diets. Performance was
measured by rat daily gain. Experimental diets
were formulated to meet the nutritional requirements established by the NRC (1972) for
laboratory rats. Complete experimental diet
formulations and analysis of diets and the sup-

TABLE 2. COMPOSITIONOF LAMB DIETS (EXP. 5)

Unt re a t e d

Ingredient

Treated
corn
cobs

corn
cobs

30.0
20.0
8.75
1.0
.25

Corn, grain (IFN 4-02-879)

Soybean, seeds (IFN 5-04-612)
Sugarcane, molasses (IFN 4-04-696)
Dicalcium phosphate
Iodized salt
Corn, cobs (untreated; IFN 1-02-783)
Corn, cobs (treated) a

30.0
20.0
8.75
1.0
.25
40.0

Dry matter
Crude protein
Crude fiber
Ether extract
N-free extract

95.4

95.1

17.3
14.3
1.5
57.0

19.5

40.0
Chemical analysisb

aTreated with Trichoderma viride.

bDry matter basis.

9.8
1.6
63.9

612

WARD AND PERRY

pleriaent were conducted according to AOAC

(1970) procedures (table 1).
The rats were housed in individual growing
cages on two-sided racks containing four levels.
Rats were randomly assigned to a treatment
and level, eight rats/treatment. The experiment
consisted of a 3-wk growth trial9
Twelve crossbred ram lambs weighing 30 kg
each were used in a digestion trial (Exp. 5)
designed to determine the effect of TV on
cellulose breakdown. A switchback design was
used. Each test consisted of a 10-d adjustment
to the diet followed by a 10-d period in the
collection crates. Three days were allowed for
adjustment to the crates, after which there was
a 7-d collection period. Daily records were kept
of feed offered, weighback and fecal output.
The corn cobs utilized in this experiment
were treated like those used in Exp. 4. Cornpositions and chemical anlayses of the diets are
summarized in table 2.
Total feces were weighed daily and a 25%
sample was composited and frozen9 Composited feed and fecal samples were dried to a constant weight at 55 C, ground in a Wiley mill and
sampled for laboratory analysis. Feed and fecal
samples were analyzed for crude protein, crude
fiber, dry matter, fat and N-free extract in
triplicate (AOAC, 1970).
Statistical analysis of experimental data was
made according to procedures of Hicks (1973)
and Anderson and McLean (1974), with significant differences between means determined by
the Newman-Keuls Sequential Range Test
(Keuls, 1952).

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Results and Discussion

Phase I. Enzymatic conversion of cellulose9

In Exp. 1 and 2, it was presumed that glucose
production might not occur at upper dilution
levels of liquor to water9 When cellulase activity
was apparent at the higher dilution levels, this
indicated the amount of cellulase produced by
TV and also its relative enzymatic activity.
In Exp. 1, clearing was recorded at all dilution levels except 1:64 (table 3). As the concentration of agar increased, there was a decrease (P<.05) in degree of clearing around the
wells. Clearing was not only a response to apparent enzymatic activity of the cellulase enzyme, but also to its capacity to diffuse
through the noble agar-corn cob substrate.
In both Exp. 1 and 2, as the dilution rate increased, there was a decrease (P<.05) in clearing around the wells (tables 3 and 4). Even so,

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614

WARD AND PERRY

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ENZYMATIC CONVERSION OF CORN COBS

the fact that the cellulase activity was yet apparent at 1:32 is interesting, indicating TV was
producing cellulases in concentrations that,
even when the liquor was diluted, degraded
fibrous matter.
In Exp. 2, no differences (P>.05) were observed among the four corn cob levels studied
(2, 3, 4 and 5% by weight). No differences were
observed in the capacity of the enzyme to
diffuse or degrade this cellulosic material (table

615

+1

4).
Results of Exp. 3 are presented in table 5.
There were differences (P<.01) in glucose production due to differences in temperature,
time, particle size, corn cob:water concentration (w/w) and inoculation with TV vs no
inoculation. The results are in ~greement with
those of Katz and Reese (1968), Mandels et al.
(1971), Reese (1976) and Sternberg (1976)9
There appeared to be a temperature effect
(P<.01). TV did not grow at the higher temperature of 37 C (Hulme and Shields, 1971).
The fact that TV was able to grow rapidly and
productively at 23 C might render the organism
more useful for agricultural use due to its lack
of need for supplemental heating 9
The effect of grinding and screening of the
cobs may be to reduce the native crystalline
state of the cellulosic fraction of the corn cobs
and increase the reactive surface area (Ghose
and Kostick, 1970)9 Fine grinding combined
with sterilization to decrease microbial contamination resulted in a loss of native crystal-

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Figure 1. Glucose production from corn cobs

treated with Trichoderma veride fungus in wk O, expressed as percentage of crude fiber fraction. Upper 2
curves are with finely ground (l-ram screen) and lower
2 curves are with Coarsely ground corn cobs (12-mm
screen), at 23 C.

+1

616

WARD AND PERRY

TABLE 7. EFFECT OF TREATMENT OF CORN COBS WITH TRICHODERMA V I R I D E
ON NUTRIENT DIGESTIBILITY BY LAMBS (EXP. 5)
Untreated

Treated

Item

cobs

cobs

No. o f lambs

12

12

Nutrient digestibility, %
Dry matter
Crude protein
Crude fiber
Ether extract
N-free extract

73.0 a
83.0
49.3 a
91.1 a
75.7 a

76.5 b
82.9
29.2 b
83.1 b
83.3 b

2.1
1.9
2.1
.9
2.7

Calculated total digestible nutrients, %

63.7 a

75.2 b

3,0

SE

a'bvalues in the same row with different superscripts differ (P<.O1).

line character of the cellulose fraction. This apparently increased the enzyme's activity severalfold (Katz and Reese, 1968).
Glucose production was maximized with a
combination of temperature at 23 C, finely
ground cobs and a 1:6 concentration of corn
cobs to water (table 5). Maximum glucose production occurred in the fourth week (figure 1).
The use of organisms to predigest cellulosic
matter seems plausible although the steps and
long-term applications are yet undefined.
Phase II. Feeding study. Differences (P<.05)
in daily gain were observed in Exp. 4 between
untreated and treated corn cobs at 60 and 80%
replacement of glucose with corn cobs (table
6). At 100% replacement of glucose (glucose
represented 69.5% o f the total diet), all rats fed
the untreated corn cob diet died. Death loss
was probably due to starvation caused by the
high amount of corn cobs (Franz, 1959).
As corn cob percentage in the diet increased,
more sorting and feed wasting were observed.
Apparently, dilution of the rat diets with a
fairly inert material, such as untreated corn
cobs, resulted in more sorting of the feed to
satisfy caloric needs (Adolph, 1947). The extreme dilution with untreated corn cobs in the
diets also contributed to increased incidence of
death, whereas none died when fed comparable ratios of treated corn cobs. These resuits are in agreement with those of Larsson
and Strom (1957) and Franz (1959).
An indication of the effectiveness of TV on
fiber breakdown is that diets containing treated
corn cobs contained lower levels of crude fiber

and higher levels of N-free extract (table 2).

In Exp. 5 apparent digestibility of a lamb
diet containing 40% ground corn cobs was affected by the treatment of the corn cobs with
TV. Apparent digestibilities of dry matter and
N-free extract were increased (P<.01; table 7)
whereas digestibilities of crude fiber and ether
extract were decreased (P<.01). Protein digestibility of the diet was not changed because of
treatment.
Chemical analyses of the two complete lamb
diets showed interesting differences due to the
different treatment o f the cobs (table 2). Treatm e n t of the ground corn cobs with TV resulted
in nearly 13% more protein in the total diet.
Apparently, the additional protein produced
during the treatment with TV was just as
digestible by the lambs as that of the remainder
of the diet because protein digestibility was
similar for the two diets. It could be postulated
the higher protein level of the treated cob diet
could contribute to the higher dry matter digestibility of the diet, but a protein level of 17.3%
would be considered to be more than adequate
for optima] digestion of the diet.
The crude fiber content of the diet containing ground corn cobs treated with TV was 31%
less than for the diet containing ground corn
cobs that had not been treated. This would
indicate that TV fungus was effective in acting
on the crude fiber portion of the ground corn
cobs. Autrey et al. (1975) reported that corn
silage that had been stored with TV contained
13% less cellulose than comparable corn silage
that had not been treated.

ENZYMATIC CONVERSION OF CORN COBS

C r u d e f i b e r digestibility was 4 0 % l o w e r for
t h e diet t h a t c o n t a i n e d t h e t r e a t e d cobs. This
e f f e c t c o u l d b e e x p l a i n e d b y t h e t r e a t m e n t of
t h e c o b s w i t h T V t h a t r e s u l t e d in a b r e a k d o w n
o f t h e m o r e readily d e g r a d a b l e fiber, leaving t h e
less d e g r a d a b l e f i b e r f o r t h e l a m b s d u r i n g t h e
digestibility s t u d y .
C a l c u l a t e d N-free e x t r a c t a n d t o t a l digestible n u t r i e n t s f a v o r e d t h e diet c o n t a i n i n g t h e
t r e a t e d cobs, in spite o f t h e fact t h a t t h e c r u d e
f i b e r p o r t i o n was m u c h l o w e r in digestibility
f o r this diet. T h e c a l c u l a t e d T D N c o n t e n t was
18% h i g h e r f o r t h e diet c o n t a i n i n g t r e a t e d
g r o u n d c o r n cobs.
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