BIOTECHNOLOGY AND BIOENGINEERING

VOL. XIII, PAGES 77-97 (1971)

Utilization of Cellulose from Waste Paper

by Myrothecium verrucaria

DAVID M. UPDEGRAFF, Chemical Division, Denver Research

Institute, University of Denver, Denver, Colorado 80.210

summary
Extensive screening studies on cellulolytic bacteria and fungi led to the selec-
tion of Myrothecium v e m m r i a as the organism producing the maximum rate of
protein biosynthesis from ball-milled newspaper. Studies in aerated stirred-jar
fermentom were carried out to determine the conditions for maximum protein
synthesis rate and maximum final protein concentration. The optimum aeration
rate was 250 to 374 mM of oxygen at 300 to 400 rpm stirring rate. The pH
optimum was broad, from 3.9 to 6.5. Urea at 0.03% and yeast autolysate a t
0.1% stimulated growth rate and protein production. The, maximum rate of
protein biosynthesis and the maximum protein yield were 0.3 glliterjday and
1.42 g/liter, respectively, from medium G3 with 4% ball-milled newspaper. The
final product, obtained by evaporation of the total culture, was 33.7 g from one
liter of medium which originally contained 40 g of ball-milled newspaper and 11.3
g of other dissolved materials. The protein content of this final product was
3.3 g, calculated from total organic N X 6.25 or 1.42 g calculated from the biuret
method. Both the synthesis rate and the final cell yield are below those obtain-
able by growing Fungi Imperjecti, yeasts or bacteria on soluble materials such as
glucose.

INTRODUCTION
Paper constitutes the largest single material in municipal solid
wastes. Not only is most of this material being wasted at present,
but it costs the nation more than 1.5 billion dollars per year just to
collect and dispose of it.' It seems clear, therefore, that research
on processes for recovering useful materials from this waste is of great
practical importance.
Most paper is made from wood pulp. Newspaper is simple ground
up softwood, and has substantially the same composition as wood (40
to SO% cellulose, 20 to 30% lignin, and 10 to 30% hemicelluloses and
xylosans).2 Lignin is highly resistant to microbial degradati~n.~By
77
@ 1971 by John Wiley & Sons, Inc.
78 UPDEGRAFF

contrast, cellulose is relatively rapidly degraded by microbial en-

zymes.2
BACKGROUND INFORMATION
The lignocellulose complex of softwood is attacked only with great
ditIiculty by cellulolytic enzymes. Thus newspaper is relatively re-
sistant to degradation by microorganisms. Paper made from chemi-
cal pulp is much more readily attacked, as the hot sulfurous acid or
alkaline sulfates used in the chemical treatment have partially
delignified the wood pulp.4 Accordingly, we have chosen to carry
out most of our work on newspaper, since any other kind of paper will
probably be more easily degraded.
Reese, Siu, and Levinsonsshowed that many fungi and bacteria are
able to hydrolyze modified cellulose, such as sodium carboxymethyl
cellulose, but few are able to attack native cellulose such as cotton
fibers. From these and other data they developed a general theory
of the dual nature of cellulase enzyme systems. According to this
theory, true cellulolytic organisms, those capable of attacking native
cellulose, produce two or more enzymes. The first, called C1, attacks
native cellulose to break up the aggregates and produce linear chains
of anhydroglucose units. These chains are then attacked by a second
enzyme C,, a,@,1, 4glucanase, which hydrolyzes them to the disac-
charide cellobiose. Cellobiose may then be assimilated directly into
the cell, or may be converted to glucose by B-glucosidase before
assimilation. Mandels and Reese6 found that woody materials, such
as newspaper, were relatively resistant to cellulases unless they were
thoroughly ground by ball-milling.
Han and Srinivasan' have isolated Cellulomonas bacteria, which
produce fair yields of high-quality protein from bagasse, and Callihan
and Dunlaps have studied the economics of producing protein for
animal feed by this process. The studies reported herein represent
an effort to develop a process for converting newspaper and other
waste paper to a high-protein animal feed supplement for mono-
gastric animals by means of fungus fermentation.

MATERIALS AND METHODS

Microorganism
The organism used in the studies reported herein, Myrothecium
verrucaria, was isolated from a soil sample during the course of an
BIOTECHNOLOGY AND BIOENGINEERING, VOL. XIII, ISSUE 1
 WASTE PAPER CELLULOSE UTILIZATION 79

extensive screening program designed to obtain microorganisms cap-

able of producing protein a t a maximum rate from cellulose. More
than 100 samples of soil, compost, decaying vegetation, sawdust,
rotting wood and paper, sewage and sewage sludge were employed as
inocula on many different media with cellulose, ball-milled newsprint
(without ink) or ball-milled newspaper as substrates. A total of 337
cultures were isolated which grew on both cellulose and ball-milled
newspaper.
The culture used herein consistently gave rates of protein synthesis
as high as or higher than any of the other organisms tested, as well as
an equal or larger final protein concentration. The runners-up were
Trichoderma Zignorum and Aspergillus fumisatus. None of the bac-
teria, isolated, even Cellul~nnonas,~
came near the protein yields and
protein synthesis rates obtained from these fungi.
Media and Inoculum
Initial isolations were made from Reese’s medium5 supplemented
with 1% of purified cellulose or ball-milled newsprint or newspaper.
Newsprint, without ink, was obtained from the Denver Post. News-
paper was from the Wall Street Journal purchased locally. The paper
was air dried at ambient temperature and humidity, and cut into
pieces one to two cm square with a hand paper cutter. Approxi-
mately 100 g of paper was placed in a ceramic ball mill (U.S. Stone-
ware Co.) size No. 1, 1.33 gallons capacity, with 125 tubular Burun-
dum grinding balls 13/16 in. 0.d. by 13/16 in. long. The mill was
sealed and placed on a roller type jar mill and rotated a t 288 rpm for
24 hours. This produced a very finely powdered material.
Enrichment cultures were streaked out on regenerated cellulose
agargfor the isolation of cellulolytic organisms. Colonies were picked
and restreaked on plates of the same agar for confirmation of purity.
They were then picked to mineral-salts agar slants containing the
original substrate for stock cultures. Transfer slants for fermenta-
tion studies were made on nutrient agar. Slants of Myrothecium
verrucaria were incubated at 30°C until heavy growth was obtained
(2 to 3 days). Then the sporulated fungus was taken up in 5 ml of
sterile deionized water and was inoculated into 100 ml of Reese’s
medium in a 500 ml shake flask with 1% ball-milled newspaper.
Shaking a t 350 rpm for 2 to 3 days gave a very heavy growth of fungus
with many microconidia. This was employed to inoculate shake
flasks of different media in order to determine the conditions of pH,
80 UPDEGRAFF

temperature, substrate concentration, accessory nutrients and shak-

ing rate which provide for the most rapid rate of growth and protein
synthesis. A series of experiments involving more than 1,0oO shake
flasks was carried out, before proceeding to stirred-jar fermentor
studies, in order to select the best culture, and the best substrate and
medium for this work. The compositions of the most frequently
employed media are listed in Table I.

Procedures
These stirred-jar fungus fermentation studies were carried out in a
New Brunswick Scientific Company Model CMF 314 microferm floor
fermentor consisting of three 14-liter fermentors in a constant tem-
perature bath. The fermentors were equipped with stirring and
aeration devices, automatic foam sensing and defoamant addition
controls, and automatic constant-pH titration equipment. The
entire fermentor, with culture media and all accessory devices and
tubing included, was autoclaved for 30 minutes a t 20 psig for steriliza-
tion. The volume of medium in each jar was 6.57 to 10 liters, and
the temperature was controlled a t 30°C. Air was sterilized by
filtration through a 15 cm by 2.0 cm column of packed sterile ab-
sorbent cotton. Samples were collected from each fermentor every
working day for analytical and pH determinations.
The culture media, type and amount of paper, initial and terminal
pH values, incubation time, stirring rate, aeration rate, inoculum,
protein yield, and cellulose utilized in each of the 12 fermentation
runs are presented in Table 11. The high stirring rate of Run 10
resulted in undesirable build-up of solids on the walls of the fermentor
above the normal liquid level. This problem was caused by foaming.
Foaming was therefore controlled in Runs 11 and 12 by the addition
of 0.9% Hodag FD-82 antifoam on demand by means of the auto-
matic electrical foam sensing and defoamant addition system. The
total amounts of Hodag FD-82 added to each fermentor in Run 11
were 7.8 g, 4.9 g, and 4.8 g, respectively, for Jars 1, 2 and 3. In Run
12 the corresponding amounts were 4.3 g, 5.4 g, and 6.8 g.
Analyses
All fermentation runs were checked periodically for fungus growth
and possible contaminants by phase contrast microscopy. The pH
was determined with a Beckman research model glass electrode pH
BIOTECHNOLOGY AND BIOENGINEERING, VOL. XIII. ISSUE 1
 WASTE PAPER CELLULOSE UTILIZATION 81

TABLE I
Composition of Frequently Used Media

Medium R: (Reese's Medium)

KHsPO4 2.0g
(NHd801 1.4g
Urea 0.3 g
MgSOI * 7Hz0 0.3 g
CaC12 0.3 g
Proteose peptone 1.og
Trace elements solution 1 ml
Distilled water to 1 liter
Trace elements solution
.
MnS04 HtO 1.56 g
FeS04 . 7H20 5.00 g
ZnClt 1.67 g
coc12 2.00g
19% HCl 1.oml
Distilled water to 1 liter
Medium RY
Same as medium R except that yeast autolysate (Amber Labs Inc.,
BYF Series 100) was substituted for the proteose peptone.
Medium F ( P H ~ . ~ ) ( N H ~ (fertilizer
) ~ S O ~ grade) 10 g
NazStOs 0.01 g
Tap water to 1 liter
Medium G (pH 5.3)(NH,)SHPO4, C. P. 10 g
NasS~001 0.01 g
Tap water to 1 liter
Medium H (pH 5.3)(NH4)2S04(fertilizer grade) 5.0 g
(NHJzHPO,, C. P. 5.0 g
Na8203 0.01 g
Tap water to 1 liter
Medium G-1 Medium G + 0.3 g/liter urea
Medium G-2 Medium G + 10 g/liter CaCOI
Medium G-3 Medium G + 0.3g/liter urea + 1.O g/liter
of yeast autolysate (Amber Labs Inc., BYF Series 100)
Medium G-4 Medium G + 0.3 g/liter urea +5.0 g/liter
of yeast autolysate (Amber Labs Inc., BYF Series 100)
 TABLE I1
StirredJar Fermentor Studies with Myrothecium verrucaria Grown on Newspaper

Aeration Rate
%
Fer- mM Cellu-
804 men- Inc. Stirring 0 2 / Protein lose
Run tor Medium Paper. Initial Term. time rate liter/ liter/ Inoculum yield uti-
No. No. (liters) (percent) pH pH (days) (rpm) min hrb (ml) (mg/ml) lized
0

54 3 1 F,10 BMW,2 5.3 3.25" 9 100 2.4 150 R,200 0.48 46

2 G,10 BMW,2 5.3 4.90 9 100 2.4 150 R,200 0.51 67 s
kU 3 H,10 BMW,2 5.3 4.15 9 100 2.4 150 R,200 0.44 62
0
G,10 BMW12 5.3 3.45 12 0 2.4 150 R,200 0.42 59 E!
G,lO BMW,2 5.3 4.65d 12 0 2.4 150 R,200 0.47 62
G, 10 BMW,2 5.2 6.50 12 0 2.4 150 R1200 0.35 64
7
GJO BMW,2 5.3 4.00 14 0 2.4 150 4-200" 0.44 58
G,10 BMW,2 5.3 4.35d 14 0 2.4 150 4-200 0.48 52
G,10 BMW,P 6.5 6.40d 14 0 2.4 150 4-200 0.30 54
2 6 1 G-1,10 BMW,2 5.3 5.50 21 0 2.4 150 R,200 0.22 63
r 2 G-1,10 BMW14 5.3 4.95 21 0 2.4 150 R,200 0.57 57
U
x, 3 G-2,10 BMW12 5.3 5.20 21 0 2.4 150 R,200 0.25 57
U

E 7 1 G-3,10 BMD,2 5.3 5.75 24 100 2.4 150 RY,200 0.25 77

2 G-3,10 BMD,4 5.3 5.30 24 100 2.4 150 RY,200 0.34 74
c.
H 3 G-3,6.57 BMD,8 5.3 5.40 24 100 2.4 228 RY,200 0.63 45
 8 1 G-1,lO BMD,2 5.3 5.65 20 100 2.4 150 RY,200 0.25 70
2 G-1,10 BMW,2 5.3 5.55 20 100 2.4 150 RY,200 0.40 81
3 G-3,lO BMD,2 5.3 5.80 20 100 2.4 150 RY,200 0.36 77
9 1 G-3,10 BMD,4 5.3 5.65 18 100 1.5 94 RY,200 0.33 34
2 G-3,10 BMD,4 5.3 5.55 18 100 3.0 187 RY,200 0.47 46
3 G3,10 BMD,4 5.3 5.35 18 100 6.0 374 RY,200 0.94 61
10 1 G-3,10 BMD,4 5.3 4.50 14 300-400 1.5 94 RY,100 1.19 63
2 G-3,10 BMD,4 5.3 4.50 14 300-400 3.0 187 RY,100 1.27 65
3 G-3,10 BMD,4 5.3 4.55 14 300-400 6.0 374 RY,100 1.36 63
11 1 G-3,10 BMD,4 5.3 4.15 11 400 3.0 187 RY,200 1.19 68
2 G-3,10 BMD,4 5.3 4.10 11 400 3.0 187 RY,200 0.97 63
3 G-3,10 BMD,4 5.3 3.60 11 400 3.0 187 RY,200 1.02 61
12 1 G-3,7.5 BMD,4 5.3 4.05 12 400 3.0 250 RY,150 1.42 62
2 G-4,7.5 BMD,4 5.3 4.15 12 400 3.0 250 RY,150 1.11 51
3 G-1,7.5 BMD,4 5.3 4.40 12 400 3.0 250 RY,150 0.89 52

* Paper BMW = ball-milled Wall Street J o u m l ; BMD = ball-milled Denver Post. The amount given aa % is grams of
paper per 100 ml of culture medium.
Assuming.an average temperature of 25OC and an average barometric pressure of 630 mm Hg.
c pH was adjusted on day 7 to 5.3 by adding sterile 1 N NaOH. Prior to this point pH values, never having been adjusted,
were: 1:3.05; 2:3.55; 3:3.25.
d These samples were pH-controlled by automatic titration.
6 These inocula consisted of 200 ml of culture from run No. 4, refrigerated until use. Jar 1 was inoculated from Jar 1 of run
4, Jar 2 from Jar 2, and Jar 3 from Jar 3.
84 UPDEGRAFF

meter. Protein was determined by the biuret method of Feinsteinlo

as modified by Bode et a1.I’ Cellulose was determined by a semi-
micro method developed by the author.’*
The finalproduct from fermentor Run 10 was isolated by evaporab
ing the entire contents by boiling, with stirring, on an electric hot
plate in a &liter stainless steel beaker. When the total volume was
reduced to 1-2 liters, the material was placed in a pyrex glass dish,
and evaporated to dryness in an oven a t 90-100°C. The dried cake
was then ball-milled into a fine powder for analysis.
Moisture content was determined by the A.O.A.C. method,la*p. s7
after equilibration of the ball-milled material with the atmosphere at
ambient temperature and humidity for several hours. Ash content
was determined gravimetrically by ignition on a Bunsen burner flame.
A suspension of this material, 1 g per 100 ml, waa prepared and an
aliquot analyzed for total protein by the biuret method. Following
centrifugation of this suspension, aliquots of supernatant were
analyzed for soluble protein in a similar manner.

TABLE 111
Analytical.Data on the Dried Ground Harvest from Stirred-Jar 3, Run 10

% (BYWeight)
Harvest Corn Wheat

Moisture content 2.4,2.5 10.6 10.5

Ash content 14, 13 1.53 1.8
Biuret, total protein 2.8,2.9 - -
Biuret, soluble protein 2.1,2.0 -
Ammonia nitrogen 4.60,4.50, 4.53
Organic nitrogen 1.01,1.05 - -
pro^^"' 6.31,6.57 10.3 11.9
Total Kjeldahl nitrogen 5.61,5.58 - -
Nitrate nitrogen <0.0005, <0.0005
Nitrite nitrogen o.Oo01,o.oO01
Total carbohydrates0 29, 31
Soluble carbohydrates 0.99,0.93 - -
Cellulose content 23,22 2.2b 1.8b
Total lipids 2.2,2.3 5.0 2.1

Organic nitrogen X 6.25.

b +
“Fiber” (cellulose lignin).
0 Cellulose used as reference standard.
BIOTECHNOLOGY AND BIOENGINEERING, VOL. XIII. ISSUE 1
 WASTE PAPER CELLULOSE UTILIZATION 85

Ammonia nitrogen and organic nitrogen were estimated by the

A.P.H.A. method,l4,PP. 240-242 and A.O.A.C.,l3#p . l6 respectively. Total
Kjeldahl nitrogen was calculated as the sum of the ammonia and
organic nitrogen values. Nitrate nitrogen was determined colori-
metrically by a brucine rea~ti0n.l~Nitrite nitrogen was determined
colorimetrically by a diazotization method.15
Total carbohydrates were determined on a sample of the material
dissolved in 67% H 8 0 4 by the anthrone reaction.lg Soluble carbo-
hydrates were measured similarly on a centrifuged sample of the
material. Cellulose content was estimated according to the method
established in this laboratory.12 Total lipids were estimated by the
A.O.A.C. method.l3. PP. 197-198 The data are summarized in Table 111.

RESULTS
General
The results in terms of final protein synthesized and amount of
cellulose consumed are summarized in Table 11. The results are also
presented graphically in Figures 1 through 18. Note that experiment
10 was not graphed because of solids build-up problems which ren-
dered many of the protein analyses inaccurate. A study of the tables
and figures leads to the following conclusions.
1. Myrothecium verrucaria grows well on ball-milled newspaper,
actively consumes cellulose and synthesizes cell material containing
protein.
2. The optimum stirring rate for protein synthesis is 300 to 400
rpm, higher stirring rates could not be employed because of excessive
splashing and foaming, which leads to excessive build-up of solids on
the walls of the fermentor above the normal liquid level.
3. The optimum aeration rate depends partially upon stirring rate,
as oxygen transfer increases with increased stirring rate. From 3 to
6 liters/min appears to be optimal a t 300 to 400 rpm. At 100 rpm,
6 liters/min is superior to lower air rates. Again, very high air rates
must be avoided because of foaming and solids build-up problems.
4. The pH optimum was broad, from 3.9 to 6.5.
5. The incorporation of 0.03%urea into the medium G prevented
the pH from dropping below pH 4.8, and maintained it within the
optimum range. Urea also increased the protein yield.
86 UPDEGRAFF

- 0.6
-sm -
E

0
3
0 -

@-Medium F
@-Medium G
@-Medium H
I

TIME, DAYS
Fig. 1. Protein production, stirred-jar run 3.

80-
@ - Medium
F
@-Medlum G

5 - -
@ Medium H
111
2

1
TIME, DAYS
Fig. 2. Cellulose utilization, stirred-jar run 3.

@-Medium F
6-
 WASTE PAPER CELLULOSE UTILIZATION 87

O6 I
-E 0.5
CL
\

0.4
d
w
u
I)

;
n
0.3
z
p 0.2
0-
NO p~ Control
0
a
a 0-pH Controlled at 4.3

0.I
0-pH Controlled at 6.5

O Y 1 I I 1 I

TIME, DAYS
Fig. 4. Protein production, stirred-jar run 4.

6. The incorporation of yeast autolysate in addition to urea greatly

stimulated both growth rate and protein yield.
7. The maximum rate of protein synthesis was obtained in Run 11,
Jar 1. A protein yield of 1.2 mg/ml was obtained in 4 days, at which
time 54% of the cellulose originally present had been consumed, as

@ - ph Controlled ot 4.3
3 20 @ -
phIControlled at 6.5
W
V

2
~
4 6 8
TIME, DAYS
Fig. 5. Cellulose utilization, stirred-jar run 4.
88 UPDEGRAFF

3-
@ - N o pH Control
2,-
IC @-pH Controlled at 6.5 1
L-u.LLLd
1 2 3 4 5 6 7 8 9 1 0 1 1 1 2
TIME, DAYS
Fig. 6. Variation in pH, stirred-jar run 4.

can be observed in Figure 17. From these data, the calculated rates
of cellulose utilization and protein biosynthesis are :
Cellulose consumed, g/liter/day 5.4
Protein produced g/liter/day 0.3
Protein, % yield as
% of cellulose consumed 5.6.
8. The protein yield increases with increasing paper concentration
up to 8 g per 100 ml of medium, the highest level tested.
The highest final protein yields, 1.42 and 1.36 mg/liter (which are
probably the same, within experimental error) were obtained in
stirred-jar No. 1of Run 12 and No. 3 of Run 10, respectively, both in

@-0.03% UREA + 4% Paper

TIME, DAYS
Fig. 7. Protein production, stirred-jar run 6.
BIOTECHNOJAGY AND BIOENGINEERING, VOL. XIII, ISSUE 1
 WASTE PAPER CELLULOSE UTILIZATION 89

@-0.03% UREA + 2% Paper

0-0.03% UREA + 4% Paper
@-I% C O C O +
~ 2 % Paper

TIME, DAYS
Fig. 8. Cellulose utilization, stirred-jar run 6.

medium G3. Jar 3 of Run 10 was a t the highest air rate employed,
374 mM of oxygen/liter/hr, and the highest stirring rate, 400 rpm
a t the start, reduced to 300 rpm after 48 hours. Thus, the oxygen
transfer rate, which is increased by both air rate and stirring rate, is
limiting growth. Higher air rates or stirring rates could not be used
because of problems encountered with foaming and solids build-up
on the walls of the fermentor above the normal liquid level. The
solids build-up problem in Run 10 was so severe that a large propor-
tion of the fermentor solids were clinging to the walls of the fermentor;

-E 12 -
\
ZlO-
d -

0-0.03% UREA + 4% Paper

@ - I *I* CaCOJ + 2% Paper

TIME, DAYS
Fig. 9. Cellulose utilization, stirred-jar run 6.
 UPDEGRAFF

5.8 - @ -0.03% UREA t 2% Poper

- @ -0.03 % UREA + 4% Paper
5.6 - @-I%CoC03 + 2% Poper

TIME, DAYS
Fig. 11. Protein production, stirred-jar run 7.

-
I-

-
@ 8% Paper
I I I I

TIME, DAYS
Fig. 12. Cellulose utilization, stirred-jar run 7.
BIOTECHNOLOGY AND BIOENGINEERING, VOL. XIIE, ISSUE 1
 WASTE PAPER CELLULOSE UTILIZATION 91

- 0 68
~ 0 . 5
1 Aeration Rate Increased-
from 150mM02/l/hr lo
300mM02/l/hr

I I M C , VATS

Fig. 13. Protein production, stirred-jar run 8.

consequently, it was impossible to obtain satisfactory protein analyses

on samples collected during the run, and all of the protein analyses
from day 3 through day 10 were very low-in the range of 0.2 to 0.5
mg/ml. At the termination of the experiment, the solids were
scraped back into the fermentor, and thoroughlyunixed, so that the
final analyses would be accurate.
Run 12 clearly indicated the beneficial effect of the yeast autolysate
in the medium G-3 on protein synthesis. In general, high-protein

5 0 r
= ,
% 40-
2 30k
- 2% Denver Post
.2% Wall Street Journal
@ - 2% Denver Post + 0 I %
Yeast Aulolysate

2-1-
2 4 6 8 10 I2 14 16 18
TIME, DAYS
Fig. 14. Cellulose utilization, stirred-jar run 8.
92 UPDEGRAFF

,,,I
0.7,
0-
Aeration Rate- 94mM02/l/hr
0-
Aeration Rate-I87mM%/l/hr
0-
Aerotion Rote-374mMO2/l/hr
7

0"
W
0
3
0
0
U
a

TIME, DAYS
Fig. 15. Protein production, stirred-jar run 9.

yield and high rate of protein synthesis correlated well with cellulose
utilization.
It is interesting to speculate whether better results could be ob-
tained a t higher substrate concentrations. Runs 8,9, and 10 indicate
that the oxygen transfer rate is limiting protein synthesis a t 4 g of
ball-milled newspaper per 100 ml of medium. Oxygen demand in-
creases with increasing substrate concentration, as does the viscosity
of the medium. The viscosity increase may decrease the oxygen
transfer rate. Thus, it is possible that further increases in the sub-
strate concentration might not give higher protein yields or higher

@ - Protein Produced Fermentor No . I

TIME, DAYS
Fig. 16. Protein production and cellulose utilization, stirred-jar run 11.
BIOTECHNOLOGY AND BIOENGINEERING, VOL. XIII, ISSUE 1
 WASTE PAPER CELLULOSE UTILIZATION 93

-E 1.61 I

\ 1.4 -
rn
1.2-

gi
3
1.0 -

E
a 0.6
o.8;

@-O.l% Yeast Autolysate

@-0.05% Yeaat Autolysate
@-No Yeast Autolysate
I I I I I I ~
5 6 7 8 9 1011 12
TIME, DAYS
Fig. 17. Protein production, stirred-jar run 12.

rates of protein synthesis. Jar 3 of Run 7, where 8 g of substrate per

100 ml of medium was employed, is a case in point. Even after
24-days’ incubation the protein yield was only 0.63 mg/ml, probably
as a result of insufficient oxygen transfer rate.

Composition of Final Product

The final product from Jar 3, Run 10 was selected for complete
chemical analysis because this run produced the highest protein yield
up to that time, because it contained no antifoam material, as did
later runs, and because the time deadline for completion of the project
demanded that the required analytical work be initiated a t once.
The analytical data are given in Table 111, along with similar data

Fig. 18. Cellulose utilized, qtirred-jar run 12.

94 UPDEGRAFF

on corn and wheat,” both of which are excellent feed materials for
cattle, although slightly low in protein to make a complete food for
human consumption. It can be seen that our material is higher in
ash and cellulose, and somewhat lower in protein. It is likely that
these factors would not limit its use as a feed for either monogastric
animals or cattle. It would also be possible to eliminate much of the
salts from the final product by harvesting by means of centrifugation
or filtration , rather than evaporation to dryness.

DISCUSSION
The screening phase of this project selected only those organisms
which were capable of a high rate of protein synthesis in submerged
culture. Myrothecium verrucaria produced the highest yields of pro-
tein, and hence the stirred-jar fermentor studies were carried out with
this organism. This is in agreement with many reports in the litera-
tures emphasizing the very active cellulolytic properties of this fungus.
By comparison with cellulose, lignin is very slowly attacked by
mi~roorganisms,~ and it is probable that little or no lignin was con-
sumed in any of our experiments, although this cannot be stated with
certainty, since direct analyses for lignin were not carried out.
Our data are not sufficient to make a material balance, but some
hypothetical calculations of this type may be given for Jar 1 Run 12,
which gave the highest protein yield.
Paper originally present, g/liter 40
Protein, g/liter (from biuret analysis) 1.42
Protein, g/liter (from organic N X 6.25) 3.3
Cellulose original present, g/liter 20.4
Cellulose consumed, g/liter 12.7.
Assume that 50% of the 12.7 g of cellulose substrate consumed was
oxidized for energy, and 50% was assimilated and converted to cell
material which contains 30% protein. (These values were taken
from Gray’s as being fairly typical for Fungi Imperfecti grown on glu-
cose.) Then the amount of cellulose converted to cell material would
be 6.35 g/liter and the protein would be 1.91 g/liter. Gray deter-
mined protein as organic N x 6.25. Thus, it can be seen that ouI
corresponding protein value, 3.3 g/liter, is considerably higher than
the example given by Gray. The true protein value of our product
BIOTECHNOLOGY AND BIOENGINEERING, VOL. XIII. ISSUE 1
 WASTE PAPER CELLULOSE UTILIZATION 95

(as well as Gray’s) is probably closer to the biuret value of 1.42

g/liter, since N X 6.25 includes in addition to true protein all other
nitrogen compounds’found in the cells such as amino acids, phos-
pholipids, purines and pyrimidines DNA and RNA. In any event,
returning to our product calculations beginning with 40 g/liter of
newspaper, it should be possible to produce 33.7 g/liter of final
product containing 3.3 g/liter of “protein” calculated as N X 6.25.
Ciegler and Lillehojl9do not list Myrothecium verrucaria among the
mycotoxin-producing fungi. However, it would be desirable to con-
duct some preliminary animal feeding studies to rule out toxicity and
establish dietary value before further efforts on scale-up. Some esti-
mates of process economics are also indicated before proceeding fur-
ther. In this connection the work of Callihan and Dunlaps on the
production of microbial protein from bagasse is pertinent. Their
process involves treatment of bagasse with hot alkali to render the
lignocellulose complex digestible by Cellulomonas bacteria, and a con-
tinuous fermentation with Cellulomonas, followed by separation of
undigested cellulose by gravity settling, and thickening of the cell
material by the addition of acid and alcohol. The thickened bacterial
cell paste is harvested by centrifugation and dried. The estimated
cost of producing such a product is 6.6 cents per Ib. The details of
growth rate and cell concentration were not given, but a growth
curve was shown which indicated a much more rapid growth rate than
for our fungi. The recent study of Meller20 gives a more detailed
economic analysis of “Conversion of Organic Safid Wastes into
Yeast.” According to Meller’s calculations Torula yeast (Cartdida
utilis) could be made from acid-hydrolyzed municipal wastes a t a cost
of 9.8 to 13.6 cents per lb. The process does not appear very attrac-
tive when the cost of soybean meal is 3.5 to 6.5 cents per lb., and fish
meal is 6.3 to 8.5 cents per lb. The feedstock was assumed to be an
acid-hydrolyzed municipal waste material containing 6.17’% glucose.
The average cell concentration in a continuous fermentation on this
feedstock was assumed to be 30.85 g/liter, and the production rate
was 3.66 g of yeast per liter-hour. The cell yield was estimated to
be 50% of the substrate (glucose) consumed. Applying this figure
to our best cellulose consumption rate, Run 11, Jar 1, where cellulose
consumption was 5.4 g/liter/day, or 0.22 g per liter-hour, the produc-
tion rate of Myrotheeium verrucaria would be 0.11 g per liter-hour,
disappointingly low compared to 3.66 g per liter-hour. Likewise our
96 UPDEGRAFF

maximum protein yield of 3.3 g/liter ( N X 6.25) obtained in Jar 1,

Run 12, does not compare very favorably with that from Torula
utilis in the above case of 30.85 X 0.55 = 17 g/liter.
Helping to counterbalance the relatively low yield and production
rate of Myrothecium verrucaria is the fact that harvesting of a fila-
mentous fungus could be carried out by means of coarse filtration, as
proposed by Church et a1.,21a more economical process than the cen-
trifugation required with bacteria or yeasts. It is evident,.however,
that our cell yields and rates of protein synthesis on waste paper are
considerably lower than those obtained from Fungi Imperfecti grow-
ing on more readily available soluble substrates by Gray1* and by
Church et a1.,21and it does not appear likely that feed material could
be produced by our process a t a price competitive with soybean meal,
yeast from acid hydrolyzed municipal wastes, or Fungi Imperfecti
grown on concentrated soluble wastes.
This research was supported in part by research grant No. 8 ROIEC, 00271-02
of the Bureau of Solid Waste Management, Environmental Control Administra-
tion, Public Health Service, U.S. Department of Health, Education and Welfare.
The author also wishes to express his appreciation to Dr. Richard H. Kinsley of
East Tennessee State University who identified the fungi, and to the hard-
working and competent technicians who carried out the work, Mr. Larry Griffin,
Mrs. Jeannette King, Mrs. Diane Mack, Mrs. Sharon Zaun, Miss L. Myra
Fuller, and Miss Sandra Hockersmith.

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2. J. A. Gascoigne and M. M. Gascoigne, Biological Degradation of Cellulose,
Butterworths, London (1960).
3. W. C. Day, M. J. Pelczar, Jr., and S. Gottlieb, Arch. Biochem., 23,360-369
(1949).
4. J. P. Casey, Pulp and Paper, Chemistry and Chemical Technology, Vol. I .
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BIOTECHNOLOGY AND BIOENGINEERING, VOL. XIII, ISSUE 1
 WASTE PAPER CELLULOSE UTILIZATION 97

11. V. C. Bode, H. Goebell, and E. Stahler, Z. Klin. Chem. u. Klin. Biochem.,

5 , 4 1 8 4 2 2 (1968).
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Received September 2, 1970